WO2019022463A2 - Système immunogène et vaccin animal le comprenant - Google Patents

Système immunogène et vaccin animal le comprenant Download PDF

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WO2019022463A2
WO2019022463A2 PCT/KR2018/008337 KR2018008337W WO2019022463A2 WO 2019022463 A2 WO2019022463 A2 WO 2019022463A2 KR 2018008337 W KR2018008337 W KR 2018008337W WO 2019022463 A2 WO2019022463 A2 WO 2019022463A2
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immunogenic
spore
protein
recombinant
base sequence
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Korean (ko)
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WO2019022463A3 (fr
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박현식
성노현
오상욱
오승준
박희용
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Jb Biotech Inc
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Jb Biotech Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides

Definitions

  • the present application relates to immunogenic substances, immunogenic systems, and animal vaccine compositions comprising them, which are directed to pathogenic agents of certain diseases of animals.
  • Animal vaccine-related technologies are used not only for the prevention of livestock diseases but also for pandemic zoonosis such as biological weapons (bruises, anthrax), SARS (Severe Acute Respiratory Syndrome) and AI (avian influenza) It has contributed greatly to the development of countermeasure technologies and is recognized as a strategic industry nationwide.
  • PCV Porcine CircoVirus
  • PMWS postweaning multi-systemic wasting syndrome
  • the PRRS virus which is an outdoors mutation, is also one of the most troublesome variant diseases. Newcastle Disease (IB), Infectious Bronchitis (IB), Infectious Bronchitis Disease (IBD) and AI (Avian Influenza) virus, which are problems in the poultry industry, have been developed in Korea as well. As a result, a new subtype of genotype and anti-circularity has been reported. The key to technological development is to reduce the disparity between outdoor disease agents and vaccine antigens
  • live vaccines are essential to compensate for ineffective vaccine use, but it takes a lot of time and money to acquire a master seed that is capable of live vaccination from pathogenic outdoor viruses.
  • Patent Document 1 Korean Patent No. 10-0578395
  • Patent Document 2 Korean Patent No. 10-0267745
  • Patent Document 3 Japanese Patent Laid-Open No. 2008-231063
  • Patent Document 4 4. WO 2016-140702
  • Non-Patent Document 1 Microbiol Spectr. 2014 Oct; 2 (5). doi: 10.1128 / microbiolspec.TBS-0011-2012.
  • Non-Patent Document 2 J Bacteriol. 2001 Nov; 183 (21): 6294-6301. Surface Display of Recombinant Proteins on Bacillus subtilis Spores
  • Non-Patent Document 3 3. Vaccine. 2004 Mar 12; 22 (9-10): 1177-87. Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner.
  • Non-Patent Document 4 4. Microbiology. 2009 Feb; 155 (Pt 2): 338-46, Phagocytosis, germination and killing of Bacillus subtilis spores presenting heterologous antigens in human macrophages.
  • Non-Patent Document 5 Sun D et al., Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein. Vet Microbiol. 2008; 131 (1-2): 73-81.
  • Non-Patent Document 6 Gallagher TM, Buchmeier MJ. Coronavirus spike proteins in viral entry and pathogenesis. Virology. 2001; 279 (2): 371-4.
  • Non-Patent Document 7 Tsuda T. Porcine epidemic diarrhea: its diagnosis and control. Proc Jpn Pig Vet Soc. 1997; 31: 21-8.
  • Non-Patent Document 8 8. Georgiou, G. et al., Display of heterologous proteins on the surface of microorganisms: From the screening of combinatorial libraries to live recombinant vaccines. Nature Biotechnology 15: 29-34 (1997)
  • the object of the present application is to provide a variety of sunscreens and various uses of immunogenic materials, immunogenic systems and animal vaccine compositions comprising them, which are directed against pathogenic agents of particular disease in animals.
  • One purpose is to provide animal vaccines against disease causing pathogens and methods for their manufacture.
  • Another goal is to provide an immunogenicity system for disease causing pathogens.
  • Another goal is to provide an immunogenic spore system.
  • Another object is to provide a vaccine against PEDV and an immunogenicity system therefor.
  • porcine disease virus is at least one of porcine epidemic diarrhea virus (PEDV) and porcine circovirus (PCV).
  • PEDV porcine epidemic diarrhea virus
  • PCV porcine circovirus
  • an immunogenic spore fusion display system characterized in that an immunogenic site derived from porcine epidemic diarrhea virus (PEDV) and porcine circovirus (PCV) is fused to spore coat protein,
  • PEDV porcine epidemic diarrhea virus
  • PCV porcine circovirus
  • concentration of the immunogenic spore fusion display system is 10 < 12 > to 10 < 14 > cells / ml.
  • the present application also provides two sets of primers selected from SEQ ID NOS: 1-4 for obtaining immunogenic regions of porcine epidemic diarrhea virus (PEDV) outdoor strains.
  • PEDV porcine epidemic diarrhea virus
  • the present application also provides two sets of primers selected from SEQ ID NOS: 41-46 for obtaining immunogenic regions of Porcine Circovirus (PCV) outdoor strains.
  • PCV Porcine Circovirus
  • the present application relates to a method for producing a spore-
  • a spore coat protein present in the outer region of the spore of the spore
  • the present application also relates to an immunogenic spore fusion display system characterized in that an immunogenic site derived from porcine epidemic diarrhea virus (PEDV) or Porcine Circovirus (PCV) is fused to spore coat protein.
  • the present invention provides a method for treating a PEDV-infected animal other than a human, which comprises administering 2 ml of the vaccine composition containing the compound at a concentration of 12 to 10 14 cells / ml.
  • an animal disease inducer (antigen) with enhanced immunogenicity and a method of using the same.
  • an animal disease inducer preferably a domestic animal, such as a pig
  • an antigen is enhanced, thereby also enhancing animal protection against certain disease infections
  • the use of the technology of this application is superior in the expression rate and the acquisition rate as compared with the existing expression system.
  • the recombinant protein produced from the recombinant cell line is used as an antigen, an immune response can be effectively induced, and it is useful for vaccines, medicines and feeds for prevention, improvement and treatment of diseases of domestic animals (for example, pigs) Can be used to make.
  • 1 is a schematic diagram showing the structure of an agent.
  • FIG. 2 is a schematic diagram showing the structure of a 1-antigen protein and a 2-antigen protein on a pathogen.
  • Figure 3 is a graph showing the location of the immunogenic site of the 1-antigen protein (spike protein) of the PEDV pathogen
  • Figure 4 is an exemplary graph of antigen homologous proteins comprising immunogenic sites.
  • Figure 5 is an exemplary graph of antigen homologous proteins comprising two or more selected from immunogenic site-1 to immunogenic site-n.
  • Figure 6 is an exemplary graph of antigen homologous proteins comprising non-immunogenic sites, including immunogenic sites.
  • Figure 7 is an exemplary graph of antigen homologous proteins comprising two or more selected from the 1-immunogenic site to the m-immunogenic site.
  • Figure 8 is an exemplary graph of an antigen homologous protein comprising a site that is homologous to an antigenic protein and a non-antigenic moiety.
  • FIG. 9 is an exemplary graph of an antigen homologous protein comprising a site and an exon portion that is homologous to the antigen protein.
  • FIG. 10 is an exemplary graphical representation of the antigen homologous portion of a protein comprising a region that is homologous to the antigen protein and a non-antigenic portion and an exogenous portion.
  • FIG. 11 is a schematic diagram showing the structure of an immunogenic inner support system in an immunogenic system.
  • FIG. 12 is a schematic diagram showing the structure of an immunogenic fused display system in an immunogenic system.
  • Figure 13 is a schematic diagram illustrating the structure of an immunogenic coupled display system in an immunogenic system.
  • Figure 14 is a schematic diagram illustrating the structure of an immunogenic attachment display system in an immunogenic system.
  • 15 is a schematic diagram illustrating the structure of an immunogenic cross-coupled display system in an immunogenic system.
  • 16 is a schematic diagram of a serial connection system that exemplary illustrates an embodiment of a fused display system in a system.
  • Figure 17 is a schematic diagram of a parallel system exemplary of an embodiment of a fusion display system.
  • FIG. 18 is an exemplary schematic diagram of an antigen-homologous protein recombinant expression vector in which the immunogenic expression objective base sequence comprises an immunogenic site.
  • 19 is an exemplary schematic diagram of an antigen-homologous protein recombinant expression vector in which the immunogenic expression objective base sequence comprises two or more base sequences selected from immunogenic site-1 to immunogenic site-n.
  • the immunogenic expression objective base sequence comprises two or more base sequences selected from the 1-immunogenic site to the m-immunogenic site.
  • 21 is an exemplary schematic diagram of a pathogen-like particle recombinant expression vector in which the immunogenicity expression objective base sequence is an immunogenic site, and the antigen-homologous portion including the base sequence of the non-invasive portion is an example of a protein recombinant expression vector.
  • FIG. 22 is an exemplary schematic diagram of an accessory immunostimulatory recombinant expression vector in which the immunogenic expression objective base sequence is an immunogenic site, and the antigen-homologous portion including a base sequence of a non-antigenic portion is an example of a protein recombinant expression vector.
  • FIG. 23 is an exemplary schematic diagram of a multivalent immunogenic recombinant expression vector in which the immunogenicity expression objective base sequence is an immunogenic site, and the antigen-homologous portion including the foreign base sequence is an example of a protein recombinant expression vector.
  • FIG. 24 is an exemplary schematic diagram of a multivalent pathogen-like particle recombinant expression vector in which the immunogenicity expression target base sequence is an immunogenic site, and the antigen-homologous portion including the foreign base sequence is an example of a protein recombinant expression vector.
  • 25 is an exemplary schematic diagram of an immunogenic serial-linkage format expression vector in which the component expression target base sequence comprises the base sequence of the x-th protein and the y-th junction;
  • a-1 first protein
  • b-1 first connection
  • a-2 second protein
  • b-2 second connection
  • a- 4 fourth protein
  • b-4 fourth junction
  • a-5 fifth protein
  • Figure 27 is a schematic diagram of an immunogenic serial linkage system expressed by the immunogenic serial-linked system recombinant expression vector of Figure 26;
  • FIG. 28 shows an example of a single immunogenic parallel system recombinant expression vector in which a system of a plurality of parallel bodies is a system expression target sequence, wherein the first parallel body is an internal carrying system, the second parallel body and the third parallel body are fusion display systems Lt; / RTI > recombinant expression vector.
  • Figure 29 is a schematic diagram of an immunogenic parallel system expressed by the immunogenic parallel system recombinant expression vector of Figure 28;
  • FIG. 30 is an exemplary schematic diagram of an immunogenic spore internal carrier system recombinant expression vector in which the spore system expression target base sequence comprises the base sequence of spore-bearing immunogenic material.
  • FIG. 31 is an exemplary schematic diagram of an immunogenic spore internal carrier system recombinant expression vector in which the spore system expression target base sequence comprises a base sequence of a substance charged with a spore-bearing immunogenic substance.
  • FIG. 32 is an exemplary schematic diagram of an immunogenic spore fusion display system recombinant expression vector in which the spore system expression target base sequence comprises a spore surface construct and a base sequence of a spore fusion immunogenic material.
  • FIG. 33 is an exemplary schematic diagram of an immunogenic spore fusion display system recombinant expression vector in which the spore system expression target base sequence comprises a plurality of spore external homology regions and a base sequence of spore fusion immunogenic material.
  • Figure 34 shows an example of an immunogenic spore fusion display system recombinant expression vector comprising a spore system expression target base sequence comprising a spore external signal sequence of a tachypse, a spore exogenous homology region, and a base sequence of a spore fusion immunogenic material It is a schematic diagram.
  • 35 shows an example of an immunogenic spore fusion display system recombinant expression vector comprising a spore system expression target base sequence comprising a spore outermost locating motif of a taut protein, a spore outer homology region, and a base sequence of spore fusion immunogenic material It is a schematic diagram.
  • FIG. 36 shows an immunogenic spore fusion display system comprising a spore system expression target base sequence comprising a spore outer locating motif of a heterosporum outer protein, a spore outer homology region, and a base sequence of a spore fusion immunogenic material.
  • a spore system expression target base sequence comprising a spore outer locating motif of a heterosporum outer protein, a spore outer homology region, and a base sequence of a spore fusion immunogenic material.
  • FIG. 37 shows an exemplary schematic diagram of an immunogenic spore fusion display system recombinant expression vector comprising a spore outline, a spore outer homology region, and a base sequence of a spore fusion immunogenic material, wherein the spore system expression base sequence is charged with a specific sign; to be.
  • FIG. 38 is an exemplary schematic diagram of an immunogenic spore-attached display system recombinant expression vector in which the spore system expression target base sequence comprises the base sequence of spore-attaching immunogenic material.
  • 39 is an exemplary schematic diagram of an immunogenic spore-attached display system recombinant expression vector in which the spore-system expression base sequence comprises a base sequence of a substance charged with a spore-bearing immunogenic substance.
  • FIG. 40 is a system diagram showing a procedure for selecting vaccine strains to be master vaccination using a phylogenetic tree by multiple sorting.
  • FIG. 41 is a fluorescence microscope photograph to determine whether Bacillus transformed when CotG was used in the production of PEDV vaccine.
  • FIG. 42 is a graph showing the results of flow cytometry experiments to determine whether Bacillus transformed when CotG was used in the production of PEDV vaccine.
  • FIG. 42 is a graph showing the results of flow cytometry experiments to determine whether Bacillus transformed when CotG was used in the production of PEDV vaccine.
  • FIG. 43 is an optical microscope photograph of Bacillus taken after Malachite green statining in order to examine whether bacillus was expressed in spores when CotG was used in the production of PEDV vaccine.
  • 45 is a graph showing the results of an immunoreaction confirmation experiment when CotG is used in the production of a PEDV vaccine.
  • FIG. 46 is a graph showing the results of immunoblotting assay for confirming the generation of an antigen-specific antibody when CotG was used in the production of PEDV vaccine.
  • FIG. 47 is a fluorescence microscope photograph to determine whether Bacillus transformed when CotB was used in the production of PEDV vaccine.
  • 48 is a graph showing the results of flow cytometry experiments to determine whether Bacillus transformed when CotB was used in the production of PEDV vaccine.
  • FIG. 49 is an optical microscope photograph of Bacillus taken after performing Malachite green statining in order to determine whether Bacillus was expressed in spores when CotB was used in the production of PEDV vaccine.
  • 50 is a graph showing the results of an immunoreaction confirmation experiment when CotB is used in the production of a PEDV vaccine.
  • 51 is a graph showing the results of an immunoblotting assay test for confirming the generation of an antigen-specific antibody when CotB is used in the production of a PEDV vaccine.
  • FIG. 52 is a fluorescence microscope photograph showing whether Bacillus transformed when CotC was used in the production of PEDV vaccine.
  • FIG. 52 is a fluorescence microscope photograph showing whether Bacillus transformed when CotC was used in the production of PEDV vaccine.
  • 53 is a graph showing the results of flow cytometry experiments to determine whether Bacillus transformed when CotC was used in the production of PEDV vaccine.
  • 55 is a graph showing the results of an immunoreaction confirmation experiment when CotC is used in the production of a PEDV vaccine.
  • FIG. 56 is a graph showing the results of immunoblotting assay for confirming the generation of an antigen-specific antibody when CotC is used in the production of PEDV vaccine.
  • FIG. 57 is a graph showing the results of flow cytometry experiments to determine whether Bacillus transformed when CotG was used in the production of a PCV vaccine.
  • FIG. 58 is a graph showing the results of flow cytometry experiments to determine whether Bacillus transformed when CotB was used in the production of PCV vaccine.
  • FIG. 59 is a graph showing the results of flow cytometry experiments to determine whether Bacillus transformed when CotC was used in the production of a PCV vaccine.
  • FIG. 60 is a graph showing the results of an immunoreaction confirmation experiment when PCV-2A was used in the production of a PCV vaccine.
  • 61 is a graph showing the results of an immunoreaction confirmation experiment when PCV-2B was used in the production of a PCV vaccine.
  • 62 is a graph showing the results of an immunoreaction confirmation experiment when PCV-m2B was used in the production of PCV vaccine.
  • 63 is a graph showing the results of an immunoblotting assay test for confirming the generation of an antigen-specific antibody when using PCV-2A in the production of a PCV vaccine.
  • 64 is a graph showing the results of an immunoblotting assay test for confirming the production of an antigen-specific antibody when using PCV-2B in the production of PCV vaccine.
  • 65 is a graph showing the results of an immunoblotting assay test for confirming the generation of an antigen-specific antibody when using PCV-m2B in the production of a PCV vaccine.
  • the present application relates to materials, systems and methods involved in the immune response.
  • it is involved in the mechanism of the immune response caused by infection by pathogens.
  • Cell-mediated immunity is an immunity that cytotoxic T cells kill cells infected individually by pathogens.
  • the cells infected with the pathogen start MHC-I protein on the surface of the pathogen, and cytotoxic T cells recognize it and induce apoptosis.
  • Humoral immunity is an immune system that produces antibodies to the antigen and kills the pathogen.
  • dendritic cells, macrophages, and B cells that have ingested antigens of pathogens begin to express antigens on the cell surface using MHC-II proteins.
  • B cells recognize and activate the antigen
  • Cytokines release B cells and cytotoxic T cells.
  • Activated B cells are differentiated into plasma cells and memory B cells and are involved in the production of antibodies.
  • B cells differentiated into plasma cells function to kill the pathogen by generating antibodies against the antigen.
  • B cells differentiated into memory B cells do not produce antibodies but are present in body fluids, and when the pathogen invades again, they differentiate into plasma cells and memory B cells.
  • the immune response exhibits a unique pattern of primary immunity and secondary immunity when the organism is first infected with a pathogen and when it is secondary infected.
  • a virus When the virus is first infected with a pathogen, a small number of plasma cells are produced because memory B cells are not present. Therefore, a primary immunity with a slowly increasing antibody concentration occurs.
  • the memory B cells formed in the first immunization process are directly differentiated into plasma cells, resulting in a second immunization, that is, a second immunization in which the antibody concentration rapidly increases. Therefore, if the organism undergoes first immunization by the first infection, it will cope with the pathogen more efficiently through the acquired immunity.
  • Vaccines function to induce host immune responses to pathogens by generating these memory B cells without infection of the pathogen.
  • Existing vaccines can be classified according to which immunogenic material they contain.
  • First there is a form that uses the pathogen itself.
  • the first vaccine was a diluted solution of a wild-type pathogen in the solution, but the risk of infection was high.
  • Second there is a form that uses only the antigen protein or surface protein of the pathogen.
  • Antigen proteins are less virulent than pathogens. Antigen proteins are either isolated from pathogens or produced by genetic engineering.
  • the present application relates to immunogenic substances, immunogenic systems, and animal vaccine compositions comprising them, which are directed to pathogenic agents of certain diseases of animals.
  • the present invention relates to a recombinant immunogenic system using only an antigenic protein or surface protein of a pathogen, and a recombinant animal vaccine composition containing the recombinant immunogenic system.
  • a recombinant immunogenic system using only an antigenic protein or surface protein of a pathogen, and a recombinant animal vaccine composition containing the recombinant immunogenic system.
  • One aspect of the present application relates to a vaccine.
  • vaccine is meant a substance or antigen that forms an acquired immunity against a particular disease in a given organism.
  • Adaptive immune system means a cellular immune system of the living body, which means immune state present in memory B cells differentiated by specific antigen.
  • Immunogen means a substance that causes immunity in vivo as a component of a vaccine.
  • &Quot Native " refers to a state in which a material is obtained in the natural world without artificial manipulation.
  • One embodiment of the present application is a vaccine comprising an immunogenic agent.
  • the immunogenic material includes all substances capable of causing an acquired immune response, and may be, for example, a pathogen, an antigen protein, an antigen homologous protein, an antigen homologous additive protein.
  • Pathogen is a term collectively referred to as a disease causing organism, including viruses, bacteria, fungi, and algae.
  • a " virulence portion” means a site that is virulent as part of a pathogen.
  • a “ maintenance portion” means a portion of a pathogen that participates in metabolism or proliferation.
  • One example of the present application may be a vaccine comprising the pathogen itself as an immunogenic substance.
  • the pathogen may disclose one or more antigenic proteins in the outer membrane of the pathogen, and the antigenic proteins may be only one kind, but various kinds of proteins may function as antigen proteins.
  • the pathogen containing such an antigen protein itself can be used as an immunogenic substance of the vaccine.
  • the pathogen may be in a wild state.
  • the pathogen may be a deletion / modification of a particular site.
  • the specific site may be a virulent site.
  • a particular site may be a viable site.
  • the specific site may be an immunogenic site within the antigen protein.
  • the pathogen may be one whose structure has been modified or engineered.
  • the modification and manipulation can be done naturally or artificially.
  • the overall structure of the pathogen may be 80% to 100% homologous to the wild-type pathogen.
  • the homology is 90 to 100%, more preferably 95 to 100%.
  • the pathogen may be a virus belonging to the same family.
  • the same term may be Coronaviridae.
  • the same may also be the Arteriviridae.
  • the same may also be the Circoviridae.
  • the same may also be Paramyxoviridae.
  • the pathogen may be a pathogen of a swine-target disease.
  • the pathogen of the swine disease can be Porcine Epidemic Diarrhea Virus (PEDV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Circovirus (PCV).
  • PEDV Porcine Epidemic Diarrhea Virus
  • PRRSV Porcine Reproductive and Respiratory Syndrome Virus
  • PCV Porcine Circovirus
  • the pathogen may be a pathogen of a poultry-related disease.
  • the pathogen of the poultry disease may be Infectious Bronchitis Virus (IBV) or Newcastle Disease Virus (NDV).
  • IBV Infectious Bronchitis Virus
  • NDV Newcastle Disease Virus
  • Antigen protein means a protein that is part of a pathogen and causes immunity in vivo.
  • Non-invasive refers to any part of the structure of the pathogen except the antigen protein.
  • immunogenic portion refers to a site that specifically causes an immune response in an antigen protein.
  • nonimunogenic portion refers to a portion of an antigenic protein that excludes an immunogenic portion.
  • the structure of the pathogen can be roughly divided into antigenic proteins and non-antigenic components that cause immunity.
  • the antigen protein may be more than one species.
  • Each antigen protein is divided into immunogenic and non-immunogenic regions.
  • the immunogenic site may be one or more species.
  • the non-antigenic portion refers to the nucleotide sequence / protein compartment of the pathogen, excluding the antigen protein portion in the full length sequence of the pathogen, and includes the capsid protein of the pathogen.
  • x-antigen protein is an antigenic protein and refers to the xth of the m kinds of antigenic proteins in a particular pathogen.
  • the " x-immunogenic site-y" is an immunogenic site and is present on the xth antigen protein of the m kinds of antigen proteins in the specific pathogen, and the yth immunity among the n kinds of immunogenicity sites on the xth antigen protein It means the original site.
  • x-non-immunogenic site refers to a non-immunogenic site present on the x-th antigen protein of the m types of antigenic proteins in a particular pathogen as non-immunogenic sites.
  • One example of the present application may be a vaccine containing the antigenic protein as an immunogenic substance.
  • the antigenic protein may be one kind of antigenic protein. Wherein the antigen protein may be in a wild state.
  • the antigen protein may be a specific site modified or modified.
  • the modification and manipulation can be done naturally or artificially.
  • the specific site may be an immunogenic site.
  • the antigen protein may be a mixture of two or more selected from 1-antigen protein to m-antigen protein.
  • the antigen protein is an antigenic protein of PEDV.
  • the antigen protein of PEDV is known as a spike protein.
  • the PEDV spike protein is an I-type glycoprotein consisting of 1,388 amino acids (aa). Spike proteins can be divided into S1 (1-794 aa) and S2 (794-1,388 aa) domains based on their homology with the spike proteins of other coronaviruses. In coronavirus, spike proteins are surface antigens, where they regulate the interaction with host cell receptor glycoproteins to mediate viral entry and serve to stimulate the induction of neutralizing antibodies in the natural host. Thus, spike glycoprotein is the primary target for the development of an effective vaccine against PEDV.
  • SEQ ID NO: 1 is the protein sequence of the spike protein of CV777 of PEDV, and unless otherwise stated, the sequence of all the antigenic proteins and the location of the immunogenic site is described in reference to CV777 (ncbi Accession No. JN599150.1) One, this is only a guideline, so the application is not limited to CV777 weeks.
  • sequence of SEQ ID NO: 17 was used as a partial sequence of the spike 1 protein.
  • sequence of SEQ ID NO: 18 was used as a partial sequence of the spike 2 protein.
  • a 1-antigen protein refers to a spike protein.
  • the M protein and N protein of PEDV can be antigen proteins, and other antigen proteins can be found.
  • the new antigen protein may be naturally contained as a 3-antigen protein or an x-antigen protein as necessary, but it should not be regarded as an object of the present application.
  • the antigen protein is an antigenic protein of PCV.
  • the antigen protein may be a capsid protein
  • the capsid protein of the PCV may include, but is not limited to, ORF1, ORF2, ORF3, ORF4, and the like.
  • the capsid protein of the PCV may be ORF2.
  • SEQ ID NO: 47 was used for the sequence of the capsid protein.
  • the PCV may be classified into genotypes such as PCV2A, PCV2B, PCVm2B, PCV2C and PCV2D, but is not limited thereto.
  • the PCV of the present application may be any one or more selected from among PCV2A, PCV2B and PCVm2B.
  • antigenic proteins other than the capsid protein can be found. Such a case is also included as an object of the present application.
  • the antigenic protein includes immunogenic regions, which are sites that strongly induce immunity.
  • the immunogenic site may be a specific site of the antigen protein and may vary in size from 6aa to 300aa. In general, immunogenic regions of pathogens can be easily found in theses.
  • the immunogenic region of PEDV is known as 504-643aa (COE), 753-760 (SS2), 769-776 (SS6), 1373-1379 (2C10) based on the CV777 strain (see Figure 3) .
  • the immunogenic portion of the spike protein of PEDV comprises 504-643 aa (COE, 1-immunogenic portion-1), 753-760 (SS2, 1-immunogenic portion- 2), 769 to 776 (SS6, 1-immunogenic site-3), 1373 to 1379 (2C10, 1-immunogenic site-4).
  • the immunogenic portion of the new antigen protein may be included as an x-immunogenic portion-1 to an x-immunogenic portion -y as needed.
  • the immunogenic site of PCV may be a fragment of at least one protein selected from ORF1, ORF2, ORF3, and ORF4 and / or a fragment of a polynucleotide.
  • it may include, but is not limited to, forms of one or more proteins selected from ORF1, ORF2, ORF3 and ORF4 and / or a part of a polynucleotide (truncation, substitution, etc.).
  • the immunogenic region of the new antigen protein may optionally include x-immunogenic site-1 to x-immunogenic site-y.
  • Antigen homologous protein means a protein having homology to the structure of an antigenic protein.
  • One example of the present application may be a vaccine containing the antigen homologous protein as an immunogenic substance.
  • the antigen homologous protein may comprise an immunogenic site.
  • the antigen homologous protein may comprise two or more selected from immunogenic site-1 to immunogenic site-n.
  • the sequence of the immunogenic site may be different from the wild-type antigen protein.
  • the antigen homologous protein may comprise a non-immunogenic region including an immunogenic site.
  • non-immunogenic portion may be located in front of the N-terminus of the immunogenic portion.
  • the non-immunogenic site may also be located after the C-terminus of the immunogenic site.
  • the non-immunogenic portion may also be located at both the N and C termini of the immunogenic portion.
  • Figure 4 is an example of an antigen homologous protein comprising a 1-non-immunogenic site at both the 1-immunogenic site-1 and the N, C -terminus of the immunogenic site.
  • the non-immunogenic site may be a specific base sequence.
  • the specific nucleotide sequence may be 18 bp, 22 bp, 28 bp, 31 bp or 36 bp.
  • the specific nucleotide sequence has a size of 18 to 22 bp.
  • the antigen homologous protein may comprise at least two selected from the 1-immunogenic site to the m-immunogenic site.
  • the antigen-homologous protein may have a non-immunogenic site at both the N and C terminals.
  • the non-immunogenic site may be a specific base sequence.
  • the specific nucleotide sequence may be 18 bp, 22 bp, 28 bp, 31 bp or 36 bp.
  • the specific nucleotide sequence has a size of 18 to 22 bp.
  • Figure 5 is an illustration of an antigen homologous protein comprising a 1-non-immunogenic site at both the 1-immunogenic sites-1 and 2 and the N, C-terminus of the 1-immunogenic site-1.
  • the antigen homologous protein may comprise one or more selected from the 1-immunogenic site to the m-immunogenic site and the 1-non-immunogenic site to the m-non-immunogenic site .
  • non-immunogenic regions may be located at both N and C termini.
  • the non-immunogenic site may be a specific base sequence.
  • the specific nucleotide sequence may be 18 bp, 22 bp, 28 bp, 31 bp or 36 bp.
  • the specific nucleotide sequence has a size of 18 to 22 bp.
  • FIG. 6 shows a schematic representation of a 1-non-immunogenic portion at the N-terminus of the 1-immunogenic portion-2 and the 2-immunogenic portion-1 and the 1-immunogenic portion- Is an example of an antigen homologous protein comprising a non-immunogenic region.
  • Figure 7 is an example of an antigen homologous protein comprising a 1-immunogenic portion-1, a 2-immunogenic portion-1 and a 2-immunogenic portion-2 and a 1-non-immunogenic portion and a 2-non-immunogenic portion .
  • the immunogenic portion may comprise the 1-immunogenic portions -1 to 4 of PEDV.
  • antigen-homologous protein of PEDV may comprise COE.
  • antigen-homologous protein of PEDV may comprise SS2 and COE in that order.
  • the antigen-homologous protein of PEDV may comprise SS2 and SS6.
  • antigen-homologous protein of PEDV may comprise the S1 domain of the spike protein.
  • the antigen-homologous protein of PEDV may comprise the COE of the spike protein and the immunogenic site of the PEDV M protein.
  • the immunogenic site may comprise a capsid protein of PCV.
  • the capsid protein of the PCV may be an antigen homologous protein.
  • the antigen-homologous protein of the PCV may comprise an ORF.
  • the antigen-homologous protein of the PCV may comprise any one or more of ORF1, ORF2, ORF3 and ORF4.
  • the antigenic homologous protein of the PCV may comprise an ORF2 of a capsid protein.
  • antigen homologous protein contained additive body means a protein that simultaneously contains a site that is not homologous to the structure of an antigen protein having some homology with the structure of the antigen protein. That is, the antigen-homologous inclusion-added protein refers to a form in which the antigen-homologous protein is additionally contained in a site that is not homologous to the antigenic protein.
  • antigen homologous inclusion addition protein is used herein interchangeably with the term " antigen homologous addition protein ".
  • Foreign portion refers to an exogenously introduced protein or base sequence that is not present in the wild-type original pathogen.
  • One example of the present application may be a vaccine comprising an antigen homologous inclusion-adding protein as an immunogenic substance.
  • the antigen-containing inclusion proteins include sites that are not homologous to the antigen protein, and sites that are not homologous may be non-antigenic or exogenous to the pathogen.
  • the antigen-homologous inclusion-adding protein may comprise a site that is homologous to the antigen protein and a non-antigenic moiety.
  • Figure 8 is an example of an antigen homology inclusion-adding protein comprising 1-immunogenic portions-1 and 2 and 1-non-immunogenic portions, and non-antigenic portion.
  • the non-antigenic portion may be homologous to the outer membrane protein of the pathogen.
  • the antigen homologous inclusion-adding protein may function as a pathogen-like particle.
  • the pathogen-like particle may be a virus-like particle.
  • the non-antigenic portion may also function as an immunosuppressive function.
  • the immune assistant function may be to suppress the secretion of cytokines that promote the secretion of cytokines related to the immune response or inhibit the immune response in the living body.
  • the immunocompetent function may be to degrade the membrane of the immune cell.
  • the outer membrane protein of the pathogen may be the E protein of PEDV.
  • the antigen-homologous inclusion-adding protein may comprise a site and an exogenous portion that is homologous to the antigen protein.
  • Figure 9 is an example of an antigen homology inclusion additional protein comprising 1-immunogenic locus-1 and 2-immunogenic locus-2 and 1-non-immunogenic locus, 2-non-immunogenic locus, and exogenous locus.
  • the foreign part may be an immunogenic site on the antigen protein of another pathogen.
  • the foreign part may be a non-immunogenic part or a non-antigenic part on the antigen protein of another pathogen.
  • the antigen homologous protein may function as a pathogen-like particle of another pathogen.
  • the pathogen-like particle may be a virus-like particle.
  • the foreign part can also function as an immune assistant.
  • the immune assistant function may be to suppress the secretion of cytokines that promote the secretion of cytokines related to the immune response or inhibit the immune response in the living body.
  • the immune-assisted function may also aid in the recognition of antigen-recognizing cells.
  • the foreign part may be an Fc variant of the antibody.
  • Immunosuppressive function may also be the degradation of the membrane of immune cells.
  • the immune helping function may be to degrade the macrophage membrane.
  • the outpatient department can be a listeria orchid.
  • the antigenic homologous protein may comprise a region that is homologous to the antigenic protein, a non-antigenic portion and an extraneous portion.
  • Figure 10 is an illustration of an antigen-homologous inclusion-adding protein comprising a 1-immunogenic site-2 and a 2-non-immunogenic site, as well as different first and second foreign and non-antigenic moieties.
  • System structure means a structure that functions to transmit a target substance such as an immunogenic substance in a living body while positioning the target substance within a certain distance.
  • System means a target material that is delivered while being located within a certain distance of the system structure; And a system structure including a system structure that functions to transfer the same into a living body, and a mechanism in which such a system operates.
  • Immunogenic system means an immune response system consisting of an immunogenic material and a system construct.
  • One example of the present application is a vaccine comprising an immunogenic system.
  • the immunogenic system has an essential constitution of a system structure for delivering an immunogenic substance and an immunogenic substance in vivo.
  • the structure and characteristics of the immunogenic substance are as mentioned above.
  • the immunogenic material and the system structure are transferred as a system, positioned within a certain distance from each other in vivo.
  • Vaccines using immunogenic systems are more stable and less risky than vaccines that only use immunogenic substances directly.
  • the effect of the system can be controlled by controlling the immunity.
  • system structure of the immunogenic system can include known elements that aid in the function of the vaccine, such as immunosuppressants.
  • the system structure functions to transmit the immunogenic material in vivo while being located within a certain distance.
  • the system structure may be, for example, a carrier for tissue engineering, a polymer, a particle, a microorganism, and the like, as long as it is capable of efficiently delivering an immunogenic substance in vivo.
  • a preferred embodiment is a microorganism such as bacteria. It is known that the matching of microorganisms with immunogenic substances of other pathogens has the advantage of generating a more persistent and powerful immune response (Georgiou et al., 1997). In addition, when the system structure is a pathogen, there is an advantage that it can act as a multivalent vaccine together with an immunogenic substance.
  • the system structure preferably has a size such that it can be administered into an animal, for example, its diameter can be on the order of about 0.02 ⁇ to about 5 ⁇ .
  • the immunogenic system has an advantage that it can cause immunity even in an extreme condition such as a stomach of a living body.
  • the system structure may be a virus.
  • the virus may cause a complex disease with the pathogen.
  • the virus may be a bacteriophage.
  • the virus may be a poxvirus.
  • the virus may be an adenovirus.
  • the virus may be a herpes virus.
  • the virus may be a Newcastle disease virus.
  • the virus may be a virus in which the infected animal is a livestock.
  • the animal is a pig.
  • the virus may be a virus in which the infected animal is a bird.
  • the alga is a chicken.
  • the system structure may be bacteria.
  • the bacterium may cause a complex disease with a pathogen.
  • the bacteria may be gram-negative homogeneous.
  • the gram negative bacteria may be homogenous gastric diseases.
  • the gastrointestinal bacterium can be E. coli.
  • the gastrointestinal disease bacterium may also be Salmonella.
  • the gram negative bacteria may be a lung disease homogeneous.
  • the gram negative bacteria may be Pasteurella multocida.
  • the bacteria may be gram positive homogeneous.
  • the gram-positive bacteria may be a lung disease homogeneous. Wherein the lung germ can be Mycoplasma hyopneumoniae.
  • the gram positive bacteria may also be lactobacillus.
  • the gram-positive bacteria may also be Bacillus. Wherein the Bacillus may be B. subtilis, B. anthrasis, B. thuringiensis, or B. cereus.
  • the system structure may also be a spore.
  • the spores may be plant spores.
  • the spores may be spores of yeast.
  • the spores may be spores of fungi.
  • the spore may be a spore of spore-forming bacteria.
  • the spore bacterium may be Clostridium, for example, C. difficile, and the like.
  • the spore bacterium may be sporolactobacillus.
  • the sporozoite bacteria may be Bacillus.
  • the Bacillus may be, for example, B. subtilis, B. anthrasis, B. thuringiensis, B. cereus, and the like.
  • the system structure may also be an artificial structure.
  • the artificial structure may be a particle or a rigid body. Further, the artificial structure may be one in which the outer membrane is coated with a phospholipid.
  • One example of the present application is a vaccine comprising an immunogenic internal support system.
  • Internal carrying system means a system having a form in which a target material is supported in a system structure as one embodiment of the system.
  • Carrying structure refers to a structure that carries an immunogenic substance carried therein as a system structure.
  • Immunogenic internal carrying system means that the immunogenic material is carried in the system structure, as shown in Fig. 11, as an example of the immunogenic system.
  • the system structure can be expressed as a "carrier”.
  • the immunogenic substance can be expressed as " supported immunogenic substance ".
  • the carrier comprises the system structure of the immunogenic system.
  • the interior of the carrier may have a specific size or larger.
  • an amount capable of containing an effective amount of an immunogenic substance capable of causing an immune response is preferred.
  • the carrier may have an internal charge. At this time, electric charge inside the carrier can cause charge and attractive force of the supported immunogenic substance.
  • the carrier may have an external charge. At this time, charges outside the carrier can cause charge and repulsion of the carrier immunogen.
  • the outer membrane of the carrier may be porous.
  • the carrier may be degradable in its outer membrane.
  • the outer membrane may contain an action site of a specific restriction enzyme.
  • the outer film may be in a state where the film is entirely deformed or manipulated in the wild state.
  • the supported immunogenic material encompasses the immunogenic material described above.
  • the carrier immunogenic material may be of a specific size or less. Preferably an amount sufficient to contain an effective amount of the supported immunogenic material capable of causing an immune response in the carrier.
  • the carrier immunogenic material may be charged. At this time, the charge of the supported immunogenic material may be opposite to the charge inside the carrier.
  • the carrier immunogenic material may include a portion which deactivates the carrier which is an organism.
  • Display system refers to a system having a form in which a target material is disclosed outside the system structure as one embodiment of the system.
  • the display system includes, but is not limited to, the fused display system, the coupling display system, and the attachment display system of the present disclosure.
  • One example of the present application is a vaccine comprising an immunogenic fusion display system.
  • Immunogenic display system refers to initiating an immunogenic substance outside the system structure as one embodiment of the immunogenic system.
  • Immunogenic display systems include, but are not limited to, immunogenic fusion display systems, immunogenic coupled display systems, immunogenic attachment display systems, as described herein.
  • fusion display system refers to a system in which a surface object and a target material, which disclose a target material outside a system structure, appear in a fused form.
  • Fusion displaying structure means a structure that fuses exogenously fused immunogenic substances to the outside as a system structure.
  • Surface displaying substructure means a substructure of a fused display system that is fused with a target material and is disclosed outside the display body.
  • Fused body means a fusion of a surface protein and an immunogenic substance.
  • Fusing part means a site where a surface construct and an immunogenic substance are fused in a fusion body.
  • An “ immunogenic fused display system” is an embodiment of an immunogenic system, which includes a surface construct introduced into the system structure by fusing with a target material as shown in Fig. 12; And a system in which the immunogenic material is expressed in a fused form.
  • fusion here refers to the direct connection of two substances as a bond of matter.
  • the system structure is defined as a " fusion display body ".
  • the immunogenic substance is defined as a "fused immunogen”.
  • Fused display bodies encompass system structures of immunogenic systems.
  • the fused display body may have an external charge. At this time, charges outside the fused display body may cause charge and attraction of the surface body. Also, charges outside the fused display body can cause charge and attraction of the fused body.
  • the fused display body may not be electrically charged outside.
  • the outer membrane of the fused display body may be porous. At this time, the pore of the outer membrane may be similar in size to or slightly larger than the surface opening body.
  • the surface construct is a sub-structure of a fused display system, the size of which is smaller than the fused display body. More preferably, the size is equal to the size of the outer membrane protein of the microorganism corresponding to the size of the fusion display body.
  • the surface construct may be a wild-state protein of the microorganism when the fusion display body is a microorganism.
  • the surface protein may be an outer membrane protein.
  • the surface protein may be one in which the wild-state protein is modified or engineered.
  • the surface construct may be one of spore exosporium protein, spore coat protein, transmembrane protein, spore appendage protein, or spore associated enzyme.
  • the surface construct may be a protein that is homologous to the structure of the wild-type protein of the fusion display body.
  • the surface construct may be a wild-state protein of the fusion display body belonging to the same genus.
  • the surface protein may be an outer membrane protein.
  • the surface protein may be a modified or manipulated wild-type outer membrane protein.
  • the surface construct may be a protein that is homologous to the structure of the wild-type protein of the fusion display body belonging to the same genus.
  • Surface constructs may include the site of action of a particular restriction enzyme.
  • the specific restriction enzyme may be, for example, a signal peptidase.
  • the surface can be charged.
  • charge on the surface construct can cause charge and attraction outside the fused display body.
  • Surface constructs may include structures in which the same sequence is repeated. Wherein the repeated structure may be homologous to the structure of the wild-type protein. Also, the repeated structure may be charged. At this time, charge of the repeated structure may cause charge and attractive force outside the fusion display body.
  • the fusion moiety refers to a site where the surface protein and the immunogenic substance are fused.
  • a fusion site may be a separate protein / base sequence but may be only a site forming a single chemical bond.
  • the fusion moiety may exhibit only the binding site between the surface construct and the immunogenic material without special function, or may have a specific structural characteristic or a specific function in the immune formation process.
  • the fusion moiety may be a chemical bond.
  • the chemical bond may be, for example, a polymer bond.
  • the polymerization bond may be a condensation polymerization bond or an addition polymerization bond.
  • the dehydration condensation polymerization bond may be, for example, an amide bond.
  • the fusion portion may have a certain length required for the desired function.
  • Surface constructs and immunogenic materials that make up the fusions each have a suitable three-dimensional structure, e.g., a folding structure, which allows the protein to function properly. If the length of the fusion site is too short, the folding structure of each material may not be formed properly due to steric hindrance between the surface construct and the immunogenic material. Therefore, it is preferable that the fusion portion necessary for the folding structure of the surface construct and the immunogenic material to be formed properly has a certain length or size.
  • the fusion moiety may be about 10 bp, 15 bp, or 20 bp or more.
  • the fusion moiety may comprise a specific amino acid sequence / base sequence.
  • the specific base sequence 5'-GGAGGTGGGGGTTCACTCCAG-3 ' was used.
  • the fusion portion may be one having a specific charge.
  • the particular charge may have the same charge characteristics as the surface charge.
  • the fusion moiety can be positively charged by (number of positively charged amino acids (arginine / histidine / lysine)): (total number of amino acids).
  • the fusion moiety may be one having activity against the cleavage enzyme.
  • the terminating enzyme may be used in combination with a restriction enzyme (restriction enzyme) or a restriction endonuclease. These enzymes recognize a specific nucleotide sequence of a double-stranded DNA molecule and cleave the part or its periphery Lt; / RTI > But may be, without limitation, proteases.
  • Fused immunogens Fused immunogens
  • Fusion immunogenic substances encompass the above-mentioned immunogenic substances.
  • the fused immunogenic material may have a specific size. Preferably greater than or equal to the effective size capable of causing an immune response. Wherein the fused immunogenic material may have a specific size ratio with the surface protein.
  • a fused immunogenic material may be one that has a specific charge.
  • the particular charge may have the same charge characteristics as the surface charge.
  • Fusions may be those with a specific charge. At this time, a specific electric charge can cause charges and attractive forces outside the fused display body. At this time, the fusions can be positively charged.
  • the charge of the fusant (number of positively charged amino acids (arginine / histidine / lysine)): (number of total amino acids). (Number of positive amino acids): (number of total amino acids) may be 0.1 to 0.8: 1. The ratio may be 0.5: 1, 0.6: 1, 0.7: 1.
  • One example of the present application is a vaccine comprising an immunogenic coupled display system.
  • Coupling display system is one embodiment of a system, comprising: a system structure having one coupling unit on the outside; And a coupling system in which a target substance having two coupling groups forms a coupling bond.
  • Coupling bond means a chemical bond formed by the interaction of a display body coupler and an immunogenic material coupling unit.
  • Coupling group means a moiety that forms a coupling bond by the interaction of two homologous or heterogeneous coupling groups.
  • Coupling group on displaying structure means that it is located on a coupling display body as a coupling unit.
  • Coupling part on immunogen means that it is located in a coupling immunogenic material as a coupling group.
  • Coupling part means a coupled site created by interaction of a display body coupling unit and an immunogenic material coupling unit.
  • Coupling display structure means a structure that is coupled to and coupled with a coupling immunogenic material as a system structure.
  • An “ immunogenic coupling display system” is an embodiment of an immunogenic system comprising: a system structure having one external coupling unit as shown in FIG. 13; And an immunogenic substance having two coupling groups form a coupling bond.
  • the system structure can be expressed as a " coupling display body ", and the immunogenic material can be expressed as a " coupled immunogen ".
  • the one coupling unit can be expressed as a " display body coupling unit ", and the two coupling unit can be expressed as an " immunogenic material coupling unit ".
  • the coupling display body encompasses the system structure of the immunogenic system.
  • Coupling bonds are generally understood to be polymeric bonds resulting from the interaction of two or more functional groups.
  • the polymerization bond may include condensation polymerization in which a part of the functional group is released, addition polymerization in which the functional group is retained, and radical polymerization in the radical.
  • the coupling bond as referred to herein is preferably a chemical bond formed by the interaction of the display body coupler and the immunogenic material coupling agent.
  • the coupling coupling may be that two or more coupling units are the same or different.
  • the coupling bond may be a polymeric bond.
  • the polymerization linkage may be condensation polymerization.
  • the condensation polymerization may be a dehydration condensation polymerization.
  • the dehydration condensation polymerization may be an amide bond.
  • the dehydration condensation polymerization may be an ester bond.
  • the dehydration condensation polymerization may be an ether bond.
  • the dehydration condensation polymerization may be an aldol bond.
  • the condensation polymerization may be a disulfide bond.
  • the polymerization linkage may be addition polymerization.
  • the addition polymerization may be maleimide-thiol polymerization.
  • the polymerization linkage may be a radical polymerization.
  • the radical polymerization may be click chemistry.
  • the display body coupling unit may include one or more kinds of functional groups for coupling coupling.
  • the display body coupling unit may contain two kinds of functional groups for coupling coupling.
  • the display body coupling unit has a first coupling unit including one kind of functional group for coupling coupling and a second coupling unit including another kind of functional group.
  • the immunogenic material coupling unit may comprise at least one functional group different from the functional group of the display body coupling group.
  • the immunogenic agent coupling group may comprise two types of functional groups for making the coupling bond.
  • the immunogenic material coupling unit may be a second coupling group including a first coupling group including one kind of functional group for coupling coupling and a different kind of functional group.
  • the coupling immunogenic material encompasses the immunogenic material mentioned above.
  • the coupling moiety refers to a site including a display body coupling unit and an immunogenic coupling unit as a coupling-coupled site of the coupling display body and the coupling immunogenic substance.
  • a coupling portion may be a separate protein / base sequence, but may be a region containing a functional group generated by one coupling bond.
  • the coupling moiety may exhibit only a binding site between the coupling display body and the coupling immunogenic material without any special function, or may have a specific structural feature or a specific function in the immune formation process.
  • the coupling moiety can refer to a functional group that forms a coupling bond or to the presence thereof.
  • the coupling portion may also have a certain length required for the desired function.
  • Coupled immunogenic materials each have a suitable three-dimensional structure, for example a folding structure, and the formation of the correct folding structure allows the protein to function properly. If the length of the coupling part is too short, the folding structure of each material may not be formed properly due to steric hindrance between the coupling display body and the coupling immunogenic material. Therefore, it is preferable that the fusion portion necessary for the folding structure of the coupling immunogenic material to be formed properly has a certain length or size.
  • the fusion moiety may be about 10 bp, 15 bp, or 20 bp or more.
  • the coupling moiety may have a specific amino acid sequence / base sequence.
  • the specific base sequence was 5'-GGAGGTGGGGGTTCACTCCAG-3 '.
  • the coupling portion may be one having a specific charge.
  • the particular charge may have the same charge characteristics as the coupling display body.
  • the coupling moiety can be positively charged by (the number of positively charged amino acids (arginine / histidine / lysine)): (the total number of amino acids).
  • the coupling moiety may be one having activity against the cleavage enzyme.
  • the cleavage enzyme can be used in combination with a restriction enzyme (restriction endonuclease) or a restriction endonuclease. These cleavage enzymes recognize a specific base sequence of a double-stranded DNA molecule and cleave the part or its periphery Lt; / RTI > But may be, without limitation, proteases.
  • One example of the present application is a vaccine comprising an immunogenic attachment display system.
  • Attached display system means that a target material is attached to the outside of the system structure as one embodiment of the system.
  • Attached displaying structure means a structure that attaches and externally attaches an attachment immunogenic material as a system structure.
  • Immunogenic attachment display system means a system in which an immunogenic material is attached to the outside of the system structure, as shown in Fig. 14, as an embodiment of the immunogenic system.
  • attachment means the bonding relationship of a substance followed by the indirect connection of two substances, eg via a specific mediator.
  • the system structure can be expressed as an " attached display body ", and the immunogenic substance can be expressed as an " attached immunogen ".
  • the attachment may be by electrostatic attraction,
  • the attachment may be by hydrogen bonding
  • the attachment may be by hydrophobic interaction.
  • the attached display body encompasses the system structure of the immunogenic system.
  • the attached display body may have electric charge inside. At this time, electric charge inside the attached display body may cause charge and repulsion of the attachment immunogenic material.
  • the attached display body may have an external charge. At this time, charges outside the attached display body may cause charge and attraction of the attached immunogenic material.
  • the attached display body may not have an external charge.
  • the attached display body may have a porous outer membrane.
  • the attachment immunogenic substance encompasses the immunogenic substance.
  • the attachment immunogenic material may be below a certain size.
  • an effective amount of the supported immunogenic material capable of causing an immune response can be of sufficient size to be adhered to the attached display body.
  • the attachment immunogenic material may be charged. At this time, charge of the attachment immunogenic material can cause charge and attraction outside the attachment display.
  • One example of the present application is a vaccine comprising an immunogenic cross-linked display system.
  • Cross-linked display system refers to a target material having a first cross-linker on the outside as one embodiment of a display system; And a polymer having two or more second cross-linking moieties cross-link to form a cross-linked network structure.
  • Cross-linked displaying structure means a system structure used in a cross-coupled display system.
  • Cross-linking means a chemical bond formed by the interaction of an immunogenic material cross-linker with a polymeric cross-linker.
  • Cross-linking group means a moiety that cross-links by the interaction of two or more homologous or heteromeric cross-linking groups.
  • Cross-linking net structure means a network structure of a target material and a polymer formed by cross-linking.
  • Polymerizing part means a moiety or bond that is polymerized between the target materials in a cross-linked network structure.
  • Cross-linking part means a cross-linked moiety made by the interaction of an immunogenic material cross-linker and a polymeric cross-linker.
  • Cross-linking group on immunogen refers to a cross-linking agent located in a cross-linking immunogenic material.
  • Polymerizing body refers to a material that has two or more polymeric cross-linkers and is capable of cross-linking with two or more immunogenic cross-linkers to effect polymerization.
  • Cross-linking group on polymerizing body means located on a polymer as a cross-linker.
  • An " immunogenic cross-linked display system” is an embodiment of an immunogenic display system in which an immunogenic material having a first cross-linker externally and a polymer having two or more second cross-linkers cross- To form a cross-linked net structure.
  • the immunogenic cross-linked display system has a high stability of the system due to the cross-linked net structure.
  • the display body is defined as a " cross-linked display body ".
  • the immunogenic substance is defined as a " cross-linked immunogen ".
  • the first cross-linker can be expressed as an " immunogenic material cross-coupler "
  • the second cross-coupler can be expressed as a " polymer cross-coupler ".
  • the immunogenic cross-linked display system is basically characterized by the cross-linked net structure itself, the form of the display system is not limited.
  • a fusion display system a coupling display system, or an attachment display system.
  • the cross-linked display body may be any one of a fused display system structure, a coupled display system structure, and an attached display system structure according to aspects of the display system.
  • Cross-linking includes various types of known physical and chemical bonds, such as the coupling bonds described above.
  • the immunogenic material cross-linker may comprise one or more functional groups for cross-linking.
  • an immunogenic material cross-linker may be two types of functional groups for cross-linking. Can be used as the first immunogenic material cross-coupler and the second immunogenic material cross-coupler, respectively, for different functional groups.
  • the immunogenic material cross-linker may comprise an active site for the cleavage enzyme.
  • the cleavage enzyme may be a protease.
  • Polymer refers to a material that causes a polymerization reaction to form a cross-linked network structure. It is preferred that the polymer comprises two or more cross-linking groups to cause a polymerization reaction.
  • the polymer may have a certain length required for the desired function.
  • the polymer In order for the cross-linked immunogenic materials to polymerize, the polymer must be able to bridge between the cross-linked immunogenic materials. If the length of the polymer is too short, only a single bond with the cross-linked immunogenic material can be formed. Therefore, it is preferable that the polymer has a certain length or size necessary to cause the desired polymerization reaction to take place.
  • the polymer may comprise an active site for the cleavage enzyme.
  • the cleavage enzyme may be a protease.
  • the polymeric cross-linker may comprise one or more functional groups for cross-linking.
  • a polymeric cross-linker may comprise two types of functional groups for cross-linking. Can be used as a first cross-linker and a second cross-linker, respectively, for different functional groups.
  • the polymeric cross-linker may comprise an active site for the cleavage enzyme.
  • the cleavage enzyme may be a protease.
  • Cross-linking moieties may refer to functional groups or sites of their occurrence resulting from cross-linking.
  • the cross-linking moiety may be one having activity against the cleavage enzyme.
  • the cleavage enzyme may be a protease.
  • the cross-connect network structure refers to the net structure formed by the cross-links.
  • the net structure refers to a structure in which points are crossed by a line composed of a polymerized portion, with the cross-linked immunogenic material as a point.
  • the net structure essentially consists of a cross-linked immunogenic material and a polymeric moiety.
  • Such a net structure may also function as a loading system that controls the size and confines the material therein.
  • the net structure may be that the mesh is a certain size, so that a larger material can not pass through it.
  • This material encompasses the following protein x.
  • the material may also be a cross-linked display body.
  • a polymeric moiety refers to a moiety that polymerizes between one cross-linked immunogenic material and another cross-linked immunogenic material. Wherein the polymeric moiety will essentially have the structure of an immunogenic material cross-linker-cross-linking-polymer-cross-linking moiety-immunogenic cross-linker.
  • the polymerized portion may also have a certain length required for the desired function.
  • the polymeric moiety should be able to bridge between the cross-linked immunogenic materials. If the length of the polymerized portion is too short, only a single bond with the cross-linked immunogenic substance can be formed. Therefore, it is preferable that the length of the polymerization portion has a certain length or size so that the desired polymerization reaction can take place. At this time, the lengths of the immunogenic material cross-linking unit, the cross-linking unit, and the polymer may be adjusted in consideration of the length of the polymerization unit.
  • the polymerized portion may be one having a specific charge.
  • This particular charge can cause charge and attraction of the cross-linked display body.
  • the specific charge can be (by the number of positively charged amino acids (arginine / histidine / lysine)) :( total amino acids).
  • the charge of the immunogenic material cross-linker, the cross-linking moiety, and the polymer can each be adjusted so that the polymeric moiety has a specific charge.
  • polymerisation moiety may comprise an active site for the cleavage enzyme.
  • cleavage enzyme may be a protease.
  • One example of the present application is a vaccine comprising an immunogenic cascade system.
  • serial connection system means a system in which a form of a target material has a form in which proteins are connected in series, as shown in FIG.
  • Serial-connected system structure means a system structure used in an immunogenic serial-connected system.
  • An " immunogenic serial-connected body" is an immunogenic substance of an immunogenic series-linked system, in which n proteins are connected in series.
  • Immunogenicity cascade system is an embodiment of the immunogenic system wherein the form of the immunogenic substance is in the form of serially connected proteins.
  • serial means a combination of two or more substances connected in series. If the form of the immunogenic substance is in-line, the immunogenic effect may be enhanced by increasing the concentration of the immunogenic substance in the vaccine.
  • the system structure can be expressed as a " serial connection system structure ", and the immunogenic material can be expressed as an " immunogenic serial connector ".
  • immunogenic serial-linking systems are characterized by the linkage structure of the immunogenic material, so that there is no limit to the mode of the system.
  • it can be any one of an internal carrying system, a fusion display system, a coupling display system, an attachment display system, and a cross-linked display system.
  • the series connection system structure may be any one selected from an internal supporting system structure, a fused display system structure, a coupling display system structure, an attached display system structure, and a cross-coupled display system structure according to aspects of the system.
  • xth protein refers to the xth protein on the immunogenic series link.
  • yth connecting part means a site connecting the yth protein and the (y + 1) protein on the immunogenicity serial link.
  • n proteins connected in series. That is, the first protein-first connecting portion-second protein-second connecting portion ... (N-1) -conjugate-n-th protein.
  • the serial link consisting of n proteins has (n-1) linkages.
  • n proteins should be included in the immunogenic substance.
  • it may be achieved with only an immunogenic substance that causes an immune response, or it may include other auxiliary factors that enhance the immune response in addition to the immunogenic substance.
  • the immunogenicity serial link consists of a first x protein of x 1 to n and a y link connecting y 1 to (n-1).
  • the x-protein may be the immunogenic substance.
  • the x-protein may be selected from among a supported immunogenic substance, a fused immunogenic substance, a coupled immunogenic substance, an adhesion immunogenic substance, and a cross-linked immunogenic substance.
  • Protein x can be a non-antigenic part of the pathogen.
  • non-antigenic portion may be homologous to the outer membrane protein of the pathogen.
  • immunogenic serially-linked construct can function as a pathogen-like particle.
  • the pathogen-like particle may be a virus-like particle.
  • the non-antigenic portion may also function as an immunosuppressive function.
  • the immune assistant function may be to suppress the secretion of cytokines that promote the secretion of cytokines related to the immune response or inhibit the immune response in the living body.
  • the immunocompetent function may be to degrade the membrane of the immune cell.
  • Protein x can be the exogenous part of the pathogen.
  • the foreign part may be an immunogenic site on the antigen protein of another pathogen.
  • the foreign part may be a non-immunogenic part or a non-antigenic part on the antigen protein of another pathogen.
  • the antigen homologous protein may function as a pathogen-like particle of another pathogen.
  • the pathogen-like particle may be a virus-like particle.
  • the foreign part may function as an immune assistant.
  • the immune assistant function may be to suppress the secretion of cytokines that promote the secretion of cytokines related to the immune response or inhibit the immune response in the living body.
  • the immune-assisted function may also aid in the recognition of antigen-recognizing cells.
  • Immunosuppressive function may also be the degradation of the membrane of immune cells.
  • Immunogenic substances among the first to n < th > proteins may comprise multiple immunogenic sites of the pathogen.
  • the first to n < th > proteins may all be the same immunogenic substance or different immunogenic substances.
  • At least one of the first proteinase n protein is an immunogenic substance.
  • the y-th linkage refers to a site connecting the y-th protein and the (y + 1) protein. Such a y-connection may be a separate protein / base sequence, but may be merely a site forming a single chemical bond.
  • the y-connection may not have a particular function, but may have a separate structural feature or specific function in the process of immunization.
  • the y-th connecting portion encompasses the fusion portion.
  • the y-th coupling portion encompasses the coupling portion.
  • the y-th connecting portion includes the above-mentioned polymerized portion.
  • first to (n-1) connecting parts may have activity on the same cleavage enzyme or may have activity on different cleavage enzymes.
  • the cleavage enzyme may be a protease.
  • One example of the present application is a vaccine comprising an immunogenic parallel connection system.
  • Parallel system is an embodiment of the system, wherein the system structure transfers n proteins in parallel as shown in FIG.
  • Parallel system structure means a system structure used in an immunogenic parallel system.
  • Parallel body refers to each of the n proteins delivered by the parallel system structure. Unless otherwise stated, "parallel” refers to any one of the n paralleles.
  • An “ immunogenic parallel system” is one embodiment of an immunogenic system wherein the system constructs deliver n proteins in parallel.
  • Parallel means that each protein is independently delivered by the system structure.
  • An immunogenic parallel system has the advantage that one system can perform several functions. In this case, the system structure can be expressed as a "parallel system structure”, and each protein delivered by the parallel system structure can be expressed as "parallel”.
  • each system may be any one of the internal supporting system, the fusion display system, the coupling display system, the attachment display system, and the cross-coupling display system.
  • the parallel connection system structure may be any one selected from among an internal supporting system structure, a fused display system structure, a coupling display system structure, an attached display system structure, and a cross-coupled display system structure.
  • the parallel body may be the immunogenic substance.
  • the conjugate may be selected from a supported immunogenic substance, a fused immunogenic substance, a coupled immunogenic substance, an adhesion immunogenic substance, and a cross-linked immunogenic substance.
  • Parallel bodies may be non-antigenic parts of the pathogen.
  • non-antigenic portion may be homologous to the outer membrane protein of the pathogen.
  • immunogenic parallel system can function as a pathogen-like particle.
  • the pathogen-like particle may be a virus-like particle.
  • the non-antigenic portion may also function as an immunosuppressant.
  • the immune assistant function may be to suppress the secretion of cytokines that promote the secretion of cytokines related to the immune response or inhibit the immune response in the living body.
  • the immunocompetent function may be to degrade the membrane of the immune cell.
  • Parallel bodies may be the exogenous part of the pathogen.
  • the foreign part may be an immunogenic site on the antigen protein of another pathogen.
  • the foreign part may be a non-immunogenic part or a non-antigenic part on the antigen protein of another pathogen.
  • the antigen homologous protein may function as a pathogen-like particle of another pathogen.
  • the pathogen-like particle may be a virus-like particle.
  • the foreign part may function as an immune assistant.
  • the immune assistant function may be to suppress the secretion of cytokines that promote the secretion of cytokines related to the immune response or inhibit the immune response in the living body.
  • the immune-assisted function may also aid in the recognition of antigen-recognizing cells.
  • Immunosuppressive function may also be the degradation of the membrane of immune cells.
  • the n parallel substances should be the immunogenic substance.
  • it may be achieved with only an immunogenic substance that causes an immune response, or it may include other auxiliary factors that enhance the immune response in addition to the immunogenic substance.
  • One example of the present application is a vaccine comprising an immunogenic spore system.
  • Spore system means in one embodiment of the system that the system structure is spore or spore-like particles.
  • Spore system structure means a spore or spore-like particle as a system structure of a spore system.
  • immunogenic spore system means an embodiment of an immunogenic system wherein the system structure is spores.
  • the immunogenic spore system consists of an immunogenic substance and a spore system structure that transmits the immunogenic substance in vivo.
  • the structure and characteristics of the immunogenic material are as described above.
  • the immunogenic material and the spore system structure are transferred as a system while being located within a certain distance in the living body.
  • Spore system structure has higher physico-chemical stability than other cell, virus type system structure, has weak virulence and is easy to concentrate.
  • the spore system structure of the immunogenic spore system may include an element that contributes to the function of the vaccine, such as an immunosuppressant.
  • the spore system structure constituting the spore system may be spore or spore-like particles.
  • the spore system structure functions to transfer the immunogenic material into a living body within a certain distance.
  • the spore system structure preferably has a size that can be administered to an animal body, and may be about the size of a general microbe, for example, about 0.02 to 5 mu m in diameter.
  • spores are generally required to survive in harsh physical / chemical environments. For this reason, most spores are characterized by high temperature, changes in pH, changes in external osmotic pressure, and external shocks.
  • the properties of these spores give immunogen spore systems the advantage of being able to cause immunity under extreme conditions than other cell, virus type system structures.
  • it has an advantage of exerting immunity due to microorganism like appearance, and in addition, when spore is a pathogen, it can act as a multivalent vaccine together with an immunogenic substance.
  • the spore system is not limited to the form described below.
  • the spores may be plant spores.
  • the spores may be spores of yeast.
  • the spores may be spores of fungi.
  • the spore may be a spore of spore-forming bacteria.
  • Spore bacterium refers to a bacterium that grows in the form of bacteria at normal times but temporarily becomes spore-forming and survivable when conditions are difficult to survive.
  • the spore bacterium may be Clostridium.
  • the Clostridium may be C. difficile.
  • the spore bacterium may be sporolactobacillus.
  • the spore bacterium may be Bacillus.
  • the Bacillus may be, for example, B. subtilis, B. anthrasis, B. thuringiensis, or B. cereus.
  • the spore may be a part of which has been deformed or manipulated.
  • the spore may be the entire structure modified or manipulated.
  • the spore of the spore-forming bacteria is in particular a spore of B. subtilis.
  • Spore-like particles are artificially created particles that are similar in structure to spores.
  • the spore-like particles may consist of a "spore-like particle nucleus” that forms the structure of the particle and a "free-desert” similar to the spore's outer membrane.
  • the spore-like particle nucleus may be a hollow spherical structure.
  • the spore-like particle nuclei may be a porous structure.
  • the spore-like particle nuclei may be a solid rigid structure.
  • the spore-like particle nuclei can be composed of organic materials.
  • the spore-like particle nuclei can be composed of inorganic materials.
  • the inorganic material may be, for example, silica (Si).
  • the spore-like particle nuclei may be composed of a mixture of an organic material and an inorganic material.
  • Spore-like particle nuclei may resemble spores of microorganisms whose diameter is in nature.
  • the diameter may be between 1 and 50 mu m.
  • the for-free desert may be a phospholipid film.
  • the spore-like particles may be composed only of spore envelope proteins.
  • One example of the present application is a vaccine comprising an immunogenic spores internal support system.
  • Spore carrying system means one embodiment of the spore system, wherein the target substance is carried in the spore system structure.
  • Carrying spore structure means a spore system structure, which is a structure that supports and transports a spore-bearing immunogenic substance therein.
  • Immunogenic spore internal support system means that, as one embodiment of the immunogenic spore system, the immunogenic substance is carried in the spore system structure.
  • the spore system structure can be expressed as "spore carrier”, and the immunogenic substance can be expressed as "spore carrier immunogen”.
  • Spore bearing bodies encompass the spore system structure of the immunogenic spore system.
  • the spore carrier includes the carrier described above.
  • the spore-bearing immunogenic material encompasses the immunogenic material described above.
  • the spore-bearing immunogenic material may be less than a specific size. Preferably an amount capable of containing an effective amount of an immunogenic substance capable of causing an immune response.
  • the spore-bearing immunogenic material may be charged. At this time, the charge of the spore-bearing immunogenic material may be opposite to the charge inside the spore carrier. For example, the charge of the spore-bearing immunogenic material can be negative.
  • the spore-bearing immunogenic material may comprise a portion that deactivates spores.
  • the spore-bearing immunogenic material may be homologous to a protein present in the spores. Where the homologous portion may be the signal sequence portion of the protein.
  • the spore internal protein may be a protein that is produced in the Spo I stage in the spore differentiation process.
  • the spore-bearing immunogenic material may be carried into the spores by the signal sequence of the protein and carried.
  • One example of the present application is a vaccine comprising an immunogenic spore fusion display system.
  • Spore display system refers to initiating a target substance outside of the spore system structure as an embodiment of the spore system.
  • An " immunogenic spore display system” refers to initiating an immunogenic substance outside of a spore system structure as one embodiment of an immunogenic spore system.
  • Spore fused display system refers to a fusion of a target substance with a spore surface construct that initiates a target substance outside a spore system structure as one embodiment of the system.
  • Spore fusion displaying structure means a spore system structure, which is a structure that externally fuses a spore fusion immunogenic substance.
  • Spore surface displaying substructure refers to a substructure of a fused display system that fuses with a target material and starts outside the display body.
  • Spore fused body means a fusion of a spore surface body and an immunogenic substance.
  • Spore fusing part means a site where a spore surface body and an immunogenic substance are fused in a spore fusion body.
  • Immunogenic fused display system means an embodiment of an immunogenic spore system, which is a fusion of an immunogenic substance with a spore surface organ initiated outside the spore system structure by fusion with a target substance.
  • fusion here refers to the direct connection of two substances as a bond of matter.
  • spore system structure is defined as “spore fusion display body”.
  • the immunogenic substance is defined as a " spore fused immunogen ".
  • the spore fusion display body encompasses the spore system structure of the immunogenic spore system.
  • the spore fusion display body may have an external charge. At this time, charges outside the spore fusion display body can cause charge and attraction of the spore surface body. In addition, charges outside the spore fusion display body can cause charge and attraction of the spore fusion body.
  • Charges outside the spore fusion display body can produce stronger negative charges than spores in the wild state.
  • the spore fusion display body may not be charged outside.
  • the outer membrane of the spore fusion display body may be porous. At this time, the hole of the outer membrane may be similar in size to the spore surface body.
  • Spore outer domain refers to the space between the double lipid membrane of the spore and the outermost perimeter of the spore.
  • Spore outer protein refers to a spore protein present in the outer region of the spore in its natural state.
  • the outer spore protein may be a protein expressed in the spore membrane.
  • the spore surface is preferably a sub-structure of the spore fusion display system and the size thereof is smaller than that of the spore fusion display body.
  • the size is the same as the size of the spore external protein corresponding to the size of the spore fusion display body.
  • the spore surface body may be a wild-state protein of spores when the spore fusion display body is a spore.
  • the spore surface construct may comprise the site of action of a particular restriction enzyme.
  • the specific restriction enzyme may be, for example, a signal peptidase.
  • Spore surface bodies may be charged. At this time, the electric charge of the spore surface openings may cause charge and attractive force outside the spore fusion display body.
  • the spore surface construct may include a structure in which the same sequence is repeated. Wherein the repeated structure may be homologous to the structure of the wild-type protein. Also, the repeated structure may be charged. At this time, the charge of the repeated structure may cause charge and attraction outside the spore fusion display body.
  • the spore surface can be an outer spore protein. Generally, spores of the same microorganism can be used as spores between the spores.
  • the spore external protein may be any one of spore exosporium protein, spore coat protein, transmembrane protein, spore appendage protein, and spore associated enzyme.
  • the spore exosporium protein may be, for example, BclA, InhA, and the like.
  • the spore coat protein may be, for example, CotC, CotD, CotB, CotE, CotF, CotG, CotN, CotS, CotT, CotV, CotW, CotX, CotY, CotZ, CotH, CotJA, CotJC, CotK, have.
  • CotB, CotC, CotE and CotG can be proteins having a high expression ratio and a high ratio in spontaneous extracellular proteins.
  • the transmembrane protein may be, for example, prkC, OppA, or the like.
  • the spore appendage protein may be, for example, P29a, P29b, P85, and the like.
  • the spore-associated enzyme may be, for example, CotA (laccase), oxdD (oxalate decarboxylase), CotQ (reticuline oxidase-like protein), tgI (transglutaminase)
  • the spore surface construct may be a modified or engineered spore protein in the wild state.
  • Spore outer homologous protein means a protein that is homologous to the structure of a particular spore-forming protein. It can have 85% to 100% homology with wild-type protein. Preferably, the homology is 90 to 100%, more preferably 95 to 100%.
  • Signal sequence refers to a protein sequence that functions to direct a protein to a specific location in a cell during the synthesis of the protein.
  • Spore outer signal sequence refers to a protein sequence that functions as a signal sequence in the spore protein and serves to direct the protein to the spore outer region.
  • outer localization motif refers to a motif that positions the outer spore protein in the outer region of the spore.
  • the spore surface opener may be a spore external homologous protein.
  • the spore outer protein may comprise a spore external signal sequence consisting of about 20 or more amino acid sequences.
  • the spore external signal sequence contained in the spore outer homologous protein may be one in which the signal sequence restriction enzyme action site has been removed.
  • spore-forming proteins it may include a spore external locating motif that allows the spore to be located in the spore outer region.
  • the spore external locating motif may be located at the N terminus of the outer spore protein, but is not limited thereto. Proteins that are homologous in these regions can be expressed in the outer region of the spore.
  • the number of amino acid sequences of the spore external homologous protein is not particularly limited as long as it is sufficient to express a protein capable of functioning as an immunogenic substance in the spore outer region. For example, 50aa, 100aa, 150aa.
  • Spore external homologous proteins may comprise spore external locating motifs.
  • Spore external homologous proteins can include both spore external signal sequences and spore external locating motifs.
  • the spore external signal sequence may be the signal sequence restriction enzyme removed region.
  • the spore outer homologous protein may include a structure in which the same sequence is repeated, wherein the repeated structure may be a full-length sequence of the spore outer protein.
  • the repeated structure may also be a spore external locating motif.
  • the repetitive structure of the protein outside the spore can also be positively charged.
  • the repeating structure may be positively charged.
  • the repeating structure may be positively charged.
  • the repeating structure may be positively charged.
  • pore outer homologous protein-contained additive or " spore outer homologous protein” refers to a protein having some homology to the structure of the outer spore protein and having homology to the structure of the outer spore protein Or a portion of the protein that contains the missing portion. That is, the spore-exogenous homologous inclusion-containing protein refers to a form containing a region which is in addition to the spore exogenous homologous protein and which is not homologous with the spore external protein.
  • Spore foreign body refers to a protein or base sequence that does not exist in wild-type spores.
  • &Quot Spore of another species " refers to a heterologous microbial spore of the same genus. &Quot; Spore outer protein of another species " refers to a protein outside the spore of heterozygous spores.
  • the spore surface construct may be a spore external homologous inclusion protein.
  • the spore external homology inclusion attachment protein may be one that is homologous to another spore external protein of the same spore other than the specific spore external protein.
  • the spore outer homologue protein may contain a spore extraneous signal sequence of the outer protein of the sponge.
  • the spore external signal sequence may be the signal sequence restriction enzyme removed region.
  • the homology region with the external protein of the other reporters may have an amino acid sequence number of, for example, 50aa, 100aa, 150aa.
  • the protein may contain spore external locating motifs of the outer sponge protein.
  • the protein may contain both spore external signal sequence and spore external locating motifs of the external protein of the external sponge.
  • the spore external signal sequence may be the signal sequence restriction enzyme removed region.
  • the protein may be homologous to two or more proteins outside the sponge in the same form.
  • the spore outer homologous portion protein may comprise a spore outgrowth and a site that is homologous to the spore outer protein.
  • the foreign part may be homologous to the heterologous spore external protein.
  • the spore outer homologous protein may contain a spore-external signal sequence of the heterospolar outer protein.
  • the spore external signal sequence may be the signal sequence restriction enzyme removed region.
  • homology sites with heterologous spore proteins can have amino acid sequences of 50aa, 100aa, 150aa.
  • the spore outer homologous protein may contain spore external locating motifs of heterologous spore external protein.
  • the homologous site with the heterologous spore protein may have the amino acid sequence numbers of 50aa, 100aa, 150aa.
  • the spore outer homologous protein may contain both the spore external signal sequence of the heterologous spore outer protein and the spore external locating motif.
  • the spore external signal sequence may be the signal sequence restriction enzyme removed region.
  • homology sites with heterologous spore proteins can have amino acid sequences of 50aa, 100aa, 150aa.
  • the spore outer homologue protein may be homologous to two or more heterospathic proteins in the same form.
  • the foreign part may be charged with a specific sign.
  • the area outside the spore is negatively charged. Therefore, when the spore surface body is positively charged, the electrostatic attraction with the outer region of the spore will be added, so that it can be positioned more stably. Therefore, it is preferable that the charge of protein of the spore external homologous portion is positive.
  • the charge of the specific code may be opposite to the charge outside the spore fusion display body.
  • the charge of the foreign part may be positive.
  • the foreign part can be positively charged by (the number of positive amino acids (arginine / histidine / lysine)): (the total number of amino acids).
  • the protein can exert a stronger positive charge than the exogenous protein in the wild state.
  • the number of amino acids in the protein (the number of positive amino acids): (the number of total amino acids) of the spore external homologous portion may be 0.1 to 0.8: 1.
  • the ratio may be 0.5: 1, 0.6: 1, 0.7: 1.
  • the spore fusion site refers to a site where a spore surface integrator and a spore fusion immunogenic substance are fused.
  • the spore fusion site may be a separate protein / base sequence but may be only a site forming a single chemical bond.
  • the spore fusion site may not have a particular function, but may have separate structural features or specific functions in the immune formation process.
  • the spore fusion unit encompasses the fusion unit described above.
  • the spore fusion immunogenic substance encompasses the above-mentioned fusion immunogenic substance.
  • the spore fusion immunogenic material may have a specific size ratio with the spore outer protein. Wherein the size ratio of the spore outer protein to the spore fusion immunogen may be from 2: 1 to 1: 2.
  • the spore fusion may be one with a specific charge.
  • the area outside the spore is negatively charged. Therefore, the spore fusions will be more stable when positively charged, due to the addition of electrostatic attraction to the area outside the spore. Therefore, the charge of the spore fusion body is preferably positive.
  • the specific charge can cause charge and attraction outside the spore fusion display body.
  • the spore fusion can be positively charged by (number of positively charged amino acids (arginine / histidine / lysine)): (total number of amino acids). (Number of positive amino acids): (number of total amino acids) may be 0.1 to 0.8: 1. The ratio may be 0.5: 1, 0.6: 1, 0.7: 1.
  • One example of the present application is a vaccine comprising an immunogenic spore-coupling display system.
  • Spore-coupling display system refers to a system having a spore system structure having a first coupler on the outside and a form having a target material having a second coupler in a coupling combination as an embodiment of the spore system.
  • Spore coupling displaying structure means a spore system structure and a structure that couples and is coupled to a spore-coupling immunogenic material.
  • spore coupling bond is meant a chemical bond formed by the interaction of a spore display body coupler and a spore immunogenic coupler.
  • Coupling group means a moiety that forms a coupling bond by the interaction of two or more homologous or heterogeneous coupling groups.
  • Spore coupling group on displaying structure means a coupler located on a coupling display body.
  • Spore coupling part on immunogen means that it is located in the coupling immunogen as a coupling group.
  • Spore coupling part means a coupled site formed by interaction of a display body coupling unit and an immunogenic material coupling unit.
  • an " immunogenic spore-coupling display system” refers to an immunogenic system having a spore system structure having a first coupling agent externally and an immunogenic material having a second coupling agent to form a coupling bond.
  • the system structure is defined as a "spore-coupling display body”.
  • the immunogenic material is defined as a " spore coupled immunogen ".
  • the first coupler is defined as a " spore display body coupler ".
  • the second coupler is defined as a " spore immunogenic substance coupling group ".
  • the spore-coupling display body encompasses the spore system structure of the immunogenic spore system.
  • the spore coupling bonds include the above-mentioned coupling bonds.
  • the spore display body coupler includes the above-mentioned display body coupler.
  • the spore immunogenic substance coupling group encompasses the above-mentioned immunogenic substance coupling group.
  • the spore-coupled immunogenic material may be homologous to the outer spore protein.
  • the homologous region may be a spore external signal sequence of the spore outer protein.
  • the spore-coupled immunogenic material is delivered to the outside of the spore by a spore external signal sequence, resulting in higher efficiency of spore-coupling binding.
  • the coupling moiety refers to a site including a display body coupling unit and an immunogenic coupling unit as a coupling-coupled site of the coupling display body and the coupling immunogenic substance.
  • a coupling portion may be a separate protein / base sequence, but may be only a functional group generated by one coupling bond.
  • the coupling part may not have a particular function, but may have a separate structural feature or a specific function in the immune formation process.
  • the spore coupling portion includes the above-mentioned coupling portion.
  • One example of the present application is a vaccine comprising an immunogenic spore-attached display system.
  • Spore-attached display system means that a target substance is attached to the outside of the spore system structure as one example of the spore system.
  • Spore attached displaying structure means a spore system structure, which refers to a structure that externally attaches and transmits spore attachment immunogenic material.
  • Immunogenic spore-attached display system means that an immunogenic substance is attached to the outside of the spore system structure as one embodiment of the spore immunogenicity system.
  • attachment means the bonding of two substances indirectly connected with each other.
  • spore system structure is defined as “spore attached display body”.
  • the immunogenic substance is defined as a " spore attached immunogen ".
  • the attachment may be by electrostatic attraction.
  • the attachment may be by hydrogen bonding.
  • the attachment may be by hydrophobic interaction.
  • the spore-attached display body encompasses the spore system structure of the immunogenic spore system.
  • the spore-attached display body includes the attached display body.
  • Spore attachment immunogenic material encompasses the immunogenic substance.
  • Spore attachment immunogenic material encompasses the attachment immunogenic substance.
  • the spore attachment immunogenic material may be homologous to the outer spore protein.
  • the homologous region may be a spore external signal sequence of the spore outer protein.
  • the spore-attach immunogenic material is delivered to the outside of the spore by the spore external signal sequence and can be attached to the spore-attached display body with higher efficiency.
  • spore systems In addition to the spore systems, other types of spore systems that deliver the target material may be used. As another example of the present application, it is possible to have aspects of a serial connection system, parallel system not mentioned above.
  • One example of the present application is immunogenic materials and their various uses made by genetic recombination methods for constructing the immunogenic system.
  • Target nucleotide sequence means a nucleotide sequence to be included in a recombinant base sequence.
  • Recombinant protein refers to a protein that is ultimately desired to be produced in a genetic engineering method.
  • Recombinant nucleotide sequence means a nucleotide sequence encoding a recombinant protein.
  • a " recombinant vector” is a carrier that functions to transfer a recombinant base sequence into living bodies, including, for example, plasmids, episomal vectors, viral vectors and the like.
  • recombinant expression vector means a vector which expresses the function of recombinant base sequence linked to a vector to express the target substance in vivo. These recombinant expression vectors can be used to confirm expression and production of recombinant proteins.
  • Such an expression vector may, for example, be in the form of a plasmid, but is not limited thereto.
  • incubating host cell means a cell that is transformed and transformed with a recombinant expression vector for the production of recombinant protein.
  • Methods for the production of recombinant proteins include obtaining proteins via genetic recombination methods.
  • the protein to be produced at this time is a desired recombinant protein, and the desired base sequence may be a sequence of such a recombinant protein.
  • a method for genetically preparing a protein comprises, as one embodiment, a first step of obtaining a target nucleotide sequence, a second step of producing a vector or polynucleotide having a recombinant nucleotide sequence containing the target nucleotide sequence, culturing the recombinant nucleotide sequence A third step of transforming host cells, a fourth step of culturing the transformed host cells, and a fifth step of obtaining recombinant proteins from the cultured host cells.
  • the first step is to obtain the desired nucleotide sequence.
  • nucleotide sequence of the protein in the wild state can be obtained by using a genbank of ncbi or by directly screening the protein.
  • the polynucleotide having the desired nucleotide sequence can be produced by conventional recombinant DNA technology.
  • the polynucleotides can be prepared by PCR cloning.
  • the second step is a step of preparing a vector or polynucleotide having a recombinant base sequence containing the desired nucleotide sequence.
  • the vector or polynucleotide may be single stranded or double stranded DNA containing the desired base sequence or RNA form.
  • the thus-produced polynucleotide is used in combination with a recombinant vector or a recombinant polynucleotide.
  • the recombinant vector or recombinant polynucleotide preparation method may comprise ligating a polynucleotide having a desired base sequence through a ligase.
  • the recombinant vector or recombinant polynucleotide may contain a promoter necessary for the expression of the recombinant base sequence.
  • the promoter may be derived from a host cell or artificially introduced separately.
  • the polynucleotide including the recombinant base sequence may be a recombinant expression vector into which the desired base sequence is inserted.
  • a method for inserting a desired nucleotide sequence into a recombinant expression vector known gene recombinant techniques can be used.
  • the third step is a step of transforming the cultured host cell with the recombinant polynucleotide or recombinant expression vector prepared in the second step.
  • the transformation may be classified as " transient transformation” in which expression of the recombinant base sequence takes place in the short term, or " permanent transformation " in which it is inserted into the genome of the cultured host cell by a recombinant expression vector.
  • Transformation can also be classified into “transformation by reactants”, “transformation by a tool”, and “transformation by a virus” by a method of inserting a base sequence into a cell membrane or a nuclear membrane.
  • Transformation by reactant refers to a conversion method in which a positively charged transformant and a nucleotide sequence are mixed to pass through a negatively charged membrane.
  • transformants include calcium phosphate, DEAE-Dextran, cationic lipid, cationic polymer, activated dendrimer, and magnetic bead.
  • Transformation by means of a tool " means a transformation method using an experimental apparatus developed for transformation.
  • transformation methods include electroporation (EP), biolistic technology, microinjection, and laserfection / optoinjection.
  • Viral transformation means a transformation using the nature of a virus that infects a host and inserts a base sequence.
  • the transformation method utilizes heat shock transfection, which is a type of electroporation.
  • Heat shock transfection refers to a method of transforming a competent cell prepared for easy transformation in advance by applying heat and electric shock. At this time, it is appropriate to convert cultured host cells into competent cells using conventional methods prior to transformation.
  • the cultured host cell may be an animal cell or a microbial cell. Vero cells and CHO cells, which are commonly used, can be used as animal cells. In case of using microbial cells, e. coli can be used.
  • the fourth and fifth steps are for culturing the transformed cultured host cells and isolating the recombinant proteins.
  • the culturing step may be carried out by using known culture conditions.
  • the step of separating the protein may be performed by a method such as cell lysis, centrifugation, gel electrophoresis or the like according to a conventional gene recombination method.
  • One example of the present application includes a recombinant expression vector for expression of the desired base sequence.
  • Expression control element means a nucleotide sequence that regulates the expression of an expression target base sequence as a component of a recombinant expression vector.
  • Expression objective base sequence means a recombinant base sequence to be expressed as a component of a recombinant expression vector.
  • One embodiment of a genetic production method of a protein is to transform a cultured host cell after preparing a recombinant expression vector.
  • the expression vector may be a plasmid, an episome vector, or a viral vector.
  • the form of the expression vector used is, but is not limited to, a plasmid. &Quot; Plasmid " means that a recombinant base sequence can be inserted as a closed circular double stranded DNA sequence.
  • the recombinant base sequence inserted into the recombinant expression vector may include an expression regulatory element and an expression target base sequence as a component.
  • the expression regulatory element is preferably an optional constituent but it is included.
  • the expression control element refers to elements that regulate expression as well as initiation of transcription of the expression target base sequence.
  • Expression control elements include promoters, enhancers, silencers, insulators, and the like.
  • Promoter means a sequence in which transcription is initiated on a base sequence.
  • Enhancer means a sequence that promotes the transcription of the base sequence when the activator is coupled as a remote control element.
  • a " silencer " is a remote control element that refers to a sequence that inhibits the transcription of the base sequence when the repressor is joined.
  • Insulator means a sequence that interrupts the interaction between the enhancer and the promoter.
  • the expression target nucleotide sequence means that the recombinant nucleotide sequence is inserted into the recombinant expression vector.
  • the expression control element in the recombinant expression vector is optimized for the host cell codon.
  • the target nucleotide sequence to be expressed in the recombinant expression vector can be inserted into the step including the first step and the second step of the genetic production method of the protein.
  • the method using the recombinant expression vector is only one example of the genetic production method. Unless otherwise stated, therefore, it is evident that the method of genetic production includes a method using a recombinant expression vector.
  • One example of the present application is a method for producing an immunogenic substance.
  • the immunogenic material produced may itself be a composition of the vaccine or may be used to produce an immunogenic system. Methods for the preparation of immunogenic materials may also be utilized in the manufacture of immunogenic systems.
  • Immunogenic target nucleotide sequence means a nucleotide sequence that is intended to be included in an immunogenic recombinant base sequence.
  • Immunogenic recombinant protein means an immunogenic protein that is ultimately desired to be produced in a genetic production method.
  • Immunogenic recombinant nucleotide sequence means a nucleotide sequence encoding an immunogenic recombinant protein.
  • immunogenic recombinant expression vector means, as an example of a vector, that the immunogenic recombinant base sequence linked to a vector is expressed in vivo. That is, when an immunogenic recombinant expression vector is inserted, production of an immunogenic recombinant protein can be confirmed. Most of such expression vectors are in the form of plasmids, but are not limited thereto.
  • Immunogen incubating host cell means a cell transformed with an immunogenic recombinant expression vector for the production of an immunogenic recombinant protein.
  • the method of manufacture provides for producing an immunogenic material through a genetic engineering method.
  • the protein to be produced at this time may be an immunogenic recombinant protein, and the immunogenic target base sequence may be an immunogenic part, a non-immunogenic part, a foreign part, or the like.
  • the method for genetically producing an immunogenic material comprises, in one embodiment, a first step of obtaining an immunogenic target nucleotide sequence, a second step of producing a vector or polynucleotide having an immunogenic recombinant nucleotide sequence containing the immunogenic target nucleotide sequence , A third step of transforming the immunogenic recombinant base sequence into an immunogenic culture host cell, a fourth step of culturing the transformed immunogenic culture host cell, a fifth step of culturing the immunogenic recombinant host cell, Step < / RTI >
  • the first step is to obtain the immunogenic target nucleotide sequence.
  • the nucleotide sequence of the wild-type immunogenic material can be obtained by using genbank of ncbi or directly screening pathogens.
  • Polynucleotides having an immunogenic target nucleotide sequence can be prepared by conventional recombinant DNA techniques.
  • the polynucleotides can be prepared by PCR cloning.
  • the second step is a step of preparing a vector or polynucleotide having an immunogenic recombinant base sequence containing the immunogenic target nucleotide sequence obtained in the first step.
  • the vector or polynucleotide may be a single-stranded or double-stranded DNA containing an immunogenic target sequence or may be in the form of RNA.
  • the method for producing the polynucleotide may be such that the polynucleotide having the immunogenic target base sequence is ligated through the ligase.
  • the immunogenic recombinant base sequence may comprise a promoter necessary for expression.
  • the promoter may be derived from a host cell or artificially introduced separately.
  • the polynucleotide including the immunogenic recombinant base sequence may also be an immunogenic recombinant expression vector inserted with an immunogenic target base sequence.
  • an immunogenic target nucleotide sequence into an immunogenic recombinant expression vector, conventional gene recombinant techniques can be used.
  • the third step is a step of transforming an immunogenic culturing host cell with the immunogenic recombinant polynucleotide or an immunogenic recombinant expression vector prepared in the second step.
  • Transformation can be classified as “transient transfection” or “permanent transfection” by its effect. In one embodiment of the present application, transient transfection was used.
  • Transformation can also be categorized as "transgenic by reaction”, “transgenic by tool” or “transgenic by virus” depending on the means. Heat shock transfection was used in one embodiment of the present application.
  • the immunogenic culture host cells include the cultured host cells.
  • the fourth and fifth steps are for culturing the transformed immunogenic cultured host cell and isolating the immunogenic recombinant protein.
  • the culturing step may be carried out by using known culture conditions.
  • the step of isolating the immunogenic recombinant protein can be carried out by conventional methods such as cell lysis, centrifugation, gel electrophoresis and the like.
  • the immunogenic recombinant expression vector The immunogenic recombinant expression vector
  • Immunogenicity expression regulatory element means a nucleotide sequence that regulates the expression of an immunogenic expression target sequence as a component of an immunogenic recombinant expression vector.
  • Immunogenic expression target nucleotide sequence means a recombinant nucleotide sequence to be expressed as a component of an immunogenic recombinant expression vector.
  • One embodiment of a method of genetically producing an immunogenic agent is to produce an immunogenic recombinant expression vector and then transform the immunogenic host cell.
  • the form of the immunogenic recombinant expression vector may be, but is not limited to, a plasmid.
  • the recombinant base sequence inserted into the immunogenic recombinant expression vector may comprise, as a component, an immunogenicity expression regulatory element and an immunogenic expression target sequence.
  • the immunogenicity expression control element encompasses the expression control element.
  • the immunogenicity expression control element is optimized in the immunogenic cultured host cell codon.
  • the immunogenic expression target nucleotide sequence refers to that inserted into the immunogenic recombinant expression vector as the above immunogenic recombinant base sequence.
  • the immunogenicity expression base sequence is the base sequence of the above immunogenic substance.
  • the nucleotide sequence for immunogenicity expression may be inserted into the step including the first step and the second step of the genetic preparation method of the immunogenic substance.
  • the method using recombinant expression vector of immunogenic material is only one example of genetic production method. Therefore, unless otherwise stated, it is clear that methods for genetic production of pathogen, antigen protein, antigen homologous protein, and antigen homologous additive protein include methods using immunogenic material recombinant expression vectors.
  • One example of the present application is a method for producing a pathogen as an immunogenic substance of a vaccine.
  • the method of manufacture comprises harvesting and culturing.
  • pathogens can be collected from the secretions, excretions, and dead bodies of diseased organisms. When these are cultured in culture medium or animal cells, they are differentiated into a plurality of pathogens. If the pathogen is sufficiently differentiated, it can be prepared as a vaccine composition. At this time, some pathogens can be stored and cultured to provide a large amount of vaccine.
  • the method of manufacture may be a method for the genetic production of a wild-type pathogen.
  • the genetic manufacturing method encompasses a genetic production method of the immunogenic substance.
  • the immunogenic target sequence may be a full-length sequence of a wild-type pathogen or a subsequence thereof.
  • the method of production may be a method of physically deleting, altering, or manipulating the amino acid / peptide / base sequence that constitutes the pathogen.
  • a known method can be used as a physical method.
  • the method may be a method of chemically deleting, altering or manipulating the amino acid / peptide / base sequence constituting the pathogen.
  • the deletion, modification, or manipulation method may include treating a pathogen with a specific chemical substance to delete, modify or manipulate a specific site.
  • a chemical e. G., A particular reagent or enzyme, that produces a response to a particular site after obtaining or producing a wild-type pathogen can be treated.
  • the method of manufacture may be a genetic production method of a deleted, transformed or engineered pathogen.
  • the genetic manufacturing method encompasses a genetic production method of the immunogenic substance.
  • the immunogenic target sequence may be a full-length sequence of a wild-type pathogen or a subsequence thereof.
  • a mutation (Point mutation) is performed by PCR in the first step described above, or a specific deletion, modification or deletion is carried out using a mutation kit for an immunogenic recombinant base sequence or an immunogenic recombinant expression vector in a second step Can cause manipulation.
  • a method for producing a pathogen with a modified overall structure is provided.
  • One example is the modification of genetically engineered pathogens.
  • the manufacturing method is a physical modification method.
  • a known method can be used as a physical method.
  • the manufacturing method is a chemical modification method.
  • a known method can be used as a chemical method.
  • the virus may be obtained and then treated with formalin, beta proprio lactone (BPL), bicomponent ethyleneimine (BEI) or gamma radiation, or by other methods known to those skilled in the art Deactivated.
  • BPL beta proprio lactone
  • BEI bicomponent ethyleneimine
  • gamma radiation or by other methods known to those skilled in the art Deactivated.
  • the inactivated virus is then mixed with a pharmaceutically acceptable carrier (such as a saline solution) and optional adjuvants. As a specific example, it may be inactivated at a final concentration of 0.1% formalin.
  • Another example is the production of modified pathogens by genetic modification methods (genetic recombination methods).
  • the genetic modification method encompasses a genetic production method of the immunogenic substance.
  • the immunogenic target nucleotide sequence may be a full-length sequence of the wild-type pathogen or a partial sequence thereof.
  • Mutation PCR may be performed in the first step described above, or, in the second step, deletion, modification, or manipulation may be performed entirely using the mutagenic kit for the immunogenic recombinant base sequence or the immunogenic recombinant expression vector.
  • One example of the present application is a method for producing an antigen protein as an immunogenic substance of a vaccine. At this time, two or more antigenic proteins of one pathogen can be produced or mixed at the same time.
  • the method of manufacture may be a method of separating the antigenic protein from a wild-type pathogen by a physical method.
  • the physical method at this time may be, but not limited to, centrifugation.
  • the physical method may be a known method.
  • the method of manufacture may be a method of isolating antigen protein from a wild-type pathogen by a chemical method.
  • the chemical method may be a known method.
  • the method of manufacture may be a method for the genetic production of a wild-type antigen protein.
  • Vaccines typically produced in this manner are termed subunit vaccines.
  • the genetic manufacturing method encompasses a genetic production method of the immunogenic substance.
  • the desired nucleotide sequence may be a full-length sequence of the wild-type antigen protein or a partial sequence thereof.
  • a method for producing an antigen protein in which a specific site has been modified or engineered is provided.
  • the method of manufacture may be a method of modifying wild-type antigenic proteins in a physical manner.
  • the physical method may be a known method.
  • the production method may be a method of chemically modifying wild-type antigen protein.
  • the chemical method may be a known method.
  • the method of production may be a method for the genetic production of an antigen protein in which a specific site has been modified or engineered.
  • the genetic manufacturing method encompasses a genetic production method of the immunogenic substance.
  • the desired nucleotide sequence may be a wild-state antigen protein. Mutagenic PCR is performed in the first step described above, or in the second step, an immunogenic recombinant base sequence or an immunogenically recombinant expression vector is subjected to a mutation kit to remove an antigen protein in which a specific site has been deleted, modified or engineered .
  • One example of the present application is a method for producing an antigen homologous protein as an immunogenic substance of a vaccine.
  • an antigen homologous protein comprising an immunogenic site.
  • the moiety comprising the immunogenic site may be a method that is made by limiting the antigen protein produced.
  • Methods for limiting antigenic proteins include physical methods such as centrifugation, and chemical methods using chemical reagents such as proteases.
  • the method for producing an antigen-homologous protein may be a genetic preparation method of a part including an immunogenic part.
  • the genetic production method encompasses the genetic production method of the immunogenic substance described above.
  • An immunogenic recombinant protein is an antigen homologous protein comprising at least one immunogenic site.
  • the immunogenic target nucleotide sequence is a nucleotide sequence encoding the immunogenic region.
  • the first step encompasses obtaining the immunogenic site.
  • the nucleotide sequence of the immunogenic region of the antigen protein can be found by using ncbi genbank or directly screening pathogens.
  • Polynucleotides containing the desired nucleotide sequence can be prepared by conventional recombinant DNA techniques.
  • the polynucleotides can be prepared by PCR cloning.
  • primers of the immunogenic target sequence can be designed through multiple alignments.
  • Common region means a sequence portion common among a plurality of protein sequences or base sequences.
  • the present inventors searched for a common region present in the antigen proteins of the existing strains and mutants, and tried to prepare the antigen by including the immunogenic region between the two common regions.
  • primers Two common regions are designed as primers.
  • an immunogenic target nucleotide sequence is obtained.
  • a primer that is designed through multiple alignments and configured as a common region is defined as a " common region primer ".
  • the common region primer by multiple alignment can eliminate the step of screening the mutant and the step of designing and producing the primer in the above step, so that it is possible to cope with the mutant more rapidly. It is well suited for the purpose of master vaccine and multiple disease master vaccine for the purpose of creating strong immunity against allogeneic or heterologous pathogens and mutants thereof.
  • the strains that are subject to multiple alignments may be strains of one or more pathogens found in one country.
  • the strains to be subjected to multiple sorting may be strains of a pathogen occurring in one country and may be of different kinds or types.
  • the multi-aligned strains may include North American and European strains.
  • the strains to be subjected to multiple sorting may include foreign strains imported from foreign countries in addition to the strains found in one country.
  • the foreign country may be an adjacent country.
  • the foreign country can be a country in a specific area. For example, East Asia, Southeast Asia, North America, and Eastern Europe.
  • the foreign country can be a country located within a certain distance from one country.
  • the foreign country can be a country where exchange is active. Preferably, it may be an active country associated with livestock. Also, the foreign country can be a country where migratory birds pass through a country.
  • strains that are subject to multiple alignments may be selected based on overall homology to a particular strain.
  • the strains may be selected so that the overall homology to a specific strain is not less than a specific value.
  • the step of selecting strains to be subjected to multiple sorting includes steps of dividing the strains according to the entire homology to a specific vaccinia, sorting the strains according to the strains, and selecting strains belonging to the strains belonging to each strains to a specific ratio .
  • the group may be divided according to the range of homology to a specific strain.
  • the common region primer by multiple alignment of the present application makes it possible to efficiently produce an antigen homologous protein as an immunogenic substance of a vaccine against these various strains and their mutant strains.
  • the common region primer may correspond to a non-immunogenic region.
  • the common region between the antigenic proteins of the mutant is mostly non-immunogenic.
  • Antigen homologous proteins produced using these common region primers will inevitably contain non-immunogenic sites, including immunogenic sites.
  • the non-immunogenic region exists at the N-terminus and C-terminus of the antigen-homologous protein and is the same as the sequence of the common region primer.
  • an immunogenic recombinant polynucleotide or an immunogenic recombinant expression vector can be prepared by inserting the immunogenic target nucleotide sequence obtained in the first step. At this time, any number of immunogenic sites can be inserted as needed.
  • the engineered antigen homologous protein may have the form of a tandem linker.
  • a method for producing an antigen-homologous protein comprising at least two selected from
  • an antigen homologous protein comprising two or more selected from immunogenic site-1 to immunogenic site-n.
  • Antigen homologous proteins comprising two or more different immunogenic regions of an antigenic protein can be prepared by a genetic engineering method. Wherein the genetic manufacturing method encompasses a genetic production method of the immunogenic substance.
  • the immunogenic recombinant protein may be an antigen-homologous protein comprising two or more of immunogenic site-1 to immunogenic site-n.
  • the immunogenic target nucleotide sequence may be a nucleotide sequence encoding each immunogenic site-x.
  • the first step encompasses obtaining each of the immunogenic sites.
  • the nucleotide sequence of the immunogenic region of the antigen protein can be found by using ncbi genbank or directly screening pathogens.
  • Polynucleotides containing immunogenic target nucleotide sequences can be prepared by conventional recombinant DNA techniques.
  • the polynucleotides can be prepared by PCR cloning.
  • the primers of the immunogenic target sequence can also be designed using the above multiple alignments.
  • the primers of the immunogenic subject sequence may be common region primers for each immunogenic site.
  • the common region primer may correspond to a non-immunogenic region.
  • the common region between the antigenic proteins of the mutant is mostly non-immunogenic.
  • Antigen homologous proteins produced using this common region primer will inevitably contain two or more selected from immunogenic site-1 to immunogenic site-n and non-immunogenic sites.
  • non-immunogenic regions are present at the 5'-end and 3'-end of each immunogenic target nucleotide sequence and are the same as those of the common region primer.
  • the second step is a step of preparing an immunogenic recombinant polynucleotide or an immunogenic recombinant expression vector by inserting the immunogenic target base sequence obtained in the first step.
  • the sequence of the inserted immunogenic region may be different from that of the immunogenic region in the wild state.
  • any number of immunogenic sites may be inserted as needed.
  • An antigen-homologous protein comprising two or more selected from the immunogenic site
  • an antigen homologous protein comprising two or more selected from a 1-immunogenic site to an m-immunogenic site.
  • Antigen homologous proteins comprising two or more immunogenic regions of different antigenic proteins can be produced by genetic engineering methods.
  • the genetic manufacturing method encompasses a genetic production method of the immunogenic substance.
  • the immunogenic recombinant protein can be an antigen-homologous protein comprising two or more of the m-immunogenic sites from the 1-immunogenic site.
  • the immunogenic target nucleotide sequence is a nucleotide sequence encoding each y-immunogenic region.
  • the first step encompasses obtaining each of the immunogenic sites.
  • the nucleotide sequence of the immunogenic region of the antigen protein can be found by using ncbi genbank or directly screening pathogens.
  • Polynucleotides containing an immunogenic target sequence can also be prepared by conventional recombinant DNA techniques.
  • the polynucleotides can be prepared by PCR cloning.
  • the primers of the immunogenic target sequence can also be designed using the above multiple alignments.
  • the primers of the immunogenic subject sequence may be common region primers for each immunogenic site.
  • the common region primer may correspond to a non-immunogenic region.
  • the common region between the antigenic proteins of the mutant is mostly non-immunogenic.
  • Antigen homologous proteins produced using this common region primer will inevitably contain two or more selected from the 1-immunogenic site to the m-immunogenic site and two selected from the 1-non-immunogenic site to the non- Or more.
  • non-immunogenic regions are present at the 5'-end and 3'-end of each target nucleotide sequence and are the same as those of the common region primer.
  • the second step is a step of preparing an immunogenic recombinant polynucleotide or an immunogenic recombinant expression vector by inserting the immunogenic target nucleotide sequence obtained in the first step.
  • the sequence of the inserted immunogenic region may be different from that of the immunogenic region in the wild state.
  • any number of immunogenic sites may be inserted as needed.
  • antigen-homologous proteins comprising a plurality of immunogenic regions of a plurality of antigenic proteins can also be prepared by the above-described genetic production methods.
  • Antigen homologous protein recombinant expression vector Antigen homologous protein recombinant expression vector
  • an antigen-homologous protein recombinant expression vector for genetic production of an antigen homologous protein in one embodiment, there is provided an antigen-homologous protein recombinant expression vector for genetic production of an antigen homologous protein.
  • the antigen-homologous protein recombinant expression vector encompasses the immunogenic recombinant expression vector.
  • the antigen-homologous protein recombinant expression vector produced according to the above-mentioned 'method for producing an antigen-homologous protein' can be provided.
  • the immunogenic expression target sequence may comprise the nucleotide sequence of the immunogenic site.
  • the immunogenicity regulating element may be a promoter.
  • the promoter may be a promoter in a wild state.
  • the promoter is optimized for immunogenic cultured host cells.
  • the promoter may be a wild-type promoter derived from an immunogenic culturing host cell, or may be a promoter introduced separately.
  • the promoter may be a wild-type promoter modified at some sites. At this time, the modified promoter may have an improved initiation efficiency.
  • the immunogenic expression objective base sequence may comprise the base sequence of the immunogenic site.
  • the nucleotide sequence of the immunogenic site may comprise a non-immunogenic region corresponding to the common region primer sequence at the 5 ', 3' terminus.
  • the immunogenic expression objective base sequence may comprise multiple base sequences of one or more immunogenic regions.
  • the arrangement of the overall configuration is preferably a promoter-immunogenic site.
  • 18 shows an example of an antigen-homologous protein recombinant expression vector comprising a promoter and a base sequence of 1-immunogenic site-1.
  • the immunogenic expression objective base sequence may comprise two or more base sequences selected from immunogenic site-1 to immunogenic site-n.
  • the immunogenicity regulating element may be a promoter.
  • the promoter includes the above-mentioned promoter.
  • the term " promoter" includes all the promoters of the first embodiment.
  • an immunogenic expression objective base sequence may comprise two or more base sequences selected from immunogenic site-1 to immunogenic site-n.
  • the nucleotide sequence of the immunogenic site may comprise a non-immunogenic region corresponding to the common region primer sequence at the 5 ', 3' terminus.
  • the immunogenic expression objective base sequence may comprise multiple base sequences of one or more immunogenic regions.
  • the arrangement of the whole constitution is a promoter- (immunogenic region-x) z.
  • z means the number of immunogenic sites contained in the vector.
  • 19 is an example of an antigen homologous protein recombinant expression vector comprising a promoter and a base sequence of 1-immunogenic site-1 and 1-immunogenic site-2.
  • sequence of the immunogenic site may be different from the wild-type antigen protein.
  • the immunogenic expression objective base sequence may comprise two or more base sequences selected from the 1-immunogenic site to the m-immunogenic site.
  • the immunogenicity regulating element may be a promoter.
  • the promoter includes the above promoter.
  • the immunogenic expression objective base sequence may include two or more base sequences selected from the 1-immunogenic site to the m-immunogenic site.
  • the nucleotide sequence of the immunogenic site may comprise a non-immunogenic region corresponding to the common region primer sequence at the 5 ', 3' terminus.
  • the immunogenic expression objective base sequence may comprise multiple base sequences of one or more immunogenic regions.
  • the overall configuration is an promoter- (y-immunogenic site) z.
  • z means the number of immunogenic sites contained in the vector.
  • 20 shows an example of an antigen-homologous protein recombinant expression vector comprising a promoter and a base sequence of 1-immunogenic site-1 and 2-immunogenic site-2.
  • the sequence of the immunogenic site may be different from the wild-type antigen protein.
  • an antigen-homologous protein recombinant expression vector in which an immunogenic expression objective base sequence comprises a plurality of immunogenic regions of a plurality of antigen proteins can also be produced.
  • One example of the present application is a method for producing an antigen homologous portion of a protein as an immunogenic substance of a vaccine.
  • immunogenic site used herein to describe a method for producing a genetic protein of an antigen includes not only the immunogenic site mentioned in the above " method for producing an antigen homologous protein " Concept.
  • immunogenic site means a y-antigen protein or a y-immunogenic site-x.
  • a method for producing an antigen homologous portion protein comprising an immunogenic portion and a nonantigen portion
  • an antigen-homologous moiety comprising an immunogenic site and a non-antigenic moiety provides a method for producing a protein.
  • An antigen homologous portion protein comprising an immunogenic portion and a non-antigenic portion can be produced by a genetic engineering method. Wherein the genetic manufacturing method encompasses a genetic production method of the immunogenic substance.
  • Immunogenic recombinant proteins are proteins that are antigen homologous, including immunogenic sites and non-antigenic sites.
  • the immunogenic subject nucleotide sequence includes the nucleotide sequence of the immunogenic region and the nucleotide sequence of the non-immunogenic region.
  • the first step encompasses obtaining the immunogenic target nucleotide sequence.
  • the nucleotide sequence of the immunogenic site can be obtained by a method comprising a genetic preparation method of the antigen protein and a first step of a genetic preparation method of the antigen homologous protein.
  • the nucleotide sequence of the noncontiguous part of the pathogen can be determined by using ncbi genbank or directly screening the pathogen.
  • Polynucleotides containing an immunogenic target sequence can also be prepared by conventional recombinant DNA techniques.
  • the polynucleotides can be prepared by PCR cloning.
  • the primers of the immunogenic target sequence can also be designed using the above multiple alignments.
  • the primers of the immunogenic subject sequence may be common region primers for each immunogenic target sequence.
  • the immunogenic target nucleotide sequence may have a sequence of a common region primer at the 5 'end and the 3' end.
  • the second step is a step of preparing an immunogenic recombinant base sequence or an immunogenic recombinant expression vector by inserting the immunogenic subject base sequence obtained in the first step.
  • the sequence of the inserted immunogenic region may be different from that of the immunogenic region in the wild state.
  • any number of immunogenic regions or non-antigenic regions can be inserted as needed.
  • the non-antigenic portion may be part of the outer membrane protein of the pathogen.
  • the non-antigenic portion may be part of a protein having immunosuppressive function.
  • an antigen homologous portion comprising an immunogenic portion and an exogenous portion provides a method for producing a protein.
  • Antigen homologous portion proteins can be produced by genetic engineering methods. Wherein the genetic manufacturing method encompasses a genetic production method of the immunogenic substance.
  • Immunogenic recombinant proteins are proteins that are antigen homologous, including immunogenic sites and foreign sites.
  • the immunogenic target nucleotide sequence is the nucleotide sequence of the immunogenic region and the nucleotide sequence of the foreign gene.
  • the foreign part can be broadly classified into those derived from proteins of other pathogens or those derived from totally different exogenous proteins.
  • the outpatient part derived from the protein of another pathogen is defined as "the outpatient part of the other pathogen”
  • the outpatient part derived from the different outpatient protein is defined as the "outpatient part of the consultation”.
  • the first step encompasses obtaining the immunogenic target nucleotide sequence.
  • the method for obtaining the base sequence of the immunogenic region encompasses the first step of the genetic preparation method of the antigen protein and the genetic preparation method of the antigen homologous protein.
  • ncbi genbank Outside sequences of other pathogens can be found using ncbi genbank or directly screening other pathogens.
  • the nucleotide sequence of the foreign part of the consultation can be found by using ncbi genbank or by directly screening the protein after separation and purification.
  • Polynucleotides containing immunogenic target nucleotide sequences can be prepared by conventional recombinant DNA techniques.
  • the polynucleotides can be prepared by PCR cloning.
  • the primers of the immunogenic target sequence can also be designed using the above multiple alignments.
  • the primers of the immunogenic subject sequence may be common region primers for each immunogenic target sequence.
  • the immunogenic target nucleotide sequence may have a sequence of a common region primer at the 5 'end and the 3' end.
  • the second step is a step of preparing an immunogenic recombinant polynucleotide or an immunogenic recombinant expression vector by inserting the immunogenic target nucleotide sequence obtained in the first step.
  • the sequence of the inserted immunogenic region may be different from that of the immunogenic region in the wild state. Also, any number of immunogenic sites or foreign parts can be inserted as needed.
  • the foreign part can be a foreign part of another pathogen.
  • the foreign part of the other pathogen may be an immunogenic part on the antigen protein of another pathogen.
  • the foreign part of another pathogen may be a non-immunogenic part or non-antigenic part of another pathogen.
  • the foreign part may be a foreign part of the consultation.
  • the antigen homologous portion is a protein recombinant expression vector
  • the antigen-homologous moiety for genetic production of a protein by an antigen-homologous moiety provides a protein recombinant expression vector.
  • the antigen-homologous protein recombinant expression vector encompasses the immunogenic recombinant expression vector.
  • the antigen-homologous portion made according to the 'method for producing an antigen-homologous portion of a protein' may provide a protein recombinant expression vector.
  • the immunogenic expression base sequence of interest may comprise an immunogenic site, a non-antigenic base sequence.
  • the immunogenicity regulating element may be a promoter.
  • the promoter may be a promoter in a wild state.
  • the promoter is optimized for immunogenic cultured host cells.
  • the promoter may be a wild-type promoter of an immunogenic culturing host cell, or may be an artificially introduced promoter.
  • the promoter may be a wild-type promoter modified at some sites. At this time, the modified promoter may have an improved initiation efficiency.
  • the immunogenic expression target base sequence may include an immunogenic site, a non-antigenic base sequence.
  • the nucleotide sequence of the immunogenic region encompasses the genetic production method of the antigen protein, the nucleotide sequence obtained in the first step of the genetic production method of the antigen homologous protein.
  • the immunogenic expression objective base sequence may include a plurality of immunogenic regions and non-antigenic regions.
  • the arrangement of the overall constitution is preferably promoter- (immunogenic site-antagonistic) z.
  • z means that the immunogenic part and non-antigenic part can be included in any order with any number.
  • the recombinant expression vector is defined as a " pathogen-like particle recombinant expression vector" when the non-antigenic part is part of the outer membrane protein of the pathogen.
  • Figure 21 is an illustration of a recombinant expression vector for a pathogen-like particle comprising a promoter, a 1-immunogenic site-1, and a base sequence of an outer membrane protein.
  • the recombinant expression vector is defined as an "auxiliary immunoaddition recombinant expression vector" when the non-antigenic portion is part of a protein having immunocompetent function.
  • Figure 22 is an illustration of a recombinant expression vector for an accessory immunostain comprising a promoter, a 1-immunogenic site-1, and a base sequence of a non-antigenic moiety having immuno-assisting function.
  • the immunogenic expression base sequence of interest may comprise an immunogenic region, an exogenous base sequence.
  • the immunogenicity regulating element may be a promoter.
  • the promoter includes the above promoter.
  • the term " promoter" includes all the promoters of the first embodiment.
  • an immunogenic expression target base sequence may include an immunogenic region, an exogenous base sequence.
  • the nucleotide sequence of the immunogenic region encompasses the genetic production method of the antigen protein, the nucleotide sequence obtained in the first step of the genetic production method of the antigen homologous protein.
  • the immunogenic expression objective base sequence may comprise a plurality of immunogenic regions, exons.
  • the arrangement of the overall constitution is preferably a promoter- (immunogenic site-foreign part) z.
  • the recombinant expression vector is defined as a " poly-immunogenic recombinant expression vector" when the foreign part is an immunogenic region on the antigen protein of another pathogen.
  • Figure 23 is an illustration of a polyimmunized recombinant expression vector comprising the promoter, the 1-immunogenic site-1, and the nucleotide sequence of the immunogenic site of the other pathogen.
  • the recombinant expression vector is defined as a " multivalent pathogen-like particle recombinant expression vector" when the foreign part is a non-immunogenic or non-antigenic part of another pathogen.
  • 24 is an illustration of a multivalent pathogen-like particle recombinant expression vector comprising the promoter, 1-immunogenic site-1, and non-immunogenic and non-antigenic sites of other pathogens.

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  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
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  • Animal Behavior & Ethology (AREA)
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  • Public Health (AREA)
  • Immunology (AREA)
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  • Virology (AREA)
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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente demande concerne divers modes de réalisation et utilisations d'une substance immunogène ciblant les pathogènes d'une maladie animale particulière, un système immunogène, et une composition vaccinale à usage animal comprenant ledit système immunogène. Dans un mode de réalisation préféré, le système immunogène selon la présente demande peut être efficacement utilisé dans un vaccin, un médicament et un aliment pour animaux pour prévenir, soulager et traiter une maladie infectieuse porcine.
PCT/KR2018/008337 2017-07-24 2018-07-24 Système immunogène et vaccin animal le comprenant Ceased WO2019022463A2 (fr)

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CN113248627A (zh) * 2021-05-21 2021-08-13 安徽农业大学 一种增强猪流行性腹泻免疫的抗原及其制备方法和应用

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KR100267745B1 (ko) 1998-01-10 2000-11-01 . 돼지 유행성설사병 바이러스의 특이 중화항체 검출방법
US20030165538A1 (en) * 2000-06-26 2003-09-04 Maxygen Incorporated Methods and compositions for developing spore display systems for medicinal and industrial applications
ES2425228T3 (es) * 2005-09-09 2013-10-14 Intervet International B.V. Vacuna contra el PCV-2
KR100578395B1 (ko) 2005-11-11 2006-05-10 주식회사 바이오리더스 면역기능이 강화된 사균화 유산균 제제 및 그 제조방법
JP2008231063A (ja) 2007-03-22 2008-10-02 Takano Foods Kk 納豆菌ワクチン
AU2015384786B2 (en) 2015-03-03 2020-08-27 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Display platform from bacterial spore coat proteins

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113248627A (zh) * 2021-05-21 2021-08-13 安徽农业大学 一种增强猪流行性腹泻免疫的抗原及其制备方法和应用
CN113248627B (zh) * 2021-05-21 2022-07-15 安徽农业大学 一种增强猪流行性腹泻免疫的抗原及其制备方法和应用

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WO2019022463A3 (fr) 2019-07-18
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