WO2021017365A1 - Cra4S1基因及其编码的蛋白和应用 - Google Patents

Cra4S1基因及其编码的蛋白和应用 Download PDF

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WO2021017365A1
WO2021017365A1 PCT/CN2019/124433 CN2019124433W WO2021017365A1 WO 2021017365 A1 WO2021017365 A1 WO 2021017365A1 CN 2019124433 W CN2019124433 W CN 2019124433W WO 2021017365 A1 WO2021017365 A1 WO 2021017365A1
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protein
cra4s1
gene
vaccine
infection
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史小菊
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Zhenjiang Yangtze Green Biotechnology Co Ltd
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Zhenjiang Yangtze Green Biotechnology Co Ltd
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Priority to US17/613,122 priority Critical patent/US12552841B2/en
Priority to GB2200745.4A priority patent/GB2602562B/en
Priority to EP19939714.2A priority patent/EP4008341A4/en
Priority to JP2022503798A priority patent/JP7396569B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/0208—Specific bacteria not otherwise provided for
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/116—Polyvalent bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P31/04—Antibacterial agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Gram-negative bacteria
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/70—Multivalent 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
    • A61K39/02—Bacterial antigens
    • A61K39/0216—Bacteriodetes, e.g. Bacteroides, Ornithobacter, Porphyromonas
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biopharmaceuticals, in particular to cra4S1 gene and its encoded protein and applications.
  • Periodontal disease is one of the most common diseases of the oral cavity worldwide, and 80-97% of adults in China have periodontal problems of varying degrees. Periodontal disease is an independent risk factor for coronary heart disease and its acute attack, and an independent risk factor for stroke. Periodontal disease-causing bacteria Porphyromonas gingivalis and Alzheimer's disease (commonly known as Porphyromonas gingivalis) have been found Alzheimer’s disease). Periodontal disease is a complication of diabetes. It is closely related to digestive and respiratory diseases and clinical diseases such as pregnant women giving birth to premature babies and low birth weight babies. It has been found to be the cause of many diseases with extremely high mortality. An important cause, it is listed by the World Health Organization as the third largest non-communicable disease after cardiovascular disease and cancer.
  • periodontal disease The development of periodontal disease is divided into different stages, and its distinctive feature is that it is easy to relapse and the local lesions are in a progressive development state.
  • the treatment of periodontal disease relies on local removal of the lesion, antibiotics, and surgery, but these measures treat the symptoms but not the root cause, and do not remove the fundamental problem of pathogen infection.
  • Porphyromonas gingivalis is the most widely studied and well-documented necessary pathogen of adult periodontal disease.
  • Candidate vaccines for Porphyromonas gingivalis including capsular polysaccharides, fimbriae, lipids, outer membrane proteins, heat shock proteins, proteases, inactivated whole bacteria, etc. are in the current research and development category, but the pathogenic bacteria have different subtypes Its molecular structure, virulence factor, DNA sequence and toxicity are different, and the distribution of different subtypes of Porphyromonas gingivalis in the population has not been found.
  • Effective vaccines must be able to induce long-term immunity to certain microbial antigens, prevent pathogenic bacteria from invading again, and reduce the harm it causes to the human body.
  • the production of vaccine/antibody products against bacterial infections of different subtypes of pathogenic bacteria can produce stable preventive and therapeutic effects, which is a difficult point in commercial development. So far, no related products have been put on the market.
  • the objective of the present invention is to develop safe, stable and effective biological products to prevent and treat diseases caused by chronic infection of Porphyria gingivalis, including but not limited to periodontal disease/peri-implantitis.
  • the key technology solved by the present invention is to provide the cra4S1 gene and its coded Cra4S1 protein.
  • the technical problem to be solved by the present invention is to provide an expression cassette, a recombinant vector or a cell, which contains the cra4S1 gene or its gene fragment.
  • the technical problem to be solved by the present invention is to provide the cra4S1 gene, the Cra4S1 protein, the expression cassette, the recombinant vector or the cell in the preparation of prevention or treatment of periodontal disease or peri-implantitis or infection with Porphyria gingivalis Application in vaccines or drugs for related diseases.
  • the technical problem to be solved by the present invention is to provide a vaccine or medicine, which contains the Cra4S1 protein or fragments thereof.
  • the technical problem to be solved by the present invention is to provide specific antibodies, which are prepared by immunizing animals with preparations containing the Cra4S1 protein or fragments thereof.
  • the present invention uses a combination vaccine and/or dual target specific antibodies to prevent and treat periodontal disease/peri-implantitis and other related diseases caused by chronic infection of Porphyria gingivalis.
  • the technical solution of the present invention is to provide a novel cra4S1 gene, and the nucleotide sequence of the cra4S1 gene is shown in SEQ ID NO:1.
  • the content of the present invention also includes the method for obtaining the cra4S1 gene.
  • the content of the present invention also includes the Cra4S1 protein encoded by the cra4S1 gene, the amino acid sequence of which is shown in SEQ ID NO.2.
  • the content of the present invention also includes an expression cassette, a recombinant vector or a cell, which contains the cra4S1 gene.
  • the content of the present invention also includes the design of the cra4S1 gene, the artificial synthesis gene, the protein, the expression cassette, the recombinant vector or the cell in the preparation of prevention or treatment of periodontal disease or peri-implantitis or gingival porphyrin Bacterial infection-related diseases in vaccines or drugs.
  • the content of the present invention also includes a vaccine or medicine, which contains the Cra4S1 protein or fragments thereof.
  • the vaccine or medicine also includes one or more of W50ragB protein, ThairagB protein, QMLragB protein and 381ragB protein of Porphyromonas gingivalis.
  • the formula of the vaccine of the present invention is as follows: the mass ratio of Cra4S1 protein to W50ragB protein in the vaccine is 1:2-8; or the mass ratio of Cra4S1 protein to ThairagB protein is 1:1-6; or The mass ratio of the Cra4S1 protein to the QMLragB protein is 1:1-6; or the mass ratio of the Cra4S1 protein to the 381ragB protein is 1:2-9; or the Cra4S1 protein and the W50ragB protein, ThairagB protein, QMLragB protein, 381ragB
  • the protein formula is optimized according to the above mass ratio.
  • the formula of the combined vaccine is Cra4S1 protein: W50ragB protein: QMLragB protein, and the mass ratio is 1:1:1.
  • the vaccines or drugs of the present invention include, but are not limited to, those used to prevent occurrence or recurrence or to treat uninfected healthy people or patients with periodontal disease or peri-implantitis or chronic infectious periodontal disease, and others related to gingival porphyrin Bacterial infection related diseases can be used.
  • the Porphyromonas gingivalis includes W50 subtype, Thai subtype, QML subtype and 381 subtype.
  • the content of the present invention also includes a specific antibody prepared by immunizing animals with a preparation containing the Cra4S1 protein or fragments thereof.
  • the combined application of the specific antibodies also includes preparations of one or more of the W50ragB protein, ThairagB protein, QMLragB protein and 381ragB protein of Porphyromonas gingivalis to guide the preparation of immunized animals.
  • the immunization dosage of the Cra4S1 protein, W50ragB protein, ThairagB protein, QMLragB protein and 381ragB protein is 0.5-50 ⁇ g
  • the immunization dosage of the Cra4S1 protein, W50ragB protein, ThairagB protein, QMLragB protein and 381ragB protein are all 0.5-5 ⁇ g.
  • the immunization dosage of the Cra4S1 protein, W50ragB protein, ThairagB protein, QMLragB protein and 381ragB protein are all 5-50 ⁇ g.
  • the amount of polyclonal antibody obtained after immunization is 100-200 ⁇ g.
  • the specific antibodies of the present invention include, but are not limited to, those used to prevent occurrence or recurrence or to treat uninfected healthy people or patients with periodontal disease or peri-implantitis or chronic infectious periodontal disease, and others related to gingival porphyrin Bacterial infection related diseases can be used.
  • the core content of the present invention is first to successfully solve the technical bottleneck of gene expression difficulties.
  • the key is to effectively utilize the molecular synergy and interdependence between co-transcribed genes to successfully manufacture the prevention and treatment of periodontal disease/periimplantitis Vaccines and antibody products.
  • the rag locus exists in the genomic DNA of Porphyromonas gingivalis bacteria, which consists of ragA and ragB genes and irregular inserts at both ends ( Figure 1).
  • the open reading frames of ragA and ragB genes are independent.
  • the RagB protein cannot be expressed normally, or when the downstream ragB gene is artificially mutated, the RagA protein cannot be expressed normally in bacteria. (Refer to PCT/GB2005/ 001976).
  • the ragA gene also has a high degree of genetic polymorphism, but there is no published literature on the related research on the encoded protein corresponding to the ragA gene DNA polymorphism.
  • cra4S1 a gene sequence named cra4S1 (see SEQ ID NO. 1), which means that there is a stable and conserved amino acid sequence in the ragA gene of Porphyromonas gingivalis, pointing out the fact that the four main bacterial subtypes share a common protein sequence.
  • the cra4S1 gene fragment was successfully cloned into an expression vector, and a recombinant protein was produced under inducing conditions.
  • Purified recombinant protein was obtained using the principle of chromatography and molecular sieve (Example 3, Figures 2, 3, 5, 6 and 7).
  • the recombinant Cra4S1 protein has a molecular weight of about 15KDa.
  • the sequencing results of the recombinant cloned gene DNA sequence are consistent with the design plan.
  • the encoded protein sequence is consistent with the theoretical sequence of the stable region ( Figure 4), and the molecular weight is consistent with the expected theoretical value.
  • Recombinant Cra4S1 protein has strong antigenicity, and high titer antibodies can be produced by immunizing animals with a small dose (described in detail in Example 8), but using Cra4S1 protein alone to immunize animals without immune protection.
  • the combination vaccine also reflects the improvement of the stability of the product, which is reflected in the data standard deviation being kept at a low level.
  • the standard deviation is a measure of the degree of dispersion of the average value of a group of data. A smaller standard deviation means that these values are more Close to the average value means that the risk of accidental dispersion is reduced. This phenomenon further proves that RagA and RagB dual target antigen antibody binding immune effect is more stable.
  • the innovation of the present invention is to use the multi-target feature of bacterial surface antigen to produce stable and efficient biological products.
  • the combination vaccine is not only used in healthy mice, but also applied to animals that have been infected with Porphyria gingivalis.
  • Experimental results show that the combination vaccine has a significant immune protection effect on the infected body (Example 9), which is another aspect of the present invention Innovation.
  • Periodontal disease/peri-implantitis caused by Porphyria gingivalis infection is a chronic and intermittent process with a progressive course. It has been observed in animal experiments that after multiple infections, not only the local lesions are getting worse, but the whole body of the animals also shows debilitating phenomena (Example 7). Clinically, most people have different degrees of periodontal disease. Therefore, the present invention controls chronic infections and can prevent chronic infections from harming the health of the whole body, and the products will have broad market prospects.
  • the passive immunization experiment also verified that the combined antibody contains the Cra4S1 protein antibody and the outer membrane protein RagB antibody, and its immunoprotective effect is stronger than the experimental group using single antibodies (Example 10) .
  • the present invention first published the cra4S1 gene related to Porphyromonas gingivalis, and cloned the cra4S1 gene for the first time and successfully expressed it in Escherichia coli to obtain the Cra4S1 protein.
  • the protein is used in combination with products such as the Porphyria gingivalis RagB vaccine, It not only enhances the immune protection effect of the body against Porphyria gingivalis infection, but also enhances the stability of the vaccine product function.
  • the combination vaccine and combination antibody both show significant immune protection in the pre-infected body, suggesting the present invention It has a wide range of application prospects, and the product can be used for healthy people as well as for patients with chronic infections.
  • the vaccine of the present invention contains the target of Porphyromonas gingivalis specific outer membrane protein and the newly discovered conserved region antigen component, which improves the efficacy of the vaccine made by using the outer membrane protein alone or the newly discovered antigen alone.
  • Porphyromonas gingivalis conservative area antigen is combined with the antibody produced by the outer membrane protein, the immune protection effect of the antibody is significantly improved.
  • the research results showed that the experimental animal group using the product of the present invention, including vaccines and bispecific antibodies, compared with the control groups, the standard deviation of the experimental data was maintained at a low level, suggesting that the product of the present invention can provide in vivo experiments. Better stability.
  • the content of the present invention points out that in anti-infective immunity, specific antibodies have interrelated target binding on the surface of bacteria.
  • the characteristics of immune molecules binding to antigens in conserved regions and specific antigens that recognize bacterial subtypes can be used to stabilize and effectively Give full play to the effect of antibodies, inhibit and eliminate pathogenic bacteria, and achieve the purpose of preventing and treating diseases.
  • FIG 1 Porphyromonas gingivalis W50 major outer membrane protein antigen receptor gene rag (rag, r eceptor a ntigen g ene) site;
  • FIG. 2 Recombinant plasmid vector construction diagram, indicating: C5361BH150-1 is the target gene cra4S1;
  • Figure 3 Recombinant plasmid identification;
  • Figure 3A Lane 1 is the recombinant cra4S1-pET-30a(+) plasmid, Lane 2 is the enzyme digestion reaction, there are plasmid DNA and cra4S1 gene bands, and M is the 1kb DNA gradient ruler (Kings Switzerland),
  • Figure 3B Comparison of 1kb DNA gradient ruler and DL3000 (GenScript) molecular weight ruler;
  • FIG. 5 Expression detection of recombinant protein after induction, lane M: protein molecular weight scale (15-120kDa); lane 9: total protein of bacteria before induction (colony 1); lane 10: total protein of bacteria after induction (colony 1); lane 11: Total protein of bacteria before induction (colony 2); Lane 12: Total protein of bacteria after induction (colony 2);
  • Figure 6 Expression detection of recombinant protein after induction, lane M: protein molecular weight scale (15-120kDa); lane 1: centrifugal supernatant; lane 2: whole bacterial lysate; lane 3: bacterial lysate supernatant;
  • Figure 7 Affinity chromatography purified protein collection, lane M: protein molecular weight scale (15-120kDa); lanes 1-10 are the protein collection liquid;
  • FIG. 8 Pictures of the animal model of mouse subcutaneous and skin soft tissue lesions, A. Taking pictures of the lesion; B. The white circle is out of the edge of the lesion. Use ImageJ to calculate the area of the lesion. See Example 4 for details;
  • FIG. 9 The relationship between the dose of bacterial challenge and local soft tissue lesions. For details, see Example 5. Damage records 15 days after bacterial challenge.
  • A Investigation of the virulence of different subtypes of Porphyromonas gingivalis.
  • the abscissa is Porphyrin gingivalis Different concentrations of bacteria attack, the different subtypes of attacking bacteria are arranged in order, the ordinate is the area of skin and soft tissue lesions square mm 2 ;
  • the dose is positively correlated with the lesion, and the abscissa is the different subtypes of Porphyria gingivalis Type, the ordinate is the area of skin and soft tissue lesions mm 2 , the doses are arranged in order, the larger the attack dose, the larger the lesion area;
  • Figure 10 The correlation between animal age and bacterial susceptibility.
  • the abscissa is time, the number of days after challenge, and the ordinate is the area of skin and soft tissue lesions in mm 2.
  • G1 (4 weeks of age), G2 (8 weeks old), G1 (12 weeks old), G4 (16 weeks old) and G5 (20 weeks old) represent animals in different age groups;
  • Figure 11 Damage caused by Porphyromonas gingivalis multiple infections See Example 7 for details.
  • A Local lesions. Comparison of soft tissue damage area between the first infection and multiple infections. The abscissa is the days after attack, and the ordinate is the skin and The area of soft tissue lesions in mm 2 ,
  • B multiple-infected animals (G148 group) recorded screenshots on the 24th day of the recovery period,
  • C first infected animals (G148.1 group) on the 24th day of the recovery period Video recording screenshot photos;
  • Figure 12 The immune protection function of the combined vaccine, see Example 8 for details, the abscissa is the number of days after challenge, and the ordinate is the area of skin and soft tissue lesions in mm 2 ;
  • FIG. 13 Local immune protection effect of using vaccine after infection. See Example 9 for details.
  • the abscissa is the number of days after challenge, and the ordinate is the area of skin and soft tissue lesions in mm 2 ;
  • Figure 14 The systemic immune protection effect of the vaccine after infection. See Example 9 for details. Video screenshots on the 24th and 42th days after bacterial challenge.
  • the G154 group uses the monovalent vaccine W50ragB
  • the G155 group uses the monovalent vaccine Cra4S1
  • G156 group used the combination vaccine W50ragB+Cra4S1, G157 group was the infection control group, no vaccine, G158 group was the first infection control;
  • Figure 16 The local immunoprotective effect of dual target-specific antibodies on animals with first infection and previous infection history.
  • G371, G372, G373 and G374 are not infected with Porphyria gingivalis, and the antibody usage is the same as in Figure 15.
  • the abscissa is the number of days after challenge, and the ordinate is the area of skin and soft tissue lesions (mm 2 );
  • Mice in the G361, G362, G363, G364 and G369 groups have a history of pre-infection, and the antibody usage is the same as Figure 15.
  • the abscissa is the number of days after attack, and the ordinate is the area of skin and soft tissue lesions in mm 2 .
  • Nanjing GenScript (hereinafter referred to as GenScript) provides nucleotide synthesis services.
  • ragA encode outer membrane protein, molecular weight about 115kDa
  • ragB encoding outer membrane protein immunogenic region, molecular weight about 55kDa protein
  • Figure 1 the applicant is in Previous invention patents and published literature have pointed out that both ragA and ragB genes have genetic polymorphism characteristics. So far, there are no published literature reports about the ragA gene and the protein it encodes.
  • the present invention uses the host's own codon preference to modify the nucleotide sequence. After the nucleotide sequence is modified, the encoded protein sequence must be consistent with the target gene to achieve the purpose of accurate expression of the target protein.
  • the synthetic gene sequence was named cra4S1, which means that there is a stable and conserved amino acid sequence in the ragA gene of Porphyromonas gingivalis, pointing out the fact that the four main bacterial subtypes share a common protein sequence.
  • all intellectual property rights of artificially designed nucleotide sequences obtained through gene synthesis methods belong to the patent applicant, and GenScript only provides nucleotide synthesis services.
  • the nucleotide sequence of the synthetic cra4S1 gene is as follows:
  • the cra4S1 gene fragment is 408bp in length and contains 393bp of target gene.
  • the 5'end restriction endonuclease is NdeI (CATATG, underlined)
  • the starting code is ATG in the restriction endonuclease
  • the 3'end restriction endonuclease Use XhoI CCGAG underlined
  • TGA in bold
  • stop codon is specially set to terminate the translation immediately to ensure the correct expression of the target gene.
  • the plasmid DNA is transformed into the host cell, the accuracy of the synthetic target gene needs to be tested.
  • Plasmid and target gene are both digested with restriction enzymes NdeI and XhoI.
  • the digestion system for the two restriction enzymes is 2 ⁇ l each, 10 ⁇ Buffer 4 ⁇ l, plasmid pET-30a(+)1 ⁇ g Or 1 ⁇ g of cra4S1 gene fragment, make up to 40 ⁇ l with ddH 2 O, and water bath at 37°C for 2h.
  • the DNA fragment was recovered and purified by agarose gel electrophoresis, and the vector and the target gene fragment were connected.
  • the connection system was: 200 ⁇ g of the vector after digestion, 280 ⁇ g of cra4S1 gene fragment, 1.0 ⁇ l of T4 DNA ligase, and 10 ⁇ Buffer 2.0 ⁇ l, make up to 20 ⁇ l with ddH 2 O, and react at 16°C for 12 to 16 hours.
  • the ligated expression vector cra4S1-pET-30a(+) plasmid was transformed into E. coli.
  • Transformation add 1 ⁇ l (100ng) of cra4S1-pET-30a(+) plasmid to 100 ⁇ l of competent E. coli BL21(DE3)plysS (purchased by KingScript), place in an ice bath for 20 minutes; heat shock at 42°C for 90 seconds, and quickly set Add 600 ⁇ l of LB broth on ice for 5 minutes; shake at 37°C and 220rpm for 1 hour. After centrifugation, the culture is spread on an LB plate containing 50 ⁇ g/ml kanamycin and incubated at 37°C overnight. A single colony was randomly selected from the transformed bacteria plate culture dish, and the plasmid DNA was extracted and purified, and the restriction enzyme digestion experiment confirmed that the plasmid inserted gene fragment (Figure 3) was sent to the plasmid DNA for sequencing.
  • the base 393bp in the bold part above is the cloned cra4S1 gene sequence, underlined is the restriction endonuclease cut point, and the stop codon TGA is at the end; the nucleotide sequence 352-744 in the recombinant gene sequencing result is the same as the designed nuclear The nucleotide sequence comparison is 100% completely consistent;
  • the DNA sequence of the target gene in the recombinant plasmid was 100% consistent with the optimally designed cra4S1 gene pairing, and the expected encoded protein sequence was 100% consistent with the target theoretical amino acid sequence.
  • IPTG induction Pick two single colonies on the transformation plate of Example 2 and inoculate them into a test tube containing 50 ⁇ g/ml kanamycin and 3ml LB broth, shake at 37°C and 200rpm overnight, and press 1:100 the next day Inoculate in 100ml LB culture medium containing 50 ⁇ g/ml kanamycin, shake at 37°C at 220rpm until the OD600 of the bacteria reaches 0.6-0.8 (about 4h), take out 1ml culture, centrifuge at 10000g at room temperature for 5min, discard the supernatant, and use 100 ⁇ l Resuspend the bacterial pellet in PBS for later use.
  • Protein expression detection Take the bacterial suspension before and after induction in the previous step, add an equal volume of 2 ⁇ SDS loading buffer after shaking, heat at 100°C for 5 min, SDS-PAGE electrophoresis, Coomassie brilliant blue staining to detect recombinant protein (Figure 5). The results showed that the transformed bacteria produced the target protein under the induction of IPTG, and showed a strong protein band at the molecular weight of 15kDa, which was in line with the theoretical value of the target protein. The uninduced bacteria did not have this protein.
  • Recombinant protein affinity chromatography purification Firstly, the bacterial cells induced by IPTG are sonicated, and 30ml Tris-HCL (PH8.0) is added per gram of wet bacterial weight, suspended evenly, and centrifuged at 4°C, 10000rpm for 30 minutes to wash the bacterial pellet After three times, the bacteria were suspended, and the bacteria were broken by ultrasonic (Ningbo Xinzhi Ultrasonic Disintegrator Y-92III) lysis method. The beaker containing the bacterial liquid was placed in an ice bath, the ultrasonic time was 3 seconds, the interval time was 5 seconds, and the ultrasonic work The total time is 8 minutes.
  • Tris-HCL PH8.0
  • the sonicated bacterial solution was centrifuged at 10,000 rpm at 4°C for 15 minutes, and the supernatant and precipitate were collected and detected by SDS-PAGE ( Figure 6).
  • the results showed that after the bacteria were broken, the recombinant protein mainly appeared in the supernatant after centrifugation, indicating that the recombinant protein was synthesized in the cell in a soluble form, and the subsequent refolding process of the inclusion bodies was avoided.
  • AKTA purifier GE company
  • affinity chromatography to purify the recombinant protein
  • the chromatography column selects DEAE column (huiyan)
  • the bacterial lysate supernatant solution containing the recombinant protein is added to the purification column
  • the sample volume can be 40ml at a time -100ml (1-5mg/ml)
  • the sample flow rate is 1.5ml/min
  • the mobile phase A solution is 50mM Tris-HCl, PH8, until the OD280 value of the effluent reaches the baseline
  • switch to the mobile phase B solution to be 50mM Tris- HCl, 1M NaCl, pH 8.0, eluted at a flow rate of 4ml/min, eluted the target protein with different gradient B solutions, 10% B, 18% B, 100% B, collected and labeled the effluent, and used SDS-PAGE electrophoresis
  • the purified protein was detected.
  • Figure 7 shows the
  • Plasmid-transformed bacteria can produce recombinant protein in the cell after induction by IPTG. After bacterial lysis, the recombinant protein is found mainly in the supernatant of the lysate, indicating that Cra4S1 exists as a soluble protein.
  • the purified Cra4S1 recombinant protein was obtained by affinity chromatography. The molecular weight of the recombinant protein was about 15kDa, which was in line with the expected theoretical value.
  • Porphyria gingivalis includes various subtypes of bacteria (Porphyromonas gingivalis W50 subtype, Thai subtype, QML subtype and 381 subtype) all grow on 5% defibrinated horse blood
  • the Fastidious Anaerobic Agar (FAA) plate is placed in an anaerobic incubator at 37°C, containing 80% nitrogen, 10% hydrogen, and 10% carbon dioxide.
  • BHI culture medium (brain heart infusion culture medium) containing 5 ⁇ g/ml hemin, grow for 18-24 hours, when the OD600nm reaches 1-1.2, the culture is centrifuged and harvested and washed Two times, use BHI culture medium to make a certain concentration of bacterial suspension (2-8x10 10 CFU/ml) for inoculation of experimental mice (Balb/c, purchased by the Animal Center of Yangzhou University). Mice are grouped according to different experimental plans, generally 6-8 weeks old, weighing 18-22 grams, each group is 8-12 mice, each mouse is inoculated with 200 ⁇ l bacterial suspension, and subcutaneously placed on both sides of the back of the mouse. injection. After the bacterial attack, the weight changes, behaviors and movements of the mice were recorded, and the skin and subcutaneous soft tissue lesions were recorded in detail.
  • Figure 8 shows that the skin and subcutaneous soft tissues of the mice were affected by bacteria, and the white circle appeared in the lesion. Calculate the area of the lesion, initially manually measure the longest longitudinal diameter and the corresponding transverse diameter of the lesion, multiply the longitudinal diameter and the transverse diameter to calculate the lesion area, and record and archive all handwritten data. The identification and area calculation methods of lesions were improved in April 2019. The method of taking pictures was used to fix the distance between the camera and the taken lesions, and then ImageJ (computer software name) was used to calculate the area of the lesions within the circle.
  • ImageJ computer software name
  • the mouse body surface soft tissue injury model used in this example dynamically observes the local lesions, including the pathological characteristics of the wound, the progress of the disease, the healing of the wound and the prognosis of the lesion, combined with the weight change, mental state and behavior of the mouse Characteristics and other indicators, comprehensively judge the harm caused by Porphyria gingivalis infection.
  • the animal experimental model used in this embodiment is in accordance with international general methods and standards have been recognized in the industry.
  • Example 5 The relationship between the dose of Porphyria gingivalis infection and soft tissue lesions in mice
  • the purpose of this example is to investigate the subtypes of Porphyromonas gingivalis corresponding to the four main outer membrane proteins, their differences in virulence, and the correlation between the dose of bacterial infection and the pathology.
  • Porphyromonas gingivalis includes W50 subtype, Thai subtype, QML subtype and 381 subtype.
  • three bacterial suspensions of different concentrations were prepared, respectively: 5x10 9 CFU/ml, 2x10 10 CFU/ml and 8x10 10 CFU/ml, experimental animals Balb/c mice, 6-8 weeks old, weighing 18-24 grams, 8-12 mice per group, each mouse inoculated 200 ⁇ l of bacterial suspension was injected subcutaneously on both sides of the vertebrae on the back of the mouse. After the bacterial challenge, the weight, behavior and local soft tissue lesions of the mice were recorded.
  • Figure 9A and Figure 9B show that the virulence of the four Porphyromonas gingivalis subtype strains tested has a certain difference.
  • PgA, PgB, PgC and PgD in Figure A represent the P. gingivalis W50 subtype and Thai subtype, respectively.
  • Type, QML subtype and 381 subtype; the three different concentrations of bacterial suspension 1, 2, and 3 in Figure B represent 5x10 9 CFU/ml, 2x10 10 CFU/ml and 8x10 10 CFU/ml, respectively.
  • Example 6 The relationship between Porphyria gingivalis infection and the age of experimental mice
  • the purpose of this example is to investigate age and porphyria gingivalis The relationship between bacteria infection.
  • Porphyromonas gingivalis W50 subtype 2*10 10 CFU/m bacterial suspension was prepared according to the method described in Example 4. 0.2ml was injected into the subcutaneous sides of the back paravertebral sides of the experimental mice of each age group, each of different ages There are 8-10 animals in the group. After bacterial attack, observe the development of the lesion area until the end of the experiment on the 35th day.
  • mice of different age groups The same dose and the same volume of bacterial suspension were used to attack experimental mice of different age groups. The results showed that animals of different age groups caused different damages to Porphyria gingivalis W50 subtype challenge. In this experiment, animals of all age groups did not die after being attacked by bacteria.
  • the animals in the G1 group (weight 15-18 grams) and G2 group (weight 22-26 grams) were 4 weeks old and 8 weeks old in their juvenile period, with rapid onset and severe disease, but the juvenile animals recovered on their own Fast and short healing period.
  • Example 6 The experimental results of Example 6 show that the animals are self-healing when they are infected with Porphyromonas gingivalis for the first time, and the younger the age, the faster the recovery. In clinical practice, periodontal disease often presents intermittent progressive development.
  • the purpose of this example is to investigate the local and systemic harm caused by multiple chronic infections to experimental animals by comparing the experimental animal models of single and multiple infections.
  • Figure 11B is the video screenshot of the systemic condition of the animals in the G148 group on the 24th day after being infected by the QML subtype of Porphyria gingivalis.
  • Figure 11C is the animal in the G148.1 group being infected by gingival porphyrin Screenshot of the video of the recovery of the QML subtype on the 24th day after the initial infection.
  • the goal of the invention is to adopt immunological methods to solve the problem of chronic infection of pathogenic bacteria and prevent pathogenic bacteria from prolonging for a long time and endangering the health of the whole body.
  • This example is the first attempt to discover the immune protection function of the combined vaccine.
  • the purpose of the initial experiment was to investigate whether there is an immune cross-protection effect between the outer membrane protein RagB of different subtypes of Porphyromonas gingivalis. Therefore, in the design of the experiment, the RagB bivalent vaccine including W50ragB protein was used first (see PCT/GB2005 for protein sequence content) /001976, protein purification and other production methods are similar to this example 3) and QMLragB protein (for protein sequence content see PCT/GB2005/001976, protein purification and other production methods are similar to this example 3) according to the mass ratio of 1:1.
  • Cra4S1 protein (the Cra4S1 protein prepared in Example 3) was added, and each protein was given three immunization opportunities.
  • the addition of the common antigen Cra4S1 shared by each subtype is to investigate whether the new combination vaccine can affect the immune protection effect of the RagB vaccine.
  • the Cra4S1 protein purified in Example 3 and the RagB protein of different subtypes were respectively immunized to animals (Table 3-1, Table 3-2).
  • the formula of the final combined vaccine in this example is Cra4S1 protein: W50ragB protein: QMLragB protein, and the mass ratio is 1:1:1.
  • each mouse was intraperitoneally injected with 100 ⁇ l of recombinant protein solution, and the first group (G11, G14 and G17) used 100 ⁇ l solution containing 5-50 ⁇ g W50ragB protein, 5-50 ⁇ g QMLragB protein and 250 ⁇ g aluminum adjuvant, according to the table 3-1 Implementation of the immunization plan; the second group (G12, G15 and G18) used 100 ⁇ l solution containing 5 ⁇ g Cra4Sl protein, 5-50 ⁇ g W50ragB protein, 5-50 ⁇ g QMLragB protein and 250 ⁇ g aluminum adjuvant, according to Table 3-2 Plan; The third largest group (G13, G16, and G19) is the control group without vaccine.
  • the immunization interval is 3-4 weeks, and the serum titer is measured before immunization, and the development of antibodies in the serum is recorded. After three immunizations, the ELISA method was used to detect the titer of the specific antibody. When the antibody titer was higher than 1:100,000, the bacterial attack experiment was performed according to the method described in Example 4.
  • This example is the initial attempt of a combination vaccine.
  • the reason for the reduced vaccine dosage is that the vaccine dose-dependent experiment results performed at the same time as this example show that, in fact, subsequent studies have found that the four RagB proteins are immunized High-titer antibodies can be produced when the dosage is 0.5-5 ⁇ g.
  • the experimental design needs to be further improved, the preliminary test results lay the foundation for subsequent research.
  • the results of this example show that the combined vaccine not only enhances the immune protection effect but also improves the stability of the product.
  • This phenomenon suggests that the combined application of two different target proteins can improve the efficacy of the product.
  • the principle of enhanced immune effect and stability of the combination vaccine may be related to the two subunits of RagA and RagB being controlled by the same transcription factor.
  • the target of two interrelated subunit molecules on the surface of the bacteria is occupied by antibodies In the future, a more stable molecular structure will be formed.
  • This combination of antigen and antibody limits the function of the outer membrane protein of the bacteria.
  • the stable combination of dual target antigen and antibody may more easily trigger the body to exert better immune coordination/ Coordinating action to eliminate and inhibit pathogens.
  • the RagB vaccine only protects the corresponding Porphyromonas gingivalis subtype
  • the combination of the Cra4S1 protein and the RagB protein can only protect the corresponding Porphyromonas gingivalis subtype.
  • the combined vaccine of Cra4S1 protein and QMLragB protein protects the body against the attack of Porphyromonas gingivalis QML subtype, but has no protective effect against the attack of Porphyromonas gingivalis Thai subtype.
  • the combination vaccine described in this example needs to contain the Porphyromonas gingivalis RagB protein (different subtypes) and Cra4S1.
  • the combination of the two molecules can enhance the immune protection function and stability.
  • the combination vaccine is a brand-new concept.
  • the following examples further Validation and application of combination vaccines.
  • mice 3-4 weeks old mice, weighing 12-16 grams, were divided into 5 groups (Table 4), 5-6 mice in each group, and 4 groups of mice were treated with low-dose Porphyromonas gingivalis W50 subtype bacterial suspension (5*10 8 CFU/ml) 200 ⁇ l was injected subcutaneously into the back, causing a slight infection.
  • the infected mice had normal diet and activities.
  • On the second day after the bacterial attack some animals showed a slight weight loss, followed by a continuous increase in weight. After infection, mice may have slight local microscopic skin lesions.
  • Group 5 mice were not infected and served as controls. After the mice continued to raise for 4 weeks, the vaccination program was started.
  • Infected animals were divided into four groups according to the plan listed in Table 4, including dose and time to immunize the animals.
  • the mice in the G154 group were immunized with W50ragB protein
  • the mice in the G155 group were immunized with the Cra4S1 protein
  • the mice in the G156 group were immunized with the combination
  • the vaccine Cra4S1 protein and W50ragB protein mixture were immunized.
  • the G157 group was a control with mild infection but no vaccine
  • the mouse in the G158 group was an uninfected control of the same age.
  • results The mildly infected experimental animal group using monovalent or combined vaccines showed that the local soft tissue damage was lower than that of the same infected control group, and the lesion damage was also lower than that of the uninfected control group (Table 5 and Figure 13).
  • the animal group has recovered from the record on the 24th day after infection.
  • the beneficial indicators of the combined vaccine group are better than those of the monovalent vaccine, while the control group has a prolonged condition.
  • Example 7 the phenomenon described in Example 7 was also observed, that is, the local damage of animals infected with Porphyromonas gingivalis multiple times was more serious (Table 5 and Figure 13, Figure 14).
  • the video data in Figure 14 observed the animal’s posture.
  • the mice using the vaccine were in good general condition, with shiny hair, well-proportioned body, and free movement, while the control group’s body surface ulcers were difficult to heal. The hair is dull and sluggish.
  • the weight of the experimental group and the control group did not change significantly.
  • Table 5 Record of average area of lesions in immune protection experiment after infection (unit: mm 2 ) and standard deviation of data
  • Example 7 The results of animal experiments in Example 7 suggest that repeated Porphyria gingivalis infections not only cause more and more severe local damage, but also affect general health.
  • Table 5 records the immunity of the animal after the combination vaccine is used for infection. When the animal is re-attacked by bacteria, the local lesions are reduced, the average lesion area (AVG) decreases, and the standard deviation (STDEV) of the experimental data is maintained at a low level. , And the animal’s overall condition is performing well.
  • the immunization protocol of the mouse specific antiserum is listed in Table 6-1 (see PCT/GB2005/001976 for the ThairagB protein sequence, and the protein purification and other preparation methods are similar to this Example 3, and the Cra4S1 protein is prepared in Example 3), After three immunizations, the titer of the specific antibody in the serum was determined by indirect ELISA, and the blood was finally collected. After the serum was separated, the polyclonal antibody was obtained and stored in the refrigerator at -20°C.
  • mice in groups G361, G362, G363, G364 and G369 have a history of pre-infection.
  • 200 ⁇ l of Porphyromonas gingivalis Thai subtype bacterial suspension (5X10 8 CFU/ml) was injected subcutaneously on the back of mice, G370, G371, G372
  • the specific process is as described in Example 4. Observe and record the weight and behavior of the mice before and after the attack, record the development of subcutaneous soft tissue lesions of the attacked animals, and measure the skin lesion area (Table 7).
  • Figure 15B in this example shows that the pre-infected control group animals had higher mortality rates in the intraperitoneal injection of normal serum group G364 and intraperitoneal injection of PBS control group G369, which were 37.5% (3/8) and 62.5% (5/ 8).
  • Figure 16A and Figure 16B show that the specific combination antibody showed a lower local lesion range in the pre-infected group and non-infected group animal experiments than other experimental group surviving animals.
  • the combined antibody was in the pre-infected group animal One death occurred.
  • the severity of local lesions in the pre-infected animals was lower than that in the control group, but in the experiment of the healthy group, only one animal survived, which affected the judgment of the entire group.
  • the anti-Cra4S1 experimental group showed a peculiar phenomenon.
  • the antibody was injected into the infected animals. The animals survived the bacterial attack with the highest survival rate without death. However, the local lesions were better than the anti-ThaiRagB experimental group and the combination antibody (anti-ThaiRagB and The anti-Cra4S1) experimental group was severe (Figure 16A and Figure 16B).
  • Example 10 The experimental results of Example 10 show that the combined antibody (anti-ThaiRagB and anti-Cra4S1) has a stable, safe and efficient function of resisting Porphyria gingivalis infection, and has an immune protection effect on both uninfected and infected experimental animals ( Figure 16A and Figure 16B). Both anti-ThaiRagB antibody and anti-Cra4S1 antibody alone have uncertain efficacy.
  • the experimental results of this example suggest that when a single antibody binds to the antigen molecule on the surface of the bacteria, there may be structural instability factors, thus causing defects in the efficacy of the antibody.
  • the combined antibody specifically occupies the dual antigen targets of the main outer membrane protein and prevents the function of the outer membrane protein, thereby achieving the effect of inhibiting and eliminating bacteria and reducing the virulence of bacteria.
  • the present invention has discovered and confirmed Cra4S1, an important outer membrane protein component of Porphyromonas gingivalis, created the expression gene of Cra4S1 protein, and used a new target Cra4S1 to bind to the specific/characteristic outer membrane protein RagB of bacteria, and proposed Combining the theory of vaccines and dual-target specific antibodies, and using experimental data to prove that innovative products have an effective and stable function of inhibiting bacteria, the new technology heralds a wide range of industrial applications.

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Abstract

提供了一种cra4S1基因、其所编码的Cra4S1蛋白及含有Cra4S1蛋白或其片段的疫苗或药物。cra4S1基因的核苷酸序列如SEQ ID NO：1所示。疫苗组合了牙龈卟啉菌外膜蛋白的特异性靶点和细菌保守区抗原成分，对机体起免疫预防保护作用。

Description

Cra4S1基因及其编码的蛋白和应用 技术领域
本发明涉及生物制药领域，具体涉及cra4S1基因及其编码的蛋白和应用。
背景技术
牙周病是全球范围内口腔最常见的疾病之一，中国有80-97％的成年人存在不同程度的牙周问题。牙周病是冠心病及其急性发作的独立危险因子，是脑卒中的独立危险因子，已发现牙周病致病菌牙龈卟啉菌(Porphyromonas gingivalis)与阿尔茨海默病(Alzheimer’s disease，俗称老年痴呆)的直接联系，牙周病是糖尿病的并发症，与消化道和呼吸道疾病以及孕妇生产早产儿和低体重儿等临床疾病有着密切关联，目前已发现是许多死亡率极高的疾病的重要诱因，被世界卫生组织列为继心血管疾病和癌症之后的第三大非传染性疾病。
牙周病发展分不同阶段，其显著特征是容易复发，局部病变情况呈进行性发展状态。目前牙周病治疗依赖局部清除病灶、抗生素和外科手术，但是这些措施治标不治本，没有去除病原菌感染的根本问题。
牙周病在人群中的广泛分布以及与全身系统性疾病的关联，引发了科学家们对疫苗研究的探索，运用免疫学方法调动机体的免疫功能，抑制和消灭病原菌，预防和治疗牙周病/种植体周围炎、阻止复发。
牙龈卟啉菌是研究最为广泛、证据最充分的成人牙周病的必要致病菌。牙龈卟啉菌的候选疫苗，包括荚膜多糖、菌毛、类脂质、外膜蛋白、热休克蛋白、蛋白酶、灭活全菌等都在目前研发的范畴中，但致病菌不同亚型其分子结构、毒力因子、DNA序列和毒性各异，尚未发现牙龈卟啉菌不同亚型在人群中的分布规律。有效疫苗必须能诱导机体对某种微生物抗原产生长期的免疫性，预防病原菌的再次入侵，降低其对人体造成的危害。制作针对致病菌不同亚型细菌感染的疫苗/抗体产品，能产生稳定的预防和治疗效果，是商业开发的难点，到目前为止，还没有相关产品上市。
发明内容
发明目的：本发明目的是开发安全、稳定、有效的生物制品，预防和治疗由牙龈卟啉菌慢性感染造成的疾病，包括但不限于牙周病/种植体周围炎。
本发明解决的关键技术是提供了cra4S1基因及其编码的Cra4S1蛋白。
本发明还要解决的技术问题是提供了表达盒、重组载体或细胞，其含有所述的cra4S1基因或其基因片段。
本发明还要解决的技术问题是提供了cra4S1基因、所述的Cra4S1蛋白、所述的表达盒、重组载体或细胞在制备预防或治疗牙周病或种植体周围炎或与牙龈卟啉菌感染相关疾病的疫苗或药物中的应用。
本发明还要解决的技术问题是提供了一种疫苗或药物，其含有所述的Cra4S1蛋白或其片段。
本发明还要解决的技术问题是提供了特异性抗体，由包含所述Cra4S1蛋白或其片段的制剂引导免疫动物制得。
本发明使用组合疫苗和/或双靶点特异性抗体预防和治疗由牙龈卟啉菌慢性感染引起的牙周病/种植体周围炎和其他相关疾病。
技术方案：为了解决上述技术问题，本发明的技术方案是提供了新颖的cra4S1基因，所述cra4S1基因的核苷酸序列如SEQ ID NO：1所示。
本发明内容还包括所述的cra4S1基因的获得方法。
本发明内容还包括所述的cra4S1基因所编码的Cra4S1蛋白，其氨基酸序列如SEQ ID NO.2所示。
本发明内容还包括表达盒、重组载体或细胞，其含有所述的cra4S1基因。
本发明内容还包括所述的cra4S1基因、人工合成基因的设计、所述的蛋白、所述的表达盒、重组载体或细胞在制备预防或治疗牙周病或种植体周围炎或与牙龈卟啉菌感染相关疾病的疫苗或药物中的应用。
本发明内容还包括一种疫苗或药物，其含有所述的Cra4S1蛋白或其片段。
其中，所述疫苗或药物还包括牙龈卟啉菌的W50ragB蛋白、ThairagB蛋白、QMLragB蛋白和381ragB蛋白中的一种或几种。
其中，本发明的疫苗的配方如下：所述疫苗中的Cra4S1蛋白与W50ragB蛋白的质量比为1∶2-8；或所述Cra4S1蛋白与ThairagB蛋白的质量比为1∶1-6；或所述Cra4S1蛋白与QMLragB蛋白的质量比为1∶1-6；或所述Cra4S1蛋白与381ragB蛋白的质量比为1∶2-9；或所述Cra4S1蛋白与W50ragB蛋白、ThairagB蛋白、QMLragB蛋白、381ragB蛋白的配方按上述质量比优化。
作为优选，所述组合疫苗的配方为Cra4S1蛋白：W50ragB蛋白：QMLragB蛋白，质量比为1∶1∶1。
本发明所述疫苗或药物包括但不仅限于用于预防发生或复发或治疗未感染健康人或已有感染的牙周病或种植体周围炎或慢性感染性牙周病患者，其他与牙龈卟啉菌感染相关疾病均可使用。
进一步的，作为优选的，所述的牙龈卟啉菌包括牙龈卟啉菌W50类亚型、Thai类亚型、QML类亚型和381类亚型。
本发明内容还包括一种特异性抗体，由包含所述的Cra4S1蛋白或其片段的制剂引导免疫动物制得。
其中，所述特异性抗体的组合应用还包括牙龈卟啉菌的W50ragB蛋白、ThairagB蛋白、QMLragB蛋白和381ragB蛋白中的一种或几种的制剂引导免疫动物制得。
其中，所述Cra4S1蛋白、W50ragB蛋白、ThairagB蛋白、QMLragB蛋白和381ragB蛋白免疫接种用量均为0.5～50μg，
作为优选，所述Cra4S1蛋白、W50ragB蛋白、ThairagB蛋白、QMLragB蛋白和381ragB蛋白免疫接种用量均为0.5～5μg。
作为优选，所述Cra4S1蛋白、W50ragB蛋白、ThairagB蛋白、QMLragB蛋白和381ragB蛋白免疫接种用量均为5～50μg。
经过免疫得到的多克隆抗体用量为100～200μg。
本发明所述特异性抗体包括但不仅限于用于预防发生或复发或治疗未感染健康人或已有感染的牙周病或种植体周围炎或慢性感染性牙周病患者，其他与牙龈卟啉菌感染相关疾病均可使用。
本发明的核心内容首先是成功解决了基因表达困难的技术瓶颈，关键是有效地利用共转录基因间的分子协同和互相依赖的特性，成功制造出预防和治疗牙周病/种植体周围炎的疫苗和抗体产品。
本发明的原理和方法：
牙龈卟啉菌细菌基因组DNA中存在rag基因位点(rag locus)，由ragA与ragB基因和两端的不规律插入片段组成(图1)，ragA与ragB基因其开放读框各自独立，实验证明两者具有共转录关系，举例说明，当ragA基因被人工突变后，RagB蛋白不能正 常表达，或者，当下游ragB基因被人工突变后，RagA蛋白不能在细菌中正常表达，(参考文献PCT/GB2005/001976)。
运用本公司已获得授权的专利(PCT/GB2005/001976)技术，按照编码细菌外膜蛋白ragB基因的序列分析，已明确了四个不同亚型致病菌在临床标本检测中占绝对优势，对应于四个不同亚型牙龈卟啉菌的外膜蛋白分别命名为W50ragB蛋白、ThairagB蛋白、QMLragB蛋白和381ragB蛋白，重组亚单位单价和多价疫苗在实验室中试生产规模的工艺流程已完成。
事实上，ragA基因同样存在高度的遗传多态性，但是，与ragA基因DNA多态性相对应的编码蛋白的相关研究未有公开文献发表。申请人尝试用常规方法从基因组DNA中克隆和表达ragA基因未能获得成功。申请人在其PCT/GB2005/001976专利技术的基础上，通过蛋白质序列比对分析发现ragA基因编码的蛋白中有一段氨基酸保守稳定序列，但是使用常规方法克隆与表达也未获得成功。
为获取RagA稳定区蛋白质分子的表达，申请人通过优化核苷酸的方法，设计了一条新的序列，使用人工合成核苷酸的方法制造出一段基因序列，命名为cra4S1(参见SEQ ID NO.1)，意指牙龈卟啉菌ragA基因中存在稳定保守的氨基酸序列，指出四个主要细菌亚型拥有一段共同蛋白质序列的事实。
本发明中cra4S1基因片段被成功地克隆到表达载体，并且在诱导条件下生成了重组蛋白。利用层析法和分子筛原理获得纯化重组蛋白(实施例3，图2、3、5、6和7)，重组Cra4S1蛋白，分子量约为15KDa，重组克隆的基因DNA序列测序结果与设计方案一致，编码的蛋白序列与稳定区理论序列一致(图4)，分子量符合预期理论值。
重组Cra4S1蛋白具有强烈的抗原性，使用微小剂量免疫动物即可产生高效价抗体(实施例8详细描述)，但单独使用Cra4S1蛋白免疫动物，未见免疫保护作用。
体外实验中，观察到Cra4S1蛋白免疫动物产生的抗体并不能与牙龈卟啉菌或/和细菌裂解物起反应，实验中还观察到，RagB抗血清以及全菌抗血清与重组Cra4S1蛋白之间也未观察到明显的免疫学反应，这些实验结果提示，Cra4S1蛋白并不是细菌表面的主要抗原。
有趣的是，如果动物用RagB疫苗免疫后又受到过牙龈卟啉菌的攻击，这时所采集的血清抗体与重组Cra4S1蛋白有不同程度的免疫交叉反应。这个现象提示当RagB抗体 与牙龈卟啉菌外膜蛋白RagB结合以后，改变了Cra4S1蛋白原有的空间结构，使其隐藏的抗原性得以暴露。
具体的实验中，当重组Cra4S1蛋白与RagB蛋白形成组合疫苗时，产生了意想不到的结果，新配方组合疫苗增强了机体抵御牙龈卟啉菌感染的免疫保护效果(实施例8，图12)，该现象提示当RagB抗体结合到细菌表面的RagB抗原时改变了rag基因位点蛋白的空间结构，让Cra4S1蛋白有暴露和被抗体识别的机会，细菌表面的双靶点同时被特异性抗体捕获，限制了主要外膜蛋白的功能，从而达到抑制和消灭细菌的效果。
组合疫苗在实验中还体现了产品的稳定性能提高，表现在数据标准差保持在低水平，标准差是一组数据平均值分散程度的一种度量，一个较小的标准差，代表这些数值更接近平均值，表示出现意外离散的危险性缩小。这个现象进一步证明RagA和RagB双靶点抗原抗体结合免疫效果更稳定。
本发明的创新点是利用细菌表面抗原多靶点特征，制作稳定、高效的生物产品。
组合疫苗不仅用于健康小鼠，还被应用于已感染牙龈卟啉菌的动物，实验结果显示组合疫苗对已感染机体有显著的免疫保护作用(实施例9)，这是本发明的另一个创新点。牙龈卟啉菌感染引起的牙周病/种植体周围炎是一个慢性和间歇性过程，病程呈进行性发展。动物实验中观察到，动物经多次感染，不仅局部病变每况愈下，动物的全身情况也表现出衰弱现象(实施例7)。临床上，绝大部分人群中都有不同程度的牙周病，因此，本发明控制慢性感染，能够阻止慢性感染对全身健康的危害，产品将具有广阔的市场前景。
继组合疫苗的免疫功效和稳定性提高试验，被动免疫实验同时也验证了组合抗体含Cra4S1蛋白抗体和外膜蛋白RagB抗体，其免疫保护作用强于分别使用单抗体的实验组(实施例10)。
组合疫苗和组合抗体对预感染动物的主动免疫和被动免疫保护实验，方法和结果也包含在本说明书中。
有益效果：本发明相对于现有技术，具有以下优点：
1、本发明首次公布了与牙龈卟啉菌相关的cra4S1基因，并首次将cra4S1基因克隆并成功在大肠杆菌中表达获得了Cra4S1蛋白，该蛋白与牙龈卟啉菌RagB疫苗等产品联合使用时，不仅增强了机体抵御牙龈卟啉菌感染的免疫保护效果，而且增强了疫苗产品功能的稳定性，同时，组合疫苗和组合抗体都在预感染的机体中表现出显著的免疫保护 作用，提示本发明有广泛的应用前景，产品可用于健康人群也可以用于慢性感染性患者。
2、本发明的疫苗含牙龈卟啉菌特异性外膜蛋白的靶点和新发现的保守区抗原成分，改善了单独使用外膜蛋白或单独使用新发现抗原制作的疫苗的功效。牙龈卟啉菌保守区抗原与外膜蛋白引导产生的抗体联合运用时，显著提高了抗体的免疫保护功效。此外，研究结果显示使用本发明产品包括疫苗和双特异性抗体的实验动物组，与各对照组相比，其实验数据的标准差均维持在低水平，提示本发明产品在体内实验中可以提供更好的稳定性。
3、本发明内容指出，抗感染免疫中，特异性抗体与细菌表面具有相互关联的靶点结合，可以利用免疫分子与保守区抗原和识别细菌亚型特异性抗原结合的特点，稳固而有效地充分发挥抗体的功效，抑制和消灭病原菌，达到预防和治疗疾病的目的。
下面将描述本发明的进一步例证，这些包括前景预测，但它们并不局限于本发明。
附图说明
图1牙龈卟啉菌W50主要外膜蛋白受体抗原基因rag(rag， receptor  antigen  gene)位点；
图2重组质粒载体构建图，注明：C5361BH150-1为目的基因cra4S1；
图3重组质粒酶切鉴定；图3A：泳道1为重组cra4S1-pET-30a(+)质粒，泳道2为酶切反应，存在质粒DNA和cra4S1基因条带，M为1kb DNA梯度标尺(金斯瑞)，图3B：1kb DNA梯度标尺与DL3000(金斯瑞)分子量标尺的对比图；
图4克隆得到的目的基因所对应的氨基酸序列与细菌理论氨基酸序列一致；
图5重组蛋白诱导后表达检测，泳道M：蛋白分子量标尺(15-120kDa)；泳道9：诱导前细菌的总蛋白(菌落1)；泳道10：诱导后细菌的总蛋白(菌落1)；泳道11：诱导前细菌的总蛋白(菌落2)；泳道12：诱导后细菌的总蛋白(菌落2)；
图6重组蛋白诱导后表达检测，泳道M：蛋白分子量标尺(15-120kDa)；泳道1：离心上清液；泳道2：全菌裂解液；泳道3：细菌裂解液上清；
图7亲和层析纯化蛋白收集，泳道M：蛋白分子量标尺(15-120kDa)；泳道柱前为过柱穿流液；泳道1-10为蛋白收集液；
图8小鼠皮下和皮肤软组织病损动物模型图片，A、病变拍照；B、白线圈出病变边缘，使用ImageJ计算病变圈内面积，详细内容见实施例4；
图9细菌攻击剂量与造成局部软组织病变的关系，详细内容见实施例5，细菌攻击 后15天病损记录，(A)牙龈卟啉菌不同亚型的毒力调查，横坐标是牙龈卟啉菌攻击时的不同浓度，不同亚型攻击菌按顺序排列，纵坐标是皮肤和软组织病损的面积平方毫米mm 2；(B)剂量与病变的正相关，横坐标是牙龈卟啉菌不同亚型，纵坐标是皮肤和软组织病损的面积mm 2，剂量按顺序排列，攻击剂量越大病变面积越大；
图10动物年龄与细菌易感性的关联，横坐标是时间，攻击后的天数，纵坐标是皮肤和软组织病损的面积mm 2，详细内容见实施例6，图中G1(4周龄)、G2(8周龄)、G1(12周龄)、G4(16周龄)和G5(20周龄)分别代表不同年龄组的动物；
图11牙龈卟啉菌多次感染造成的损害，详细内容见实施例7，(A)局部病变，初次感染与多次感染的软组织损伤面积比较，横坐标是攻击后天数，纵坐标是皮肤和软组织病损的面积mm 2，(B)多次感染的动物(G148组)恢复期第24天的录像记录截屏照片，(C)初次感染的动物(G148.1组)恢复期第24天的录像记录截屏照片；
图12组合疫苗的免疫保护功能，详细内容见实施例8，横坐标是攻击后天数，纵坐标是皮肤和软组织病损的面积mm 2；
图13感染后使用疫苗的局部免疫保护作用，详细内容见实施例9，横坐标是攻击后天数，纵坐标是皮肤和软组织病损的面积mm 2；
图14感染后使用疫苗的全身免疫保护作用，详细内容见实施例9，细菌攻击后第24天和42天的视频截图照片，G154组使用的是单价疫苗W50ragB，G155组使用的是单价疫苗Cra4S1，G156组使用的是组合疫苗W50ragB+Cra4S1，G157组是感染对照组，无疫苗，G158组是初次感染对照；
图15双靶点特异性抗体被动免疫功能调查，详细内容见实施例10，(A)G370、G371、G372、G373和G374未预感染牙龈卟啉菌，细菌攻击后动物死亡率调查。抗体使用的情况分别为：G370使用抗ThairagB血清，G371使用组合抗体抗ThairagB和抗Cra4S1血清，G372使用抗Cra4S1血清，G373使用正常小鼠血清，G374使用PBS对照；(B)G361、G362、G363、G364和G369组的小鼠有预感染史，细菌攻击后动物死亡率调查，各组使用抗体的情况分别为：G361使用抗ThairagB血清，G362使用组合抗体抗ThairagB和抗Cra4S1血清，G363使用抗Cra4S1血清，G364使用正常小鼠血清，G369使用PBS对照；
图16双靶点特异性抗体对初次感染和既往有感染史的动物产生的局部免疫保护作用，(A)G371、G372、G373和G374均未感染牙龈卟啉菌，抗体使用情况同图15， 横坐标是攻击后天数，纵坐标是皮肤和软组织病损的面积(mm 2)；(B)G361、G362、G363、G364和G369组的小鼠有预感染史，抗体使用情况同图15，横坐标是攻击后天数，纵坐标是皮肤和软组织病损的面积mm 2。
具体实施方式
下面结合附图对本发明作更进一步的说明。
实施例1：cra4S1基因核苷酸序列设计
申请人提供DNA序列和设计要求，南京金斯瑞生物科技公司(以下简称金斯瑞)提供核苷酸合成服务。
人工创建cra4S1基因，思路源于牙龈卟啉菌ragA基因。ragA(编码外膜蛋白，分子量约115kDa)与ragB(编码外膜蛋白免疫源区域，分子量约55kDa蛋白)组成rag基因位点(rag locus)，在同转录因子中(图1)，申请人在之前的发明专利和发表的文献中已指出ragA与ragB基因都存在遗传多态性特征。迄今为止，有关ragA基因和其编码的蛋白未有公开文献报导。
申请人的研究成果发现ragA基因编码的蛋白中有一段相对保守的稳定区序列，下图RagA序列中，下划线部分是四个主要牙龈卟啉菌亚型拥有的共同氨基酸序列；
>W50ragA2334-5387-
Figure PCTCN2019124433-appb-000001
不同亚型细菌共有相同蛋白质序列给研究广谱疫苗指出了前景，但是尝试使用常规方法从细菌基因组DNA中提取目的基因片段未能成功获得克隆。
本发明运用宿主本身的密码子偏好对核苷酸序列进行了修饰，核苷酸序列改良后，其编码的蛋白序列必须与目的基因保持一致，能达到目标蛋白准确表达的目的。人工合成的基因序列被命名为cra4S1，意指牙龈卟啉菌ragA基因中存在稳定保守的氨基酸序列，指出四个主要细菌亚型拥有一段共同蛋白质序列的事实。本发明中，人工设计的通过基因合成方法获得的核苷酸序列，所有知识产权归专利申请人所有，金斯瑞仅提供核苷酸合成服务。
人工合成的cra4S1基因的核苷酸序列如下：
Figure PCTCN2019124433-appb-000002
该cra4S1基因片段长度为408bp，含393bp目的基因，5’端限制性内切酶采用NdeI(CATATG，下划线部分)，起始密码为限制性内切酶中ATG，3’端限制性内切酶采用XhoI(CTCGAG下划线部分)。在目的基因翻译完成后专门设置插入TGA(黑体字)终止密码子立即终止翻译，确保目的基因正确表达。质粒DNA转化到宿主细胞后需要检测人工合成的目标基因的准确度。
实施例2：cra4S1基因表达载体的构建和鉴定
含有Cra4S1蛋白开放读框载体的原核表达载体构建方法：将实施例1人工合成的cra4S1基因片段连接到载体质粒pET-30a(+)(从南京金斯瑞购买)中，重组质粒构建如图2所示。质粒和目标基因均使用限制性内切酶NdeI和XhoI双酶切，两者酶切体系为：2种限制性内切酶各2μl，10×Buffer缓冲液4μl，质粒pET-30a(+)1μg或cra4S1基因片段1μg，用ddH 2O补足至40μl，37℃水浴2h。DNA片段经琼脂糖凝胶电泳，回收纯化，连接载体和目的基因片段，连接体系为：分别酶切回收后的载体200μg，cra4S1基因片段280μg，T4 DNA连接酶1.0μl，10×Buffer缓冲液2.0μl，用ddH 2O补足至20μl，16℃反应12～16h。连接成功后的表达载体cra4S1-pET-30a(+)质粒转化到大肠杆菌。
转化：将cra4S1-pET-30a(+)质粒1μl(100ng)加入100μl感受态大肠杆菌BL21(DE3)plysS(金斯瑞购买)中，置冰浴20分钟；42℃热激90秒，迅速置冰中5分钟，加入600μl LB培养液；37℃，220rpm振摇1小时，离心后培养物涂布于含50μg/ml卡 那霉素的LB平板上，37℃培养过夜。在转化菌平板培养皿中随机挑选单个菌落，质粒DNA提取纯化后酶切实验确认质粒插入基因片段(图3)送质粒DNA测序。
测序结果如下：
Figure PCTCN2019124433-appb-000003
以上黑体部分的碱基393bp为克隆cra4S1基因序列，划线为限制性内切酶切点，终止密码子TGA在结尾处；重组基因测序结果中第352-744位核苷酸序列与设计的核苷酸序列对比100％完全吻合；
理论氨基酸序列如下：
rag A保守氨基酸序列(切除前导肽20AA)
Figure PCTCN2019124433-appb-000004
将上述的测序结果翻译的蛋白质对应的氨基酸序列与理论氨基酸序列比对，比对结果如图4。
结果：重组质粒中目标基因DNA序列与优化设计的cra4S1基因配对100％相符合，预期编码的蛋白序列与目标理论氨基酸序列100％相符。
实施例3 Cra4S1蛋白的诱导、表达与纯化
IPTG诱导：挑取实施例2的转化平板上的两个单菌落分别接种到含50μg/ml卡那霉素的3ml LB培养液的试管中，37℃200rpm振摇过夜，次日按1∶100接种于50μg/ml卡那霉素的100ml LB培养液中，37℃220rpm振摇至菌体OD600达0.6-0.8(约4h)，取出1ml培养物，10000g室温离心5min，弃上清，用100μl PBS重悬菌体沉淀，备用。向剩余的培养物中加入IPTG至终浓度为1mM，37℃220rpm振摇4小时诱导重组蛋白表达，取1ml培养物，10000g室温离心5min，弃上清，用100μl PBS重悬菌体沉淀，备用。余培养物离心沉淀后弃上清，称湿重并记录存档，置-20℃冰箱保存。
蛋白表达检测：取上一步骤中诱导前和诱导后的细菌悬液，震荡后加入等体积的2×SDS上样缓冲液，100℃加热5min，SDS-PAGE电泳，考马斯亮蓝染色检测重组蛋白(图5)。结果显示转化菌在IPTG的诱导下产出目的蛋白，在分子量15kDa的位置显示较强的蛋白条带，符合目的蛋白的理论值，未诱导菌无此蛋白出现。
重组蛋白亲和层析纯化：首先将IPTG诱导的菌体超声破碎，按照每克细菌湿重加30ml Tris-HCL(PH8.0)，混悬均匀，4℃，10000rpm离心30分钟，清洗细菌沉淀物三次后，悬浮细菌，超声波(宁波新芝超声破碎仪Y-92III)裂解法破碎菌体，将装有菌液的烧杯置于冰浴中，超声时间3秒，间隔时间5秒，超声工作总时间8分钟。超声破碎的菌液在4℃条件下，10000rpm离心15分钟，收集上清液和沉淀，SDS-PAGE检测(图6)。结果显示细菌破碎后，重组蛋白主要出现在离心沉淀后的上清液中，说明重组蛋白以可溶性形式在细胞内合成，避免了后续包涵体的复性过程。
使用AKTA纯化仪(GE公司)，亲和层析纯化重组蛋白，层析柱选择DEAE柱(汇研)，含重组蛋白的细菌裂解液上清溶液加入到纯化柱中，一次上样体积可以40ml-100ml(1-5mg/ml)，上样流速1.5ml/min，使用流动相A溶液为50mM Tris-HCl，PH8，至流出液OD280值到达基线，转为使用流动相B溶液为50mM Tris-HCl，1M NaCl，pH8.0，以4ml/min流速洗脱，使用不同梯度B溶液洗脱目的蛋白，10％B，18％B，100％B，收集流出液并标记，运用SDS-PAGE电泳检测纯化的蛋白，图7显示第一梯队洗脱收集 到的蛋白，进一步纯化使用Phenyl Sepharose FF柱，去除杂质，-20℃保存。
结论：质粒转化菌经IPTG诱导后可在细胞内产生重组蛋白，菌体裂解后，发现重组蛋白主要存在于裂解液上清中，说明Cra4S1以可溶性蛋白形式存在。通过亲和层析的方法获得纯化的Cra4S1重组蛋白，重组蛋白分子量约15kDa，符合预期理论值。
实施例4 小鼠软组织病变部位的面积计算方法
建立小鼠表皮和皮下软组织病损模型，用于检测牙龈卟啉菌的致病毒力。
简述如下：牙龈卟啉菌包括各种不同亚型细菌(牙龈卟啉菌W50类亚型、Thai类亚型、QML类亚型和381类亚型)均生长在含5％去纤维马血的Fastidious厌氧琼脂(FAA)平板上，放置在37℃厌氧培养箱中，含80％氮气，10％氢气，10％二氧化碳。挑取菌落转种到新鲜配制的BHI培养液(脑心浸液培养液)中含5μg/ml氯化血红素，18-24小时生长，当OD600nm达1-1.2时，培养物离心收获，洗涤2次，使用BHI培养液制成一定浓度的细菌悬浮液(2～8x10 10CFU/ml)，用于接种实验小鼠(Balb/c，扬州大学动物中心购买)。小鼠按不同实验计划分组，一般取6-8周龄，体重18-22克，每组8～12只，每只小鼠接种200μl细菌混悬液，在小鼠背部脊椎旁两侧的皮下注入。细菌攻击后，小鼠的体重变化、行为和动作被记录在案，皮肤和皮下软组织的病损情况有详细记录。
具体的，当采用牙龈卟啉菌W50亚型攻击Balb/c小鼠实验时，准备3*10 10CFU/m细菌悬液，0.2ml注入小鼠背部脊椎旁两侧皮下。
结果：图8显示小鼠受细菌攻击后，皮肤和皮下软组织发生病变，白线圈出病变部位。计算病变面积，初时使用人工手动测量病变的最长纵径和相对应的横径，纵径和横径相乘计算出病变面积，所有手写数据记录存档。病变部位认定和面积计算方法在2019年4月改进，使用拍照的方法，固定相机和所摄病变部位的距离，然后照片使用ImageJ(计算机软件名)计算圈入范围内的病变面积。
小结：本实施例使用的小鼠体表软组织损伤模型，动态观察局部病变情况，包括创面的病理特征，病程进展，创口愈合情况和病变的预后状态，结合小鼠的体重改变，精神状态和行为特征等指标，综合判断牙龈卟啉菌感染造成的危害。使用拍照方法和计算机软件定位和计算病变面积，减少人为误差，保留影像记录，更利于溯源和进一步研究原始资料。本实施例使用的动物实验模型是按照国际通用方法，标准获得行业内的认可。
实施例5 牙龈卟啉菌感染的剂量与小鼠软组织病损的关系
本实施例的目的是调查与四种主要外膜蛋白相对应的牙龈卟啉菌亚型，它们的毒力差异以及细菌感染的剂量与病变的关联性。
具体的，四种不同亚型牙龈卟啉菌包括W50亚型、Thai亚型、QML亚型和381亚型，参照实施例4描述的方法，制成三种不同浓度的细菌悬液，分别为5x10 9CFU/ml，2x10 10CFU/ml和8x10 10CFU/ml，实验动物Balb/c小鼠，6-8周龄，体重18-24克，每组8～12只，每只小鼠接种200μl细菌混悬液，在小鼠背部脊椎旁两侧的皮下注入。细菌攻击后，记录小鼠的体重、行为和局部软组织病变情况。
结果：随着攻击菌浓度的增加，动物的全身和局部病变情况都在不断加重，表现在高浓度菌侵犯引起的体重损失最严重，小鼠病态状，局部病损面积最大。四种不同牙龈卟啉菌亚型在受到相同剂量的细菌攻击时，局部损害也不一致(图9A和图9B)。
结论：图9A和图9B显示测试的四个牙龈卟啉菌亚型菌株的毒力有一定的差异，图A中的PgA、PgB、PgC和PgD分别代表牙龈卟啉菌W50亚型、Thai亚型、QML亚型和381亚型；图B中三个不同浓度细菌悬液1、2、3分别代表5x10 9CFU/ml，2x10 10CFU/ml和8x10 10CFU/ml。
但是，约20个不同牙龈卟啉菌菌株(分属于四个主要牙龈卟啉菌亚型)用同样的方法攻击动物，发现即使在同一个亚型内菌株之间也存在不同的毒力(数据没有显示)，因此，本实施例的菌株毒力差异不具备亚型的代表菌特征。也就是说本实施例的结果并不能说明牙龈卟啉菌哪个亚型的毒力最强或最弱。但是毒力与剂量的关系在四个测试的亚型中都得到重复，细菌攻击剂量越大造成的危害也越大。结合临床，牙周病患者的局部慢性感染的细菌聚集到一定浓度时引起急性发作。
实施例6 牙龈卟啉菌感染与实验小鼠年龄的关系
实验过程中，曾发现25周龄以上未曾受到过牙龈卟啉菌感染的小鼠对不同亚型牙龈卟啉菌的攻击仅产生局部轻微病损，本实施例的目的是调查年龄与牙龈卟啉菌感染的关系。
具体的，按照实施例4描述的方法制作牙龈卟啉菌W50亚型2*10 10CFU/m细菌悬液，0.2ml注入各年龄组实验小鼠的背部脊椎旁两侧皮下，每个不同年龄组有8-10只动物，细菌攻击后，观察病变面积发展情况直至第35天实验结束。
结果：使用同剂量和同体积细菌悬液攻击不同年龄组的实验小鼠，结果显示不同年龄组的动物对牙龈卟啉菌W50亚型攻击造成的损害有区别。本次实验，各年龄组动物 受细菌攻击后未发生死亡现象。G1组(体重15-18克)和G2组(体重22-26克)动物分别是4周龄和8周龄处于幼龄期，起病快，病变程度严重，但是幼龄组动物自愈恢复快，愈合期短。成龄鼠从G3组的12周(体重28-32克)、G4组(体重32-36克)的16周到G5组(体重33-38克)的20周病变过程相似，病损的面积明显低于幼龄组动物，但是成龄组动物的预后有迁延现象(表1，图10)。
结论：初次遭受牙龈卟啉菌感染的动物有自愈现象，幼龄组动物感染后可快速愈合，但成龄组动物有形成慢性感染病灶的趋势。这个实验现象对临床实际情况有一定的参考价值，也就是说在人群中，或许青少年更容易罹患牙龈卟啉菌感染引起的牙周病，但是因为感染后可以快速修复，所以并没有在青少年期引起严重的局部损害和得到更多的关注。牙周病在成年人中发病率高可能的解释是因为成年人感染后病程迁延，预后差，因此局部损害也不断加重，后期甚至引起局部牙槽骨的破坏和吸收，造成牙齿松动。本实施例提示预防牙龈卟啉菌感染从幼年到青少年开始。
表1，不同年龄组小鼠受牙龈卟啉菌感染后造成的局部病损调查
Figure PCTCN2019124433-appb-000005
实施例7 牙龈卟啉菌多次感染引发局部病情加重和全身状况衰弱
实施例6的实验结果表明动物初次感染牙龈卟啉菌有自愈现象，年龄越小恢复越快。临床实践中，牙周病病情常常呈现的是间隙性进行性发展状况。本实施例的目的是通过比较单次和多次感染的实验动物模型，调查多次慢性感染对实验动物造成的局部和全身的危害。
具体的，同年龄小鼠8-10周龄，体重26-30克，分成两组，编号第148组(G148)采用5*10 10CFU/m细菌悬液牙龈卟啉菌QML亚型攻击，编号第148.1组(G148.1)不攻击。40天后，第148组(G148)受细菌攻击的小鼠已痊愈，这时与第148.1组(G148.1)动物一起接受5x10 10CFU/ml细菌悬液牙龈卟啉菌QML亚型感染，方法同实施例4所述。
结果：动态观察动物受到第二次细菌攻击时，局部病变损伤面积大于初次感染组(表2)，此外，二次感染的动物其全身情况呈衰弱趋势，表现在毛发黯然，耸刺，尾部枯萎，恢复缓慢，预后差。多次感染实验在牙龈卟啉菌的四种主要亚型中都已重复多次，得到相似的结果。局部病变见图11A，动物全身状况见视频截图，图11B是G148组动物被牙龈卟啉菌QML亚型感染后第24天的全身状况视频截图，图11C是G148.1组动物被牙龈卟啉菌QML亚型初次感染后第24天的恢复情况视频截图。
表2牙龈卟啉菌多次感染与初次感染造成的软组织损害面积比较(单位：mm 2)
组别/感染后天数 2 4 6 8 10 12 14 18 24 31
G148二次感染 19.85 176.52 258.84 234.19 177.54 196.02 134.32 113.04 70.34 35.53
148.1初次感染 20.66 136.01 172.69 238.30 196.87 208.50 95.06 54.00 0.00 1.38
结论：动物实验结果显示，牙龈卟啉菌持续感染不仅使局部病变情况越来越差，同时慢性感染可以影响到机体的全身健康状态。临床上，牙周病不仅造成牙齿松动脱落，牙周病与全身性疾病的关联已有大量临床和统计学数据公布，牙周病是冠心病及其急性发作的独立危险因子，是脑卒中的独立危险因子，已发现牙周病致病菌牙龈卟啉菌(Porphyromonas gingivalis)与阿尔茨海默病(Alzheimer’s disease，俗称老年痴呆)的直接联系。慢性反复牙龈卟啉菌感染是否可以直接造成或引起心血管疾病或阿尔茨海默病，需要进一步数据证明和研究。本发明创造的目标是采用免疫学方法解决病原菌慢性感染的问题，阻止病原菌长期迁延而危害全身健康。
实施例8 组合疫苗的发现
本实施例是发现组合疫苗免疫保护功能的最初尝试。起初实验的目的是调查牙龈卟啉菌不同亚型外膜蛋白RagB之间是否存在免疫交叉保护作用，所以实验设计时，首先 使用的是RagB二价疫苗包括W50ragB蛋白(蛋白序列内容参见PCT/GB2005/001976，蛋白纯化等制作方法类同本实施例3)和QMLragB蛋白(蛋白序列内容参见PCT/GB2005/001976，蛋白纯化等制作方法类同本实施例3)按照质量比为1∶1。两次免疫后，又加入了Cra4S1蛋白(实施例3制备的Cra4S1蛋白)，每种蛋白都给予三次免疫机会。加入各亚型都拥有的共同抗原Cra4S1是调查新的组合疫苗是否可以影响RagB疫苗的免疫保护效果。
免疫方案，实施例3纯化得到的Cra4S1蛋白与不同亚型RagB蛋白分别免疫动物(表3-1、表3-2)。小鼠6-7周龄，体重18-22克，分三大组，第一大组(分为三个组别：G11、G14和G17)用QMLragB蛋白和W50ragB蛋白免疫；第二大组(分为三个组别：G12、G15和G18)用组合疫苗含QMLragB蛋白、W50ragB蛋白和Cra4S1蛋白免疫；第三大组(分为三个组别：G13、G16和G19)是未免疫对照组。本实施例最终组合疫苗的配方为Cra4S1蛋白：W50ragB蛋白：QMLragB蛋白，质量比为1∶1∶1。
具体的，每只小鼠腹腔注射100μl重组蛋白溶液，其中第一大组(G11、G14和G17)使用100μl溶液中含5-50μg W50ragB蛋白、5-50μg QMLragB蛋白和250μg铝佐剂，按照表3-1免疫计划执行；第二大组(G12、G15和G18)使用100μl溶液中含5μg Cra4Sl蛋白、5-50μg W50ragB蛋白、5-50μg QMLragB蛋白和250μg铝佐剂，按照表3-2免疫计划；第三大组(G13、G16和G19)是对照组，不加疫苗。免疫间隔期为3-4周，加强免疫前测血清效价，记录血清中抗体的发展过程。三免后使用ELISA方法检测特异性抗体的效价，当抗体效价高于1∶10万后，按照实施例4描述的方法做细菌攻击实验。
表3-1，G11/G14/G17免疫计划
Figure PCTCN2019124433-appb-000006
表3-2，G12/G15/G18免疫计划
Figure PCTCN2019124433-appb-000007
表3-3，组合疫苗与RagB疫苗的免疫保护功能对比
Figure PCTCN2019124433-appb-000008
结果：经W50ragB和QMLragB蛋白免疫的动物，当组合疫苗中加入Cra4S1蛋白，针对相对应的牙龈卟啉菌亚型(W50亚型和QML亚型)的攻击，动物的免疫保护功能明显增强，表现在软组织病变面积比对照组小(表3-3)。图12中，横坐标为G17(W50ragB和QMLragB混合疫苗)，G18(组合疫苗含Cra4S1蛋白、W50ragB蛋白和QMLragB蛋白)，G19(未免疫小鼠对照组)，攻击菌为牙龈卟啉菌QML亚型。
表3-3中，W50ragB和QMLragB二价疫苗或W50ragB、QMLragB和Cra4S1三价组合疫苗免疫的动物，都没有显示对牙龈卟啉菌Thai亚型的免疫保护作用，其中三价 组合疫苗的局部损害甚至较其他两组更严重。
结论和分析：本实施例是组合疫苗的最初尝试，疫苗用量降低的原因是因为与本实施例同时进行的疫苗剂量依赖实验结果显示，事实上，后续的研究发现，四种RagB蛋白的免疫接种用量在0.5-5μg时也均可产生高滴度抗体，虽然实验设计需要进一步完善，但是初步试验结果为后续研究打下了基础。
本实施例的结果显示组合疫苗不仅增强了免疫保护效果同时也提高了产品的稳定性。该现象提示两个不同靶点的蛋白联合应用，可以提高产品的功效。组合疫苗增强免疫效果和稳定性的原理可能与RagA和RagB两个亚单位受制于同一个转录因子的调控有关，一方面，当细菌表面的两个相互关联的亚单位分子的靶点被抗体占据以后，形成更稳定的分子结构，这种抗原抗体的结合，限制了细菌的外膜蛋白的功能，另一方面，双靶点抗原抗体的稳固结合可能更容易触发机体发挥更好的免疫协同/协调作用，消灭和抑制病原体。
在起初的研究中，当重组Cra4S1蛋白获得成功表达后，用单价Cra4S1蛋白免疫动物，可以获得高效价特异性抗体，但是多次细菌攻击实验结果均显示被免疫动物发生的软组织病变比未免疫对照组强烈，这个现象提示Cra4S1分子可能参与了超敏反应，因此认为Cra4S1蛋白并不是一个良好的候选疫苗。在两年多的时间里，体外实验发现，如果动物用RagB疫苗免疫后又受到牙龈卟啉菌的攻击，这时所采集的血清抗体与重组Cra4S1蛋白有不同程度的免疫交叉反应。这个现象提示当RagB抗体与牙龈卟啉菌外膜蛋白RagB结合以后，改变了Cra4S1蛋白原有的空间结构，使其抗原性得以暴露，为双靶点抗原抗体结合创造了机会。
本实施例还证明了RagB疫苗仅保护与其对应的牙龈卟啉菌亚型，Cra4S1蛋白与RagB蛋白联合使用，仅对相对应的牙龈卟啉菌亚型起免疫保护作用。举例说明，Cra4S1蛋白与QMLragB蛋白的组合疫苗保护机体防御牙龈卟啉菌QML亚型的攻击，但对牙龈卟啉菌Thai亚型的攻击没有保护作用。
因此，本实施例所述组合疫苗需要包含牙龈卟啉菌RagB蛋白(不同亚型)和Cra4S1，两个分子配合才能增强免疫保护功能和稳定性，组合疫苗是一个全新的概念，以下实施例进一步验证和应用组合疫苗。
实施例9 感染后使用组合疫苗对动物的免疫保护作用调查
临床上，绝大部分牙周病/种植体周围炎的患者出现不适症状就医时常常感染和感染 造成的局部病变情况已经比较严重。本实施例从多个角度探讨组合疫苗在已经有牙龈卟啉菌感染的机体中的免疫保护作用。
具体的，3-4周龄小鼠，体重12-16克，分成5组(表4)，每组5-6只，其中4组小鼠用低剂量牙龈卟啉菌W50亚型细菌悬液(5*10 8CFU/ml)在背部皮下注射200μl，造成轻微感染，受感染的小鼠饮食和活动如常，细菌攻击后第二天，部分动物出现轻微体重下降，随后体重呈持续增长，一周后感染小鼠可出现轻微的局部微小皮损。第5组小鼠未受感染，作为对照。小鼠继续饲养4周后，开始疫苗免疫计划。
已感染动物分成四组按表4所列的计划包括剂量和时间免疫动物，编号G154组的小鼠使用W50ragB蛋白免疫，编号G155组的小鼠使用Cra4S1蛋白免疫，编号G156组的小鼠使用组合疫苗Cra4S1蛋白和W50ragB蛋白混合物免疫，编号G157组是有轻微感染但没有注射疫苗的对照，编号G158组的小鼠是同年龄未感染对照。
三次免疫后，每组取一只小鼠采血，检测抗体效价，然后按照实施例4描述的方法使用牙龈卟啉菌W50亚型做细菌攻击实验，细菌悬液浓度为5X10 10CFU/ml，200μl注入背部皮下。细菌攻击后，记录小鼠的体重、行为和局部软组织病变情况，定期全身状况录像被记录在案。
结果：受轻微感染的实验动物组使用单价或组合疫苗都表现出局部软组织损害低于同样感染的对照组，同时病变损害也低于未感染的对照组(表5和图13)，接受疫苗的动物组在感染后第24天的记录中已经痊愈，其中组合疫苗组的各项有益指标均优于单价疫苗，而对照组则病情迁延。
本实施例同时也观察到实施例7所描述的现象，即多次受牙龈卟啉菌感染的动物局部损害情况更加严重(表5和图13、图14)。特别关注的是，在动物恢复过程中，图14录像资料观察到动物的体态，使用疫苗的小鼠全身状况良好，毛发光泽，身体匀称，运动自如，而对照组小鼠体表溃疡难愈，毛发暗淡无光泽，动作迟缓。实验组与对照组的体重变化不明显。
表4，2019年G154-G158感染后免疫试验计划
Figure PCTCN2019124433-appb-000009
Figure PCTCN2019124433-appb-000010
表5感染后免疫保护实验病变面积平均值记录(单位：mm 2)和数据标准差
Figure PCTCN2019124433-appb-000011
结论：实施例7的动物实验结果提示反复牙龈卟啉菌感染不仅造成越来越严重的局部损害，事实上还影响到全身健康。表5记录了组合疫苗用于感染后动物的免疫，当动物受到细菌的再次攻击时表现出局部病变程度减低，病变面积平均值(AVG)下降，实验数据的标准差(STDEV)维持在低水平，而且动物的全身状况表现良好。这些事实说明组合疫苗对健康机体或已经有感染的机体均可起到有效的免疫保护作用。四个主要牙龈卟啉菌亚型都开展了类似实验，预示着本发明专利技术组合疫苗的使用有益于全身健康，具有广泛的市场需求。
实施例10调查特异性双靶点抗体的被动免疫保护功能
本实验的目的是调查被动免疫(即注射特异性抗体)是否可以起到安全有效的免疫保护作用。事实上，抗体作为治疗性药物，针对的患者通常是已经有感染的，所以本实施例对未感染和已感染的动物同时做了分析。
具体的，小鼠特异性抗血清的免疫方案列于表6-1(ThairagB蛋白序列参见PCT/GB2005/001976，蛋白纯化等制作方法类同本实施例3，Cra4S1蛋白为实施例3制备)，三次免疫后，通过间接ELISA方法确定血清中特异性抗体的效价，最终采血，血清分离后得到多克隆抗体置放在-20℃冰箱储藏。
表6-1，抗血清制备免疫计划得到多克隆抗体
Figure PCTCN2019124433-appb-000012
动物实验分组情况如表6-2所示。编号G361、G362、G363、G364和G369组的小鼠有预感染史，用200μl牙龈卟啉菌Thai亚型细菌悬液(5X10 8CFU/ml)在小鼠背部皮下注入，G370、G371、G372、G373和G374共5组小鼠为未感染牙龈卟啉菌对照组。
具体的，小鼠9-10周龄，体重28-32克，细菌攻击前4天和前1天，按照表6实验计划在实验小鼠的腹腔注入100μl抗体溶液含200μg小鼠血清，随后按计划执行细菌攻击实验，具体过程如实施例4所述，观察和记录小鼠被攻击前后的体重和行为状态，记录受攻击动物的皮下软组织病变发展情况和测量皮肤病变面积(表7)。
表6-2抗体被动免疫保护实验动物分组信息
组别 数量 轻微感染 多克隆抗体 剂量 体积
G361 8 是 抗ThairagB血清 200μg/小鼠 100μl
G362 8 是 抗ThairagB+Cra4S1血清 200μg/小鼠 100μl
G363 8 是 抗Cra4S1血清 200μg/小鼠 100μl
G364 8 是 小鼠正常血清 200μg/小鼠 100μl
G369 8 是 PBS / 100μl
G370 4 否 抗ThairagB血清 200μg/小鼠 100μl
G371 4 否 抗ThairagB+Cra4S1血清 200μg/小鼠 100μl
G372 5 否 抗Cra4S1血清 200μg/小鼠 100μl
G373 6 否 小鼠正常血清 200μg/小鼠 100μl
G374 6 否 PBS / 100μl
表7注射抗体被动免疫保护实验病变面积平均值数据记录(单位：mm 2)
组/天数 0 2 4 6 8 10 12 14 17 21 28 32
G361 0 184.27 124.91 166.74 236.72 197.79 156.33 106.04 61.23 31.05 13.88 0.00
G362 0 195.08 95.38 180.54 186.96 152.41 97.08 44.43 12.53 4.37 2.02 0.00
G363 0 239.75 194.29 249.85 265.03 221.80 141.58 90.64 28.71 3.51 0.00 0.00
G364 0 251.98 183.93 230.06 212.13 164.39 99.88 59.84 13.06 10.28 4.05 0.00
G369 0 206.68 139.09 219.30 210.55 202.80 140.63 86.03 21.47 2.77 0.00 0.00
G370 0 116.42 109.95 118.07 136.79 113.62 82.43 70.07 71.08 0.00 448.97 110.89
G371 0 155.34 131.54 172.22 195.88 217.62 157.67 101.65 43.61 8.39 0.00 0.00
G372 0 291.57 246.22 252.10 285.31 282.30 207.49 140.88 81.25 8.66 0.84 5.07
G373 0 457.47 294.04 324.44 433.62 419.36 341.65 171.24 55.08 7.16 0.00 0.00
G374 0 243.82 262.59 270.86 297.03 307.68 151.93 90.18 19.20 0.00 0.00 0.00
结果：首先，本实施例中，使用常规剂量攻击动物，实验动物死亡率高于预期，需要指出的是本实施例中所有动物都有腹腔注射的步骤。本项目所设计的牙龈卟啉菌攻击剂量经多次重复实验，受感染动物发生死亡的情况比较少见。本实施例中图15A显示未感染对照组的动物，没有人为的牙龈卟啉菌感染史，抗ThaiRagB实验组G370、抗Cra4S1实验组G372、腹腔注射正常血清的G373和注射PBS的G374对照组均有动物死亡，死亡率分别为75％(3/4)、40％(2/5)、33.3％(2/6)和50％(3/6)，而组合抗体组(抗ThaiRagB和抗Cra4S1)血清注射的G371动物组未发生动物死亡。本实施例中图15B显示预感染对照组动物中腹腔注射正常血清组G364和腹腔注射PBS的对照组G369均有较高的死亡率，分别为37.5％(3/8)和62.5％(5/8)，注射特异性抗体的实验组中，抗ThaiRagB抗体G361、组合抗体(抗ThairagB和抗Cra4S1)G362的死亡率分别为25％(2/8)和12.5％(1/8)，抗Cra4S1抗体实验组G363的动物全部存活。
图16A和图16B显示特异性组合抗体在预感染组动物和未感染组动物实验中都表现出局部病损范围低于其他实验组存活动物，两组实验中，组合抗体在预感染组动物中发生1例死亡现象。抗ThaiRagB血清实验组在预感染的动物中局部病损严重度低于对照组，但在健康组的实验中，仅存活1只动物，影响整组的判断。抗Cra4S1实验组显示的是一个奇特的现象，抗体注入已感染的动物体内，动物受细菌攻击后存活率最高，没有发生死亡现象，但是局部病变情况比抗ThaiRagB实验组和组合抗体(抗ThaiRagB和抗Cra4S1)实验组严重(图16A和图16B)。
结论：实施例10的实验结果显示，组合抗体(抗ThaiRagB和抗Cra4S1)具有稳定、 安全和高效的抵御牙龈卟啉菌感染的功能，对未感染和已感染的实验动物都起到免疫保护作用(图16A和图16B)。单独使用抗ThaiRagB抗体和抗Cra4S1抗体都存在不确定的疗效。本实施例实验结果提示当单一抗体与细菌表面的抗原分子结合以后，有可能存在结构不稳定的因素，因此造成抗体疗效有缺陷。当使用双靶点特异性抗体时，组合抗体特异性地占据了主要外膜蛋白的抗原双靶点后，阻止了外膜蛋白的功能，从而达到抑制和消灭细菌，减低细菌毒力的功效。
综上所述，本发明发现并证实牙龈卟啉菌的重要外膜蛋白成分Cra4S1，创造了Cra4S1蛋白的表达基因，并且利用新靶点Cra4S1结合细菌的特异/特征性外膜蛋白RagB，提出了组合疫苗和双靶点特异性抗体的理论，并且用实验数据证明创新产品具有有效和稳定的抑制细菌功能的功效，新技术预示着广泛的工业应用前景。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (11)

  1. cra4S1基因，其特征在于，所述cra4S1基因的核苷酸序列如SEQ ID NO：1所示。
  2. 权利要求1所述的cra4S1基因所编码的Cra4S1蛋白，其特征在于，其氨基酸序列如SEQ ID NO.2所示。
  3. 表达盒、重组载体或细胞，其含有权利要求1所述的cra4S1基因或其基因片段。
  4. 权利要求1所述的cra4S1基因、权利要求2所述的Cra4S1蛋白、权利要求3所述的表达盒、重组载体或细胞在制备预防或治疗牙周病或种植体周围炎或与牙龈卟啉菌感染相关疾病的疫苗或药物中的应用。
  5. 一种疫苗或药物，其含有权利要求2所述的Cra4S1蛋白或其片段。
  6. 根据权利要求5所述的疫苗或药物，其特征在于，所述疫苗或药物还包括牙龈卟啉菌的W50ragB蛋白、ThairagB蛋白、QMLragB蛋白和381ragB蛋白中的一种或几种。
  7. 根据权利要求5所述的疫苗或药物，其特征在于，所述疫苗中的Cra4S1蛋白与W50ragB蛋白的质量比为1:2-8；或所述Cra4S1蛋白与ThairagB蛋白的质量比为1:1-6；或所述Cra4S1蛋白与QMLragB蛋白的质量比为1:1-6；或所述Cra4S1蛋白与381ragB蛋白的质量比为1:2-9。
  8. 权利要求5或6所述的疫苗或药物，其特征在于，所述疫苗或药物用于预防发生或复发或治疗未感染健康人或已有感染的牙周病或种植体周围炎或与牙龈卟啉菌感染相关的患者。
  9. 一种特异性抗体，其特征在于，由包含权利要求2所述Cra4S1蛋白或其片段组成的制剂引导免疫动物制得。
  10. 根据权利要求9所述的特异性抗体，其特征在于，所述特异性抗体还包括牙龈卟啉菌的W50ragB蛋白、ThairagB蛋白、QMLragB蛋白和381ragB蛋白中的一种或几种或其片段组成的组合制剂引导免疫动物制得。
  11. 权利要求9或10所述的特异性抗体，其特征在于，所述特异性抗体用于预防发生或复发或治疗未感染健康人或已有感染的牙周病或种植体周围炎或与牙龈卟啉菌感染相关的疾病的药物应用。
PCT/CN2019/124433 2019-07-30 2019-12-11 Cra4S1基因及其编码的蛋白和应用 Ceased WO2021017365A1 (zh)

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US17/613,122 US12552841B2 (en) 2019-07-30 2019-12-11 CRA4S1 gene, encoded CRA4S1 protein, and application
GB2200745.4A GB2602562B (en) 2019-07-30 2019-12-11 CRA4S1 gene, encoded CRA4S1 protein, and application
EP19939714.2A EP4008341A4 (en) 2019-07-30 2019-12-11 CRA4S1 GENE, ENCODED CRA4S1 PROTEIN AND USAGE
JP2022503798A JP7396569B2 (ja) 2019-07-30 2019-12-11 Ｃｒａ４Ｓ１遺伝子及びそれによってコードされるタンパク質と応用

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