WO2014190838A1 - 一种猪伪狂犬病病毒、疫苗组合物及其制备方法和应用 - Google Patents

一种猪伪狂犬病病毒、疫苗组合物及其制备方法和应用 Download PDF

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WO2014190838A1
WO2014190838A1 PCT/CN2014/076691 CN2014076691W WO2014190838A1 WO 2014190838 A1 WO2014190838 A1 WO 2014190838A1 CN 2014076691 W CN2014076691 W CN 2014076691W WO 2014190838 A1 WO2014190838 A1 WO 2014190838A1
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vaccine
pseudorabies virus
strain
vaccine composition
virus strain
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French (fr)
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张许科
孙进忠
伍锐
谭菲菲
白朝勇
田克恭
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Pulaike Biological Engineering Co Ltd
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Pulaike Biological Engineering Co Ltd
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Priority to EP14804814.3A priority Critical patent/EP2985288B1/en
Priority to US14/779,132 priority patent/US20160137700A1/en
Priority to JP2016504478A priority patent/JP6236144B2/ja
Priority to ES14804814T priority patent/ES2762535T3/es
Publication of WO2014190838A1 publication Critical patent/WO2014190838A1/zh
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Priority to US14/980,673 priority patent/US9650424B2/en
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    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
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    • A—HUMAN NECESSITIES
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
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    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16771—Demonstrated in vivo effect

Definitions

  • the present invention relates to a vaccine composition which belongs to the field of animal virology. Background technique
  • Pseudorabies also known as Aujeszky's disease
  • Aujeszky's disease is a breed of livestock, poultry and wild animals such as pigs, cattle, sheep, etc. caused by the Sued herpesvirus 1 strain in the alpha subfamily of the Herpesviridae family.
  • Pseudorabies in pigs is widespread in China and is a serious hazard. It is one of the major diseases that restrict the production of large-scale pig farms. It can cause abortion of pregnant sows, stillbirth or mummified fetuses and piglets with neurological symptoms, paralysis, and high mortality.
  • PRV has strong ubiquitous, neurotropic and latent infection characteristics, and can be latently infected in the peripheral nervous system. When the latent virus is activated to become an infectious virus, the latently infected host will develop disease.
  • Subunit vaccines can provide corresponding protection to immunized animals.
  • Subunit vaccines are vaccines that use genetic engineering methods to clone pathogen protective antigen genes into prokaryotic or eukaryotic expression systems for efficient expression. It has been found that gB, gC, and gD in pseudorabies virus glycoproteins can produce neutralizing antibodies in the body. These antibodies have the ability to neutralize PRV in vivo, in vitro, and in the presence or absence of complement. "Progress in the research of pseudorabies subunit vaccine" (Yang Chengwei, Yan Gaoming, Yan Nanhui, "Jiangxi Animal Husbandry and Veterinary Journal", No.
  • gB, gC among the 11 glycoproteins currently found in pseudorabies virus.
  • gD can induce the body to produce neutralizing antibodies.
  • Monoclonal antibodies to gB, gC, and gD neutralize PRV without the involvement of complement.
  • Pigs and mice injected with anti-gB, gC, gD monoclonal antibodies are resistant to PRV virulent attacks. Therefore, gB, gC, and gD are the preferred proteins for the development of PRV subunit vaccines.
  • Glycoprotein gD is an important neutralizing antigen and a major target of protective antibodies, which induces a better protective response.
  • 6,512,231 disclose the use of the porcine pseudorabies virus gD protein to prepare vaccines for the prevention of pseudorabies in pigs.
  • Porcine PRV has only one serotype. It is generally considered that the cross-protection of the strain is very strong, but there are still typical pseudorabies in pigs after the commercial vaccine is injected, such as prolonged body temperature, depression, loss of appetite, respiratory tract and / or neurological symptoms. It is highlighted that pigs of any age can be infected, can spread at the level of pigs, have a short incubation period (1 to 2 days), the incidence rate is between 10% and 100%, and the mortality rate of sick pigs is between 10% and 100%.
  • Piglet mortality can be as high as 100%), can cause pigs with high fever (40 ⁇ 42 ° C for more than 3 days), dyspnea, diarrhea, asthma, cough, sneezing, hind limb paralysis, dog sitting, suddenly falling , convulsions, side lie, angulation, stroke, stroke, and finally death, and can cause boar semen quality decline, pregnant sow abortion (up to 35%), premature delivery, stillbirth, weak babies ( Symptoms of reproductive disorders such as death of all weak children before the age of 14 days.
  • the prior art vaccine can not completely resist the wild poison attack after immunizing the pig, and there will still be symptoms such as high fever, depression, loss of appetite or abolition.
  • the infection rate is over 80%, the incidence rate is over 30%, and the mortality rate is 10% ⁇ Between 20%.
  • the present invention aims to provide a pseudorabies virus strain which can be used for preparing a vaccine, and the vaccine has good immunity against porcine pseudorabies by animal examination.
  • the present invention provides a nucleotide sequence which substantially encodes the protein of SEQ ID NO.
  • the present invention provides a nucleotide sequence which substantially encodes the protein shown in SEQ ID NO.
  • the present invention provides a nucleotide sequence which substantially encodes the protein shown in SEQ ID NO.
  • Coding sequence refers to a DNA sequence that is capable of being transcribed to the corresponding RNA sequence.
  • the main object of the present invention is to provide a pseudorabies virus strain, wherein the pseudorabies virus strain has the gD sugar encoded by the nucleotide sequence shown in SEQ ID NO. protein.
  • the term "gD glycoprotein" of the present invention is a structural protein essential for infection by Porcine Pseudorabies Virus and is one of the major glycoproteins on the surface of the mature virion membrane, also known as gp50 protein.
  • the term "homology" as used in the present invention refers to the degree of similarity between two amino acid sequences or two nucleotide sequences.
  • the amino acid sequence or nucleotide sequence homology can be calculated by any suitable method known in the art, for example, the target amino acid (or nucleotide) sequence can be sequenced with a reference amino acid (or nucleotide) sequence. Alignment, if necessary, a gap can be introduced to optimize the number of identical amino acids (or nucleotides) between the two aligned sequences, and on the basis of which the two amino acid (or nucleotide) sequences are identical The percentage of amino acids (or nucleotides).
  • the alignment of amino acid (or nucleotide) sequences and the calculation of homology can be accomplished by software well known in the art, such as, but not limited to, BLAST software (available on the National Center for Biotechnology Information (NCBI) website. : http: ⁇ blast. ncbi.nlm.nih.gov/Blast.cgi, or see, 1*, Altschul SF et al, J. Mol.
  • the pseudorabies virus strain has a gB glycoprotein encoded by the nucleotide sequence shown in SEQ ID NO.
  • the pseudorabies virus has the gB glycoprotein of the amino acid sequence of SEQ ID NO.
  • the pseudorabies virus of the present invention which has the gC glycoprotein of the amino acid sequence of SEQ ID NO.
  • the pseudorabies virus strain is HN1201 strain (Pseudorabies virus, strain HN1201) or a culture thereof, and the preservation number is CCTCC NO. V 201311; preserved in the China Center for Type Culture Collection; the deposit address is Wuhan City, Hubei province Wuhan University, the preservation date is May 20, 2013.
  • culture is a different generation of subcultures of the virus, and those skilled in the art know that only minor variations may occur in the sequence of the gene between different generations.
  • the culture is 5-35 generations. Cultures within.
  • Another object of the present invention is to provide a pseudorabies virus vaccine composition, wherein the vaccine composition comprises an attenuated live vaccine, an inactivated vaccine, a subunit vaccine, a synthetic vaccine or an immunological amount of a pseudorabies virus strain of pigs Genetic engineering vaccine.
  • the vaccine composition comprises an immunological amount of the live attenuated vaccine, inactivated vaccine, subunit vaccine, synthetic peptide vaccine or genetically engineered vaccine of the pseudorabies virus strain HN1201 strain or a culture thereof.
  • the vaccine composition of the present invention comprises the pseudorabies virus or antigen thereof as an active ingredient.
  • the pseudorabies virus of the composition for vaccine has the gD glycoprotein represented by the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having 98% or more homology thereto.
  • the porcine pseudorabies virus of the vaccine composition is HN1201 strain or a culture thereof.
  • the antigen used in the present invention means an antigenic portion of a viral component which elicits an immune response, and may comprise an amino acid sequence having the same as SEQ ID NO.
  • the antigen may comprise a gB protein having a nucleotide sequence homology of 95% or more with the amino acid sequence of SEQ ID NO. 2 or a fragment thereof.
  • the antigen may comprise a gC protein having 95% or more nucleotide homology with the amino acid sequence of SEQ ID NO. 3 or a fragment thereof.
  • live vaccine refers to a vaccine prepared from a virus whose virulence has been attenuated but which is still replicable in the host or on the cell.
  • attenuated is used to artificially reduce the toxicity of a gene by mutating the gene in a manner that renders the pathogen pathogenic but maintains immunogenicity. Typically, by UV radiation, chemical treatment, or continuous high in vitro Subculture Attenuate the poison. Artificial genetic alterations, such as deletion of specific nucleotides in known sequences to attenuate virulence.
  • inactivated vaccine also referred to as inactivated vaccine, refers to a suspension of inactivated virus that is used as an antigen to produce immunity.
  • inactivated vaccines include whole virus vaccines and lytic vaccines.
  • Inactivated vaccines can be readily produced using known methods. For example, a whole virus inactivated vaccine can be obtained by treating the virus with a furfural solution. The split vaccine can be prepared from the viral envelope after treatment with ether.
  • subunit vaccine refers to a vaccine prepared by genetically engineering a pathogen's protective antigen gene into a prokaryotic or eukaryotic expression system for efficient expression. It is less likely to cause side effects than a whole virus vaccine.
  • the expressed gD or gC protein of Pseudorabies virus can be used to prepare a subunit vaccine.
  • synthetic peptide vaccine refers to a small peptide containing only immunological determinant components, i.e., a vaccine prepared by artificially synthesizing a protective short peptide in the amino acid sequence of a natural protein, and attaching an adjuvant to a carrier.
  • the vaccine composition comprises a porcine pseudorabies virus strain HN1201 strain of >10 6 G TCID 5 o/ml or a culture inactivated vaccine thereof.
  • the vaccine composition of the invention may comprise a porcine pseudorabies virus in an amount of 10 6 () TCID 5 () per head.
  • the pseudorabies virus is used in an amount of less than 10 6 G TCID 5 () , the vaccine is not effective in stimulating antibody production. On the other hand, the excess may be uneconomical.
  • the vaccine composition comprises 25 to 10 ( ⁇ g/dose gD protein antigen) of the pseudorabies virus strain HN1201 strain or a culture thereof.
  • combined vaccine is used to mean a vaccine prepared from a virus mixture of the pseudorabies virus of the present invention and at least one different virus.
  • complex vaccine refers to a vaccine prepared from viruses and bacteria.
  • the pseudorabies virus of the present invention may be mixed or combined with classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus and/or Haemophilus parasuis, mycoplasma.
  • the vaccine composition further comprises a medium, an adjuvant, an excipient.
  • the vaccine compositions of the invention may also comprise a vehicle, an adjuvant and/or an excipient.
  • Saline or Agent or complete adjuvant aluminum hydroxide gel, vegetable oil or mineral oil, and the like.
  • the excipient include aluminum linolate, aluminum hydroxide, and potassium aluminum citrate, but are not limited thereto.
  • the method is: (1) inoculating a pseudorabies virus vaccine strain into respective susceptible cells, and culturing the inoculated susceptible cells; harvesting the cell culture;
  • step (2) treating the virus from step (1) with a furfural solution, BPL (P-propiolactone) or BEI (diethyleneimine);
  • the susceptible cell may be a passage cell line or a primary cell.
  • Susceptible cells suitable for pseudorabies virus include, but are not limited to, ST cell line (ATCC number: CRL-1746), PK-15 cell line (ATCC number: CCL-33), African green monkey kidney cell Marc-145 cell Department (ATCC No.: CRL-12219), Bovine Kidney Cell MDBK Cell Line (ATCC No.: CCL-22), Bovine Testicular Passage Cell BT Cell Line (ATCC No.: CRL-1390), Vero Cell Line (ATCC No.: CCL- 81), BHK-21 cell line (ATCC number: CCL-10), porcine kidney passage cell line (eg, IBRS-2, see for example, DECASTRO, MP1964.
  • ST cell line ATCC number: CRL-1746
  • PK-15 cell line ATCC number: CCL-33
  • African green monkey kidney cell Marc-145 cell Department ATCC No.: CRL-12219
  • Bovine Kidney Cell MDBK Cell Line ATCC No.: C
  • the vaccine composition according to the invention may be prepared in an oral dosage form or a parenteral dosage form.
  • parenteral dosage forms which can be administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or epidural routes.
  • a further object of the present invention is to provide a method for preparing the vaccine composition, the method comprising: (1) cloning the recombinant gene of the pseudorabies virus gD protein; (2) expressing the pseudorabies virus gD of the pig Recombinant protein; (3) the pseudorabies virus gD The protein antigen is mixed with the adjuvant in proportion and emulsified.
  • pseudorabies virus-associated disease refers to a disease caused by infection with a pseudorabies virus. Examples of it include sick piglets exhibiting obvious neurological symptoms, lethargy, barking, vomiting, diarrhea, and body temperature rise. High, once the disease occurs, pregnant sows can have miscarriage, mummy or stillbirth or reproductive disorders, but not limited to this.
  • pigs of any age are infected, can be transmitted at the level of pigs, have a short incubation period (1 to 2 days), and the incidence rate is 10% to 100%. Between the disease, the mortality rate of pigs is between 10% and 100% (the mortality rate of piglets can be as high as 100%).
  • the pigs After infection, the pigs can cause high fever (40 ⁇ 42 °C for more than 3 days), difficulty breathing, diarrhea, Asthma, coughing, sneezing, hind limb paralysis, dog sitting, suddenly falling to the ground, convulsions, side lying, angulation, squatting, and finally dying, can cause boar semen quality decline, pregnancy Sow abortion (up to 35%), premature delivery, stillbirth, weak babies (all deaths before 14 days of age), but not limited to this.
  • the above symptoms are different from those of the prior art when infected with the common porcine pseudorabies virus.
  • the infection may cause adult pigs after infection (weights above 50kg) can cause high fever in pigs (40 ⁇ 42 °C, continuous) 3 days or more), difficulty breathing, diarrhea, asthma, coughing, sneezing, paralysis of hind limbs, sitting, suddenly falling to the ground, convulsions, sideways, angulation, swimming strokes, and finally exhaustion; And piglets within 4 weeks of sudden onset, a large number of deaths, mortality rate of more than 90%; the incidence of piglets mainly manifested as a rise in body temperature above 41 °C, loss of appetite, accompanied by obvious neurological symptoms and diarrhea; piglets before and after weaning Mainly for respiratory symptoms, showing difficulty breathing, coughing, runny nose and so on.
  • prevention refers to all acts of inhibiting a pseudorabies infection or delaying the onset of a disease by administering a vaccine composition according to the invention.
  • treating refers to all actions that alleviate or divert the symptoms caused by infection with the pseudorabies virus by administering a vaccine composition according to the present invention.
  • DRAWINGS Figure 1 is a result of homology analysis of the amino acid sequence of the HN1201 strain gC and the amino acid sequence of the SA215 strain gC;
  • Figure 2 shows the results of homologous analysis of the amino acid sequence of the HN1201 strain gD and the SA215 strain gD amino acid sequence.
  • Sequence 1 is the amino acid sequence of the pseudorabies virus HN1201 strain gD;
  • Sequence 2 is the amino acid sequence of the pseudorabies virus HN1201 strain gB;
  • Sequence 3 is the amino acid sequence of the pseudorabies virus HN1201 strain gC;
  • Sequence 4 is the gD nucleotide sequence of the pseudorabies virus HN1201 strain
  • Sequence 5 is the gB nucleotide sequence of the pseudorabies virus HN1201 strain
  • Sequence 6 is the gC nucleotide sequence of the pseudorabies virus HN1201 strain. detailed description
  • head portion in the present invention means the amount of vaccine injected per pig.
  • the "TCID 50 ,” (50% tissue culture infective dose) described in the present invention refers to the amount of infection of half of the cell culture, and is an expression indicating the infectivity of the virus.
  • the MEM liquid medium (liquid) was prepared in accordance with the instructions using MEM dry powder medium purchased from Life Technologies, USA.
  • the DMEM medium of the present invention is prepared by referring to the preparation method of Appendix A of GB/T18641-2002.
  • the "PBS” referred to in the present invention means the abbreviation of Phosphate Buffer Saline, and the PBS of pH 7.4 of O.OlmM is used in the present invention, as described in "Molecular Cloning", Third Edition. Formulated.
  • Example 1 virus collection and separation
  • the porcine brain tissue was aseptically collected from a sample of suspected pseudorabies infection from Henan, and was added to the MEM medium at a ratio of 1:10 (by volume), ground to prepare a tissue suspension, and after repeated three freeze-thaw cycles, 2000r After centrifugation at /min for 15 min, the supernatant was collected, filtered through a 0.2 ⁇ filter filter, and cultured at 37 ° C for 1 h in PK-15 cells, and then added to a MEM medium containing 2% yak serum, 37. C culture for 5 days. The toxic culture solution was harvested, and after two freeze-thaw cycles, the virus was collected and replaced with a MEM medium containing 2% yak serum.
  • the pig pseudorabies virus PCR test kit (Beijing Century Yuanheng Animal Epidemic Prevention Technology Co., Ltd.) was used to detect the pseudorabies virus, and the result was positive.
  • the isolated virus was detected by the PCR kit for the detection of the foreign virus (porcine blue ear disease).
  • the virus RT-PCR detection kit, the porcine parvovirus PCR detection kit, the swine fever virus RT-PCR detection kit are all Beijing Century Yuanheng animal epidemic prevention technology limited public products), and the PCR test results are all negative, indicating that the poisonous species are pure.
  • the isolated pseudorabies virus is submitted for preservation.
  • the pseudorabies virus strain is 1201 strain (Pseudorabies vims, strain HN 1201), and the accession number is CCTCC NO. V 201311; it is preserved in the China Center for Type Culture Collection; It is Wuhan University of Wuhan, Hubei province, and the preservation date is May 20, 2013.
  • Example 2 Genetic characteristics of isolated virus
  • Example 1 The genetic characteristics of the virus isolated in Example 1 were determined by genetic analysis.
  • the porcine pseudorabies virus genomic DNA isolated on PK15 cells was used as a template, and the primers shown in Table 1 were used for PCR.
  • Primer Premier 5.0 was used to design primer sequences for amplifying the gB, gC, and gD genes, respectively.
  • the PCR amplification system was prepared as follows: template DNA 100 g, PrimerSTAR HS DNA Polymerase (2.5 ⁇ / ⁇ 1) 0.5 ⁇ 1, 2 PrimerSTAR GC Buffer 25 ⁇ 1, 1 ⁇ l each of the upstream and downstream primers (10 ⁇ m/ ⁇ ) , dNTP Mix (2.5 mM each ) 4 ⁇ 1, and make up the volume by 50 ⁇ 1 with distilled water.
  • a two-step PCR reaction was carried out: denaturation at 98 ° C for 10 sec, followed by annealing and extension at 68 ° C (required time calculated in lkb / min) for a total of 30 cycles.
  • the PCR reaction was terminated at 4 °C.
  • the resulting PCR product was analyzed by electrophoresis on a 1% agarose gel containing ethidium bromide.
  • the PCR product was sequenced.
  • the resulting sequence data was analyzed using Lasergene software. Table 1 PCR primer sequences
  • test group and 2 heads/group (control group), inoculated with pig pseudorabies virus HN1201 strain (the challenge dose was 2x l0 8O TCID 5 o/head), the control group was inoculated with DMEM medium; Day-old piglets were cultured for 3 generations of virulent HN1201 strain (the challenge dose was 2x l0 8 G TCID 5 / head), and 35-day-old piglets were still used as controls. After virus inoculation, the body temperature of piglets was measured daily to observe clinical symptoms and death. The specific results are shown in Table 2. Pathogenicity of pig pseudorabies virulent HN1201 strain to piglets of different ages
  • A1 has elevated body temperature and died 4 days after challenge
  • Seeds of different generations of the isolated strains were inoculated in a PK-15 cell culture according to Table 4 to form a seed batch, and then 1% (V/V) of the amount of the virus culture medium was added to form a monolayer.
  • the culture was carried out at 37 ° C.
  • the toxic cell culture solution was harvested, and after two freeze-thaw cycles, the virus was collected and the toxic value was determined.
  • the virus solution was diluted with PBS solution of pH 7.4 to the virus content shown in Table 4 and then mixed with 206 adjuvant (product of French SEPPIC) at a volume ratio of 54:46 at 30 °C. Stir for 15 minutes at 120 rpm.
  • 206 adjuvant product of French SEPPIC
  • 16 21-day-old PRV antibody-negative piglets were randomly divided into 4 groups, 4 heads/group.
  • the vaccine-inactivated vaccine group was injected with the vaccine prepared in Example 4 to immunize pig pseudorabies inactivated vaccine 2 ml/head, and the control vaccine was used.
  • the porcine pseudorabies live vaccine SA215 strain prepared by the method of CN101186902 was used according to the method for determining the immunogenicity of the patent specification, and the control group was inoculated with 2 ml/head of DMEM medium. After 28 days of immunization, the challenge was HN1201 strain of pseudorabies virus 2x l0 8 G TCID 5 . / Head, daily measurement of piglet body temperature after challenge, observe clinical symptoms and death (see Table 5 for results) Group of immunogenic test animals
  • Vaccination dose Inactivated vaccine A The vaccine group prepared in Example 4 A 2 ml/head inactivated vaccine B
  • Live vaccine SA215 Porcine pseudorabies virus live vaccine 10 60 TCID 50 / head control DMEM medium 2ml / head vaccine immunization, weekly reference GB / T 18641-2002 method serum neutralization test method to determine the inactivated vaccine group Neutralizing antibody titers, the results are shown in Table 6.
  • RNA/DNA Extraction Kit Ver.3.0 kit extracts PRV genomic DNA. Take ⁇ ⁇ genomic DNA as a template and use gD-specific primers: gDSF: 5' ATGCTGCTCGCAGCGCTATTGGC 3' and
  • gDSR 5' CTACGGACCGGGCTGCGCTTTTAG3'
  • PCR amplification was performed using TAKARA's high-purity enzyme Prime STAR® HS DNA Polymerase with GC Buffer. The amplification conditions were: 94 ° C for 3 min; 94 ° C for 30 s, 68 ° C for 90 s, 30 cycles; 72 ° C for 5 min. .
  • the PCR product was named gD. Its nucleotide sequence is shown in SEQN0.4, and its amino acid sequence is deduced as SEQNO.L.
  • the PCR product gD obtained by amplification of the high-fidelity enzyme was cloned into the pFastBac/HBM-TOPO vector (purchased from Invitrogen, Cat. No. A1 1339), and the cloning system was as follows: PCR product gD 4 l, Salt solution ⁇ ⁇ , TOPO Vector ⁇ ⁇ , a total of 6 ⁇ 1. Mix well, incubate for 5 min at room temperature, transform One ShotR Machl TM TlR competent cells, coat ampicillin-resistant plates, pick the monoclonal antibody to identify the insertion direction of gD gene, insert the correct orientation plasmid into Invitrogen, and identify the gD sequence. Correctness. The correct plasmid was sequenced as pFastBac/HBM-TOPO-gD.
  • pFastBac/HBM-TOPO-gD plasmid transforms DHlOBac competent cells (source), pFastBac/HBM-TOPO-gD and shuttle plasmid Bacmid in competent cells for transposition, recombinant plasmid, and pUCM13 Forward/pUCM13 Reverse primer to identify gD Insertion, positive Bacmid named Bacmid-gD.
  • the cell culture supernatant was collected and recorded as the P0 generation recombinant virus vBac-gD.
  • the P0 generation recombinant virus vBac-gD was infected with sf cells, and after 3 generations of expanded culture, the obtained P3 generation vBac-gD was used for recombinant protein expression.
  • the P3 generation recombinant baculovirus vBac-gD was inoculated with High-five cells (purchased from Invitrogen, Cat. No. B85502). High-five cells were suspended and cultured in a 500 ml flask, and after the cell density reached 7.0 ⁇ 10 5 cells/ml, the virus was inoculated according to the amount of 1 MOI, and the cell culture supernatant was collected 72 hours after infection. The volume was concentrated to 1/10 of the original volume using Millipore's tangential flow filtration system.
  • the baculovirus was inactivated with 1% (by volume) of Triton X-100 (purchased from sigma), and the protein content was determined by SDS-PAGE densitometry to be 20 ( ⁇ g/ml.
  • Triton X-100 purchased from sigma
  • the protein content was determined by SDS-PAGE densitometry to be 20 ( ⁇ g/ml.
  • Example 7 pig pseudorabies gave g D Preparation of subunit vaccine
  • the subunit antigen prepared in Example 6 was diluted with PBS solution (pH 7.4) to a volume of Table 9 and 206 adjuvant (product of SEPPIC, France) was mixed at a volume ratio of 54:46 at 120 rpm at 30 °C. Stir for 15 minutes in minutes.
  • Example 2 Twelve 21-day-old PRV antibody-negative piglets were randomly divided into 3 groups, 4 heads/group, and the vaccine prepared in Example 2 was injected according to Table 2, and the pig pseudorabies virus subunit vaccine was immunized with 2 ml/head.
  • the control group was inoculated with 2 ml/head of DMEM medium.
  • the challenge was porcine pseudorabies virus HN1201 strain 2x l0 8 G TCID 5 . / head, daily determination of piglet body temperature after the attack, observe the clinical Symptoms and death (see Table 10 for results).
  • Table 12 Changes in body temperature of piglets after piglet vaccination with pig pseudorabies virus subunit vaccine

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Abstract

本发明提供了一种疫苗组合物，该疫苗组合物包括免疫量的猪伪狂犬病病毒株的减毒活疫苗、灭活疫苗、亚单位疫苗、合成疫苗或基因工程疫苗。该疫苗组合物能有效诱发抗体产生，预防猪伪狂犬强毒株的感染，对猪产生有效的保护。

Description

一种猪伪狂犬病病毒、 疫苗组合物及其制备方法和应用 技术领域
本发明涉及一种疫苗组合物， 属于动物病毒学领域。 背景技术
伪狂犬病， 又称 Aujeszky氏病， 是由危渗病毒科（ Herpesviridae ) α 亚科中的猪疱疹病毒 I型 ( Suid herpesvirus 1 strain ) 所引起的猪、 牛、 羊等多种家畜、 家禽和野生动物的一种以发热、 奇痒 (除猪外)及脑脊髓 炎为主症的急性传染病。 猪的伪狂犬病在我国广泛存在， 危害严重， 是 制约规模化猪场生产的主要疫病之一。 它能引起妊娠母猪流产、 死胎或 木乃伊胎和仔猪出现神经症状、 麻痹， 死亡率高。 PRV具有较强的泛嗜 性、 神经嗜性及潜伏感染特性， 在外周神经系统可以长期潜伏感染， 当 潜伏病毒被激活变成有感染力的病毒， 被潜伏感染的宿主就会发病。
研究表明亚单位疫苗能够给免疫动物提供相应的保护， 亚单位疫苗 是利用基因工程方法将病原保护性抗原基因克隆到原核或真核表达系 统中，使其高效表达而制成的疫苗。目前发现伪狂犬病毒糖蛋白中的 gB、 gC、 gD 均能使机体产生中和抗体， 这些抗体无论是在体内、 体外， 还 是在有无补体存在的情况下都有中和 PRV的能力。 "伪狂犬病亚单位疫 苗研究进展"（杨承槐， 娄高明， 谌南辉《江西畜牧兽医杂志》 2004 年 第 3期)公开了在伪狂犬病病毒目前已发现的 11 种糖蛋白中， gB、 gC、 gD 均能诱导机体产生中和抗体。在没有补体的参与下， gB、 gC、 gD 的 单克隆抗体能中和 PRV。 猪和小鼠注射抗 gB、 gC、 gD 的单克隆抗体 均能抵抗 PRV 强毒的攻击。 因此， gB、 gC、 gD是研制 PRV 亚单位疫 苗的首选蛋白。 糖蛋白 gD是一种重要的中和抗原， 也是保护性抗体的 主要目标， 能诱导较好的保护反应。 US6858385和 US6521231公开了利 用猪伪狂犬病病毒 gD蛋白可以制备疫苗用于猪伪狂犬病的预防。 猪 PRV只有一个血清型，通常认为毒株的交叉保护力很强，但目前 仍存在仔猪注射商品化疫苗后发生典型猪伪狂犬病， 例如长时间体温升 高， 精神沉郁， 食欲减退， 出现呼吸道和 /或神经症状。 突出表现为任何 年龄的猪都会感染， 可在猪群水平传播， 潜伏期短（1 ~ 2天） ， 发病率 在 10% ~ 100%之间， 发病猪死亡率在 10%〜100%之间 （仔猪死亡率可 高达 100% ) ， 感染后可引起猪只高热 （40 ~ 42°C， 持续 3 日以上） ， 呼吸困难， 腹泻， 喘， 咳嗽， 打喷嚏， 后肢麻痹， 犬坐， 突然倒地， 抽 搐， 侧卧不起， 角弓反张， 泳状划水， 最后衰竭而死， 并可引起种公猪 精液质量下降， 怀孕母猪流产 （高达 35% ) ， 早产， 死胎， 弱仔（弱仔 14日龄前全部死亡）等繁殖障碍症状。 现有技术的疫苗免疫猪只后不能 完全抵抗野毒攻击， 依然会出现高热， 精神沉郁， 食欲下降或废绝等症 状， 感染率超过 80%， 发病率超过 30%， 死亡率在 10% ~ 20%之间。 现 有技术还没有疫苗能够解决针对猪伪狂犬变异株引起的伪狂犬病。 发明内容
为解决现有技术的不足， 本发明旨在提供一种猪伪狂犬病病毒株， 该猪伪狂犬病病毒株可用于制备疫苗， 通过动物实检证明该疫苗对于猪 伪狂犬病具有良好的免疫作用。
本发明提供了一种核苷酸序列， 所述核苷酸序列实质上编码序列 表 SEQ ID NO. l所示的蛋白。
本发明提供了一种核苷酸序列， 所述核苷酸序列实质上编码序列 表 SEQ ID NO.2所示的蛋白。
本发明提供了一种核苷酸序列， 所述核苷酸序列实质上编码序列 表 SEQ ID NO.3所示的蛋白。
"编码序列"在本申请中是指一种 DNA序列， 其能够被转录得到相 应的 RNA序列。
为解决现有技术的不足， 本发明的主要目的是提供一种猪伪狂犬病 病毒株， 其中， 所述猪伪狂犬病病毒株具有序列表 SEQ ID NO.4所示的 核苷酸序列编码的 gD糖蛋白。 本发明术语" gD糖蛋白"， 是猪伪狂犬病病毒进行感染必需的结构蛋 白， 是成熟的病毒粒子嚢膜表面的主要糖蛋白之一， 也称为 gp50蛋白。
本发明术语"同源性 "在本申请中是指两条氨基酸序列或两条核苷酸 序列的相似程度。 氨基酸序列或核苷酸序列的同源性可以通过本领域公 知的任何适当的方法计算得到， 例如， 可以将目标氨基酸 (或核苷酸）序 列与参比氨基酸（ 或核苷酸）序列进行序列比对， 必要时可以引入空缺， 使得两条比对的序列间相同的氨基酸（ 或核苷酸）数目达到最优化， 并 在此基础上计算两条氨基酸（ 或核苷酸） 序列之间相同氨基酸（ 或核苷 酸） 的百分比。 氨基酸（ 或核苷酸） 序列的比对和同源性的计算可以通 过本领域公知的软件实现， 例如， 但不限于， BLAST 软件（ 在美国国 立 生 物 技 术 信 息 中 心 （NCBI) 的 网 址 上 可 获 得 ： http：〃 blast. ncbi.nlm.nih.gov/Blast.cgi， 或者见， 1*列 口， Altschul S.F.et al， J.Mol.Biol , 215 ： 403-410(1990) ； Stephen F.et al, Nucleic Acids Res. , 25 ： 3389-3402(1997)), ClustalW2 软件（在欧洲生物信息研究所网址上 可获得： http://www.eji.ac.uk/Toolsa/clustalw2/,另见，例 口， Higgins D.G.et al, Methods in Enzymology, 266 ： 383-402(1996) ； Larkin M.A.et al, Bioinformatics(Oxford， England), 23(21) ： 2947-8(2007)) ；和 TCoffee 软 件等（在瑞典生物信 息 学研究所的 网 站上可 以 获得： http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi, 另见， 例^口， Poirot O.et al, Nucleic Acids Res. , 31(13) ： 3503-6(2003) ； Notredame C.et al, J.Mol.Boil , 302(1) ： 205-17(2000))。 使用软件进行序列比对时， 可 以使用软件提供的默认参数， 或者也可以根据实际情况对软件提供的参 数进行调整， 这些都在本领域技术人员的知识范围内。
优选地， 所述猪伪狂犬病病毒株具有序列表 SEQ ID NO.5所示的核 苷酸序列编码的 gB糖蛋白。
优选地， 所述的猪伪狂犬病病毒， 它具有 SEQ ID NO.2的氨基酸序 列的 gB糖蛋白。
优选地， 居本发明的所述猪伪狂犬病病毒， 它具有 SEQ ID NO.3 的氨基酸序列的 gC糖蛋白。 优选地， 所述猪伪狂犬病病毒株为 HN1201株（Pseudorabies virus, strain HN1201 )或其培养物， 保藏号为 CCTCC NO. V 201311 ; 保藏于中 国典型培养物保藏中心； 保藏地址为湖北省武汉市 .武汉大学， 保藏日 期为 2013年 5月 20曰。
所述术语"培养物 "是病毒的不同代次传代培养物， 本领域技术人员 知晓不同代次之间其基因序列仅可能会发生微小的变异， 优选地， 所述 培养物是 5-35代以内的培养物。
本发明的另一个目的是提供一种猪伪狂犬病病毒疫苗组合物，其中， 所述疫苗组合物包括免疫量的猪伪狂犬病病毒株的减毒活疫苗、 灭活疫 苗、 亚单位疫苗、 合成疫苗或基因工程疫苗。
优选地， 所述疫苗组合物包括免疫量的所述猪伪狂犬病病毒株 HN1201 株或其培养物的减毒活疫苗、 灭活疫苗、 亚单位疫苗、 合成肽 疫苗或基因工程疫苗。
优选地， 本发明的疫苗组合物包含所述猪伪狂犬病病毒或其抗原作 为活性成分。 用于疫苗的组合物的猪伪狂犬病病毒具有 SEQ ID NO. l 的氨基酸序列或与其共有 98%以上同源性的氨基酸序列表示的 gD糖蛋 白。
优选地， 所述疫苗组合物的猪伪狂犬病病毒为 HN1201株或其培养 物。
用于本发明的抗原是指病毒组分的抗原部分， 它引起免疫应答， 并 可包含具有与 SEQ ID NO. l的氨基酸序列。
可选择地，抗原可包含具有 SEQ ID NO.2的氨基酸序列或其片段共 有 95%以上核苷酸同源性的 gB蛋白。
可选择地，抗原可包含具有 SEQ ID NO.3的氨基酸序列或其片段共 有 95%以上核苷酸同源性的 gC蛋白。
本发明所用的术语 "活疫苗"指的是以毒力已经减弱但仍可在宿主体 内或细胞上复制的病毒制备的疫苗。 本发明所用的术语 "减毒，，用于指以 使病原丧失致病性、 但保持免疫原性的方式对基因进行突变来人工降低 病原体毒性。 通常， 通过 UV辐射、 化学处理或体外连续高阶继代培养 实现减毒。 人工的基因改变， 例如将已知序列中的特定核苷酸缺失以使 毒力减弱。
所用的术语"灭活疫苗，，， 也称作失活疫苗， 指的是用作抗原以产生 免疫力的灭活病毒的混悬液。 灭活疫苗的例子包括全病毒疫苗和裂解型 疫苗。 使用已知方法可以很容易地产生灭活疫苗。 例如， 通过用曱醛溶 液处理病毒可获得全病毒灭活疫苗。 裂解型疫苗可在用醚处理后由病毒 包膜制备得到。
术语"亚单位疫苗"指的是利用基因工程方法将病原体的保护性抗原 基因克隆到原核或真核表达系统中， 使其高效表达而制成的疫苗。 它比 全病毒疫苗引起副反应的可能性小。例如，表达的猪伪狂犬病病毒的 gD 或 gC蛋白可用于制备亚单位疫苗。
术语"合成肽疫苗"指的是一种仅含免疫决定簇组分的小肽， 即用人 工方法按天然蛋白质的氨基酸顺序合成保护性短肽， 与载体连接后加佐 剂所制成的疫苗。
优选地，所述疫苗组合物包括 > 106 GTCID5o/ml的猪伪狂犬病病毒株 HN1201株或其培养物灭活疫苗。
优选地，本发明的疫苗组合物可包含每头份 106 ()TCID5()量的猪伪狂 犬病病毒。 当猪伪狂犬病病毒以少于 106 GTCID5()的量使用时， 疫苗不能 有效刺激抗体产生。 另一方面， 超过的量可能是不经济的。
优选地， 所述疫苗组合物包括猪伪狂犬病病毒株 HN1201株或其培 养物的 25〜10(^g/剂量 gD蛋白抗原。
所用的术语"联合疫苗"用于指从本发明的猪伪狂犬病病毒与至少一种不 同病毒的病毒混合物制备的疫苗。 术语"复合疫苗"指的是从病毒和细菌 制备的疫苗。 例如， 本发明的猪伪狂犬病毒可与猪瘟病毒、 猪繁殖与呼 吸综合征病毒、 猪圓环病毒和 /或副猪嗜血杆菌、 支原体混合或组合。
优选地， 所述疫苗组合物进一步包括介质、 佐剂、 赋形剂。
本发明的疫苗组合物还可包含介质、 佐剂和 /或赋形剂。 生理盐水或 剂或完全佐剂、 氢氧化铝凝胶、 植物油或矿物油等。 赋形剂的例子包括 麟酸铝、 氢氧化铝和;琉酸铝钾， 但不限于此。 在实践中， 本领域技术人 本发明的再一目的是提供一种制备所述疫苗组合物的方法， 所述制 备方法包括增殖培养所述猪伪狂犬病病毒株 HN1201株， 灭活， 加入佐 剂， 搅拌均匀。
具体地， 所述方法为： （1) 将猪伪狂犬病病毒疫苗株接种于各自的 易感细胞， 并培养所述接种后的易感细胞； 收获细胞培养物；
(2)用曱醛溶液、 BPL(P-丙内酯）或 BEI(二乙烯亚胺)处理来自步骤 ( 1 ) 的病毒；
所述易感细胞可以是传代细胞系， 也可以是原代细胞。 适合于猪伪 狂犬病病毒的易感细胞包括但不限于， ST细胞系（ATCC 编号： CRL-1746)、 PK-15细胞系（ATCC编号： CCL-33)、 非洲绿猴腎细胞 Marc-145细胞系（ATCC 编号： CRL-12219)、 牛腎细胞 MDBK细胞系 (ATCC编号： CCL-22)、 牛睾丸传代细胞 BT细胞系（ATCC编号： CRL-1390)、 Vero 细胞系（ATCC编号： CCL-81)、 BHK-21 细胞系（ATCC 编号： CCL-10)、 猪腎传代细胞系（如: IBRS-2, 见例如， DECASTRO, M.P.1964. Behavior offoot and mouth disease virus in cell culture ： susceptibility of the IB-RS-2swine cell line.Arquivos Instituto Biologica 31 ： 63-78)、 兔腎传代细胞系（RK， 如： ATCC 编号： CCL-106) 等传 代细胞系， 或者鸡胚成纤维细胞和猪腎细胞等原代细胞。 原代细胞可以 通过本领域公知的方法， 用动物体内的组织进行分离和制备。
可将根据本发明的疫苗组合物制备成口服剂型或非口服剂型。 优选 的是可通过皮内、 肌肉、 腹膜内、 静脉内、 皮下、 鼻内或硬脑膜外途径 给予的非口服剂型。
本发明的又一目的在于提供一种制备所述疫苗组合物的方法， 所述 方法包括： （ 1 )克隆所述猪伪狂犬病病毒 gD蛋白重组基因； （2 )表 达所述猪伪狂犬病病毒 gD重组蛋白； （3 )将所述猪伪狂犬病病毒 gD 蛋白抗原与佐剂按比例混合， 乳化。
本发明的还一目的在于提供所述的疫苗组合物在制备预防和治疗 猪伪狂犬病病毒相关疾病的药物中的应用。
本文所用的术语 "猪伪狂犬病病毒相关疾病，，用于指由猪伪狂犬病毒 感染引起的疾病。它的例子包括发病仔猪表现出明显的神经症状、 昏睡、 呜叫、 呕吐、 拉稀、 体温升高， 一旦发病， 怀孕母猪可发生流产、 产木 乃伊胎儿或死胎或繁殖障碍， 但不限于此。
本文所用的术语"猪伪狂犬病病毒相关疾病"可以进一步用于指表现 为任何年龄的猪都会感染， 可在猪群水平传播， 潜伏期短 （1 ~ 2天） ， 发病率在 10% ~ 100%之间， 发病猪死亡率在 10%〜100%之间（仔猪死亡 率可高达 100% )， 感染后可引起猪只高热（40 ~ 42°C， 持续 3日以上） ， 呼吸困难， 腹泻， 喘， 咳嗽， 打喷嚏， 后肢麻痹， 犬坐， 突然倒地， 抽 搐， 侧卧不起， 角弓反张， 泳状划水， 最后衰竭而死， 并可引起种公猪 精液质量下降， 怀孕母猪流产 （高达 35% ) ， 早产， 死胎， 弱仔（弱仔 14日龄前全部死亡）等繁殖障碍症状， 但不限于此。 上述症状与现有技 术中感染了普通猪伪狂犬病病毒后产生的症状差异在于： 感染了后会造 成感染后成年猪（体重在 50kg以上猪）可引起猪只高热 （40 ~ 42°C， 持 续 3日以上） ， 呼吸困难， 腹泻， 喘， 咳嗽， 打喷嚏， 后肢麻痹， 犬坐， 突然倒地， 抽搐， 侧卧不起， 角弓反张， 泳状划水， 最后衰竭而死； 新 生及 4周龄以内的仔猪突然发病， 发生大批死亡， 死亡率达 90%以上； 发 病仔猪主要表现为体温上升达 41 °C以上， 食欲废绝， 伴有明显的神经症 状和腹泻； 断奶前后仔猪主要为呼吸系统症状， 表现呼吸困难、 咳嗽、 流鼻涕等。
本文所用的术语 "预防 "指通过给予 ^^据本发明的疫苗组合物抑制猪 伪狂犬感染或推迟疾病发作的所有行为。 术语"治疗"指通过给予根据本 发明的疫苗组合物使猪伪狂犬病病毒感染引起的症状减轻或转好的所 有行为。 附图说明 图 1为 HN1201株 gC氨基酸序列与 SA215株 gC氨基酸序列同源 分析结果；
图 2为 HN1201株 gD氨基酸序列与 SA215株 gD氨基酸序列同源 分析结果。
序列表中：
序列 1为猪伪狂犬病病毒 HN1201株 gD氨基酸序列；
序列 2为猪伪狂犬病病毒 HN1201株 gB氨基酸序列；
序列 3为猪伪狂犬病病毒 HN1201株 gC氨基酸序列；
序列 4为猪伪狂犬病病毒 HN1201株 gD核苷酸序列；
序列 5为猪伪狂犬病病毒 HN1201株 gB核苷酸序列；
序列 6为猪伪狂犬病病毒 HN1201株 gC核苷酸序列。 具体实施方式
下面结合具体实施例来进一步描述本发明， 本发明的优点和特点 将会随着描述更为清楚。 但这些实施例仅是范例性的， 并不对本发明 的范围构成任何限制。 本领域技术人员应该理解的是， 在不偏离本发 明的精神和范围下可以对本发明技术方案的细节和形式进行修改或 替换， 但这些修改和替换均落入本发明的保护范围内。
本发明中的术语"头份"是指每头猪注射的疫苗量。
本发明中所述的 "TCID50，， ( 50% tissue culture infective dose )是指半 数细胞培养物感染量， 是表示病毒感染力的一种表示方式。
MEM液体培养基（液）用购自美国 Life Technologies公司的 MEM干 粉培养基按照其说明书配制。
本发明的 DMEM培养基参照 GB/T18641-2002附录 A配制方法配制。 本发明中所述的 "PBS"是指磷酸盐緩沖液（ Phosphate Buffer Saline ) 的英文缩写， 本发明中使用的是 O.OlmM的 pH7.4的 PBS， 按《分子克隆》 第三版所述配制。 实施例 1、 病毒的采集分离 从来自河南的疑似猪伪狂犬感染的样本中分离样本无菌采集猪脑 组织， 以 1： 10 (体积比）加入 MEM培养液， 研磨， 制备组织悬液， 经反复 3次冻融后， 2000r/min离心 15min， 收集上清液， 再经 0.2μηι滤 膜滤器过滤， 在 PK-15细胞上传代 37°C培养 lh，换加含 2 %犊牛血清的 MEM培养液， 37。C培养 5日。 收获含毒培养液， 经 2次冻融后， 收毒， 换加含 2 %犊牛血清的 MEM培养液。 采用猪伪狂犬病病毒 PCR检测试 剂盒 (北京世纪元亨动物防疫技术有限公司）检测猪伪狂犬病病毒， 结果 为阳性； 对分离的病毒利用利用 PCR试剂盒进行外源病毒的检测 (猪蓝 耳病病毒 RT-PCR检测试剂盒、 猪细小病毒 PCR检测试剂盒猪瘟病毒 RT-PCR检测试剂盒均为北京世纪元亨动物防疫技术有限公产品）， PCR 检测结果均为阴性， 表明毒种纯净。
将分离到的伪狂犬病毒提交保藏， 所述猪伪狂犬病病毒株为 丽 1201株（Pseudorabies vims, strain HN 1201 )， 保藏号为 CCTCC NO. V 201311 ; 保藏于中国典型培养物保藏中心； 保藏地址为湖北省武汉武 汉大学， 保藏日期为 2013年 5月 20 曰。 实施例 2、 分离病毒的遗传特性
通过基因分析来确定实施例 1 中分离的病毒的遗传特性。 利用在 PK15细胞上分离的猪伪狂犬病病毒基因组 DNA做模板， 使用表 1所示 的引物进行 PCR。 分别使用 Primer Premier 5.0来设计用于扩增 gB、 gC、 gD基因的引物序列。
以提取的基因组 DNA为模板， 制备 PCR扩增体系如下： 模板 DNA 100 g， PrimerSTAR HS DNA Polymerase (2.5υ/μ1) 0.5μ1， 2 PrimerSTAR GC Buffer 25μ1,上下游引物各 1 μ1( lOpmol/μΙ )， dNTP Mix( 2.5mM each ) 4μ1，并用蒸熘水将体积补足 50μ1。进行两步 PCR反应：在 98°C变性 10 sec, 然后在 68°C退火和延伸（按照 lkb/min计算所需时间）， 共 30个循环。 在 4°C终止 PCR反应。 通过在含有溴化乙锭的 1%的琼脂糖凝胶上进行电泳 分析所得的 PCR产物。 PCR产物进行序列测定。 用 Lasergene软件分析所 得到的序列数据。 表 1 PCR引物序列
Figure imgf000012_0001
实施例 3、 病毒的致病性试验
3.1不同日龄仔猪的致病性
(试验组）和 2头 /组 (对照组） ， 滴鼻接种猪伪狂犬病病毒 HN1201株 (攻毒剂量为 2x l08OTCID5o/头） ， 对照组接种 DMEM培养基； 同时将 4头 49 日龄仔猪接种保藏后培养 3代的强毒 HN1201株（攻毒剂量为 2x l08 GTCID5。/头） ， 仍以 35 日龄仔猪作为对照。 病毒接种后， 每日测 定仔猪体温， 观察临床症状和死亡情况， 具体结果见表 2。 猪伪狂犬强毒 HN1201株对不同日龄仔猪的致病性 组 编
曰龄 接种剂量 临床症状 死亡情况
别 号
A1 体温升高， 攻毒后 4天死亡
A2 精神沉郁， 攻毒后 5天死亡
A3 35 曰 2x l08 0TC 食量降低， 攻毒后 5天死亡
1
龄 ID50/头 出现呼吸道
A4 和 /或神经系 攻毒后 6天死亡 统症状
2 B1 49 曰 2x l08 0TC 体温升高， 攻毒后 7天死亡
B2 龄 ID50/头 精神沉郁， 攻毒后 7天死亡 B3 食量降低， 攻毒后 5天死亡
出现呼吸道
B4 和 /或神经系 耐过存活
统症状
CI 35曰 DMEM对 存活
3 正常
C2 龄 昭、、、 存活
结果显示， 猪伪狂犬病病毒 HN1201株接种不同日龄的仔猪均可导 致仔猪发病， 且可导致 3/4以上的仔猪死亡。
3.2不同剂量对仔猪的致病性
将 49 日龄猪伪狂犬抗体阴性仔猪 8头随机分成 2组， 每组 4头， 另取 2 头仔猪作为对照。 实验组分别滴鼻接种 2x l07 GTCID5。/头和 2x l08 GTCID5。/头猪伪狂犬病病毒 HN1201株， 对照组接种 DMEM培养 基。 病毒接种后， 每日测定仔猪体温， 观察临床症状和死亡情况， 具体 结果见表 3。
不同剂量猪伪狂犬病病毒 HN1201株对仔猪的致病性
Figure imgf000013_0001
结果显示， 猪伪狂犬病病毒临床分离 HN1201 以不同剂量接种 49 日龄的仔猪， 均可导致仔猪发病， 且 4/4的仔猪死亡。 实施例 4、 猪伪狂犬病病毒病灭活苗的制备
将按照表 4将所分离的毒株的不同代次的培养物接种于 PK-15细胞 培养物先形成种子批， 然后按病毒培养液量的 1 % ( V/V)接入形成单层 的 PK细胞培养物中， 置 37°C旋转培养， 当病变达到 80 %时， 收获含毒 细胞培养液， 经 2次冻融后， 收毒， 测定毒价。 分别向不同代次病毒液 中加入 10% ( v/v)曱醛溶液使曱醛的终浓度为 0.2 % (V/ V)， 37°C灭活 I8 小时， 每 4小时搅拌 1次， 每次搅拌 10min， 灭活后病毒液用 PH7.4的 PBS液稀释至表 4所示的病毒含量然后与 206佐剂 （法国 SEPPIC公司 产品）按照体积比 54:46混合， 在 30°C条件下 120转 /分钟搅拌 15分钟。 各组别猪伪狂犬疫苗的制备
Figure imgf000014_0001
实施例 5、 灭活疫苗免疫原性实验
将 21 日龄 PRV抗体阴性仔猪 16头随机分成 4组， 4头 /组，按照表 4 注射疫苗灭活苗组注射实施例 4 制备的疫苗免疫猪伪狂犬灭活疫苗 2ml/头，对照疫苗采用 CN101186902方法制备的猪伪狂犬活疫苗 SA215 株， 按照其专利说明书免疫原性测定方法的使用， 对照组接种 DMEM 培养基 2ml/头。 免疫后 28 日后攻毒， 攻毒剂量为 HN1201株猪伪狂犬 病病毒 2x l08 GTCID5。/头， 攻毒后每日测定仔猪体温，观察临床症状和死 亡情况（结果见表 5 ) 免疫原性试验动物分组
注射疫苗 免疫剂量 灭活苗 A 实施例 4制备的疫苗组别 A 2ml/头 灭活苗 B 实施例 4制备的疫苗组别 B 2ml/头
活疫苗 SA215 猪伪狂犬病病毒活疫苗 106 0TCID50/头 对照组 DMEM培养基 2ml/头 疫苗免疫后， 每周参照 GB/T 18641-2002方法血清中和试验的方法 测定灭活疫苗组的中和抗体效价， 结果见表 6。
Figure imgf000015_0001
Figure imgf000015_0002
表 6的结果显示， 猪伪狂犬灭活疫苗免疫仔猪后， 能产生较高的中 和抗体， 且随免疫时间逐渐升高。
免疫后 28日后攻毒，攻毒剂量为猪伪狂犬病病毒 HN1201株猪伪狂 犬病病毒 2x l08 GTCID5。/头， 观察临床症状和死亡情况见表 7， 攻毒后每 日测定仔猪体温见表 8。 猪伪狂犬灭活疫苗免疫仔猪后的攻毒情况
组别 临床症状及死亡情况 保护率
体温升高 2-3天， 食欲正常，
灭活苗 A 100% ( 4/4 )
基本没有精神症状， 存活
体温升高 2-3天， 食欲正常，
灭活苗 B 100% ( 4/4 )
基本没有精神症状， 存活
体温升高 7-10天，食欲降低，
活疫苗 100% ( 4/4 )
有明显的精神症状， 存活 有明显的症状， 攻毒后 2天
对照组 100% (0/4)
死 2头， 3天全部死亡 表 8 猪伪狂犬疫苗免疫的仔猪攻毒后仔猪体温变化
Figure imgf000016_0001
表 7和表 8的结果显示， 猪伪狂犬灭活疫苗免疫仔猪后， 虽然不能 阻断病毒感染 （出现临床症状） ， 但能为仔猪提供 100% (4/4)保护， 而对照仔猪攻毒后 4日后全部死亡， 因此， 猪伪狂犬灭活疫苗具有艮好 的保护力。 另外相对于对照组活疫苗， 灭活疫苗免疫组的仔猪体温升高 时间更短， 食欲基本正常， 并且基本没有临床症状， 显示出很好的免疫 保护。 实施例 6、 猪伪狂犬 gD蛋白的制备
l.PRVgD基因的扩增 在生长良好的 PK15细胞上接种 PRV HN1201病毒或其不同代次的 培养物 （猪伪狂犬病病毒株为 HN1201 株（ Pseudorabies virus , strain HN1201 ) , 保藏号为 CCTCC NO. V 201311 ; 保藏于中国典型培养物保 藏中心；保藏地址为中国武汉.武汉大学，保藏曰期为 2013年 5月 20曰）， 该不同代次的培养物为 5-35代以内的培养物， 收获病毒后用 TAKARA 公司 MiniBEST Viral RNA/ DNA Extraction Kit Ver.3.0 试剂盒提取 PRV 基因组 DNA。 取 Ι μΐ基因组 DNA作为模板， 利用 gD特异性引物： gDSF: 5' ATGCTGCTCGCAGCGCTATTGGC 3'和
gDSR: 5' CTACGGACCGGGCTGCGCTTTTAG3'
进行 PCR扩增， 利用 TAKARA 的高保真酶 Prime STAR® HS DNA Polymerase with GC Buffer, 扩增条件为： 94°C 3min; 94 °C 30 s, 68 °C 90 s，30 cycles; 72 °C 5min。 PCR产物命名为 gD。其核苷酸序列见 SEQN0.4 , 推导其氨基酸序列为 SEQNO. L
2. 重组 Bacmid的获取及鉴定
将高保真酶扩增获得的 PCR产物 gD克隆至 pFastBac/HBM-TOPO 载体（购自 Invitrogen公司， 货号 A1 1339 ) ， 克隆体系如下： PCR产物 gD 4 l， Salt solution (盐溶液） Ι μΐ, TOPO vector Ι μΐ, 共 6μ1。 混合均 匀， 室温孵育 5min， 转化 One ShotR MachlTMTlR感受态细胞， 涂布氨 苄青霉素抗性平板， 挑取单克隆鉴定 gD基因的插入方向， 插入方向正 确的质粒送 Invitrogen公司测序， 鉴定 gD序列的正确性。 测序正确的质 粒命名为 pFastBac/HBM-TOPO-gD。
pFastBac/HBM-TOPO-gD质粒转化 DHlOBac感受态细胞 (来源）， pFastBac/HBM-TOPO-gD和感受态细胞中的穿梭质粒 Bacmid进行转座， 重组质粒，并用 pUCM13 Forward/pUCM13 Reverse引物鉴定 gD的插入， 阳性 Bacmid命名为 Bacmid-gD。
3. 转染获得重组杆状病毒
按照 Invitrogen公司 Bac-to-Bac HBM TOPO Secreted Expression System的说明书提供的方法进行。 6孔板每孔铺 8χ 105 个 sf 细胞， 待 细胞贴壁后按照 Cellfectin ll转染试剂的说明书进行转染： 分别稀释 8μ1 Cellfectin II和 Bacmid-gD DNA到 100 ul SF-900 II培养基中， Vortex混匀，混合稀释后的 DNA与稀释后的 Cellfectin II (总体积〜 210μ1) 混合均匀室温孵育 15〜30min， 一滴滴加到细胞中。 转染后 72h待出现细 胞病变后， 收集细胞培养上清， 记为 P0代重组病毒 vBac-gD。 P0代重 组病毒 vBac-gD感染 sf 细胞，经 3代扩大培养后，获得的 P3代 vBac-gD 用于重组蛋白表达。
4. 重组杆状病毒感染 High-five细胞获得重组蛋白
将 P3代重组杆状病毒 vBac-gD接种 High-five细胞（购自 Invitrogen, 货号 B85502 ) 。 在 500ml三角瓶中悬浮培养 High-five细胞， 至细胞密 度达到 7.0χ 105 cell/ml后， 按照 1 MOI的量接种病毒， 感染后 72h收取 细胞培养上清。利用 Millipore的切向流过滤系统将体积浓缩为原来体积 的 1/10。 用 1% (体积比）的 Triton X-100 (购自 sigma )灭活杆状病毒， SDS-PAGE光密度法测定蛋白含量为 20(^g/ml。 实施例 7、 猪伪狂犬给 gD亚单位疫苗的制备
将实施例 6制备的亚单位抗原， 用 PBS液（ pH 7.4 )稀释至表 9的 体积与 206佐剂 （法国 SEPPIC公司产品）按照体积比 54:46混合， 在 30°C条件下 120转 /分钟搅拌 15分钟。
表 9 各组别猪伪狂亚单位疫苗的制备
Figure imgf000018_0001
实施例 8、 疫苗对猪的免疫原性实验
将 21日龄 PRV抗体阴性仔猪 12头随机分成 3组， 4头 /组， 按照表 2注 射实施例 2制备的疫苗， 免疫猪伪狂犬病病毒亚单位疫苗 2ml/头。对照组 接种 DMEM培养基 2ml/头。 免疫后 28日后攻毒， 攻毒剂量为猪伪狂犬病 病毒 HN1201株 2x l08 GTCID5。/头， 攻毒后每日测定仔猪体温， 观察临床 症状和死亡情况（结果见表 10 ) 。
免疫原性试验动物分组
Figure imgf000019_0001
免疫后 28 日后攻毒， 攻毒剂量为猪伪狂犬病病毒 HN1201 株 2x l08 GTCID5。/头，观察临床症状和死亡情况见表 11， 攻毒后每日测定仔 猪体温见表 12。 猪伪狂犬病病毒亚单位疫苗免疫仔猪后的攻毒情况
Figure imgf000019_0002
表 12猪伪狂犬病病毒亚单位疫苗免疫的仔猪攻毒后仔猪体温变化
Figure imgf000019_0003
攻毒后 8天 39.5 39.4
攻毒后 9天 39.5 39.6
攻毒后 10天 39.2 39.3
攻毒后 11天 39.3 39.4
攻毒后 12天 39.4 39.4
攻毒后 13天 39.2 39.4
攻毒后 14天 39.2 39.1
表 11和表 12的结果显示，猪伪狂犬病病毒亚单位疫苗免疫仔猪后， 虽然不能阻断病毒感染（出现临床症状）， 但能为仔猪提供 100% ( 4/4 ) 保护， 而对照仔猪攻毒后 4日后全部死亡， 显示出很好的免疫保护。 以上所述仅为本发明的较佳实施例而已， 并不用以限制本发明， 凡在本发明的精神和原则之内， 所作的任何修改、等同替换、 改进等， 均应包含在本发明的保护范围之内。

Claims

权 利 要 求 书
1、 一种核苷酸序列， 所述核苷酸序列实质上编码序列表 SEQ ID NO. l所示的蛋白。
2、 一种核苷酸序列， 所述核苷酸序列实质上编码序列表 SEQ ID NO.2所示的蛋白。
3、 一种核苷酸序列， 所述核苷酸序列实质上编码序列表 SEQ ID NO.3所示的蛋白。
4、 一种猪伪狂犬病病毒株， 其中， 所述猪伪狂犬病病毒株具有序 列表 SEQ ID NO.4所示的核苷酸序列编码的 gD糖蛋白。
5、 根据权利要求 4所述的猪伪狂犬病病毒株， 其中， 所述猪伪狂 犬病病毒株具有序列表 SEQ ID NO.5所示的核苷酸序列编码的 gB糖蛋 白。
6、根据权利要求 5所述的猪伪狂犬病病毒株， 其中， 所述猪伪狂犬 病病毒株为 HN1201株或其培养物， 保藏号为 CCTCC NO. V 201311 ; 保藏于中国典型培养物保藏中心； 保藏地址为湖北省武汉 ·武汉大学， 保藏日期为 2013年 5月 20曰。
7、 一种疫苗组合物， 所述疫苗组合物包括免疫量的权利要求 4〜6 任一项所述的猪伪狂犬病病毒株的减毒活疫苗、灭活疫苗、亚单位疫苗、 合成疫苗或基因工程疫苗； 优选地， 所述疫苗组合物包括免疫量的所述 猪伪狂犬病病毒株 HN1201株或其培养物的减毒活疫苗、 灭活疫苗、 亚 单位疫苗、 合成疫苗或基因工程疫苗。
8、一种疫苗组合物， 所述疫苗组合物包括免疫量的权利要求 5所述 的猪伪狂犬病病毒株的减毒活疫苗、 灭活疫苗、 亚单位疫苗、 合成疫苗 或基因工程疫苗； 优选地， 所述疫苗组合物包括免疫量的所述猪伪狂犬 病病毒株 HN1201株或其培养物的减毒活疫苗、灭活疫苗、亚单位疫苗、 合成疫苗或基因工程疫苗。
9、根据权利要求 8所述的疫苗组合物， 其中， 所述疫苗组合物包括 > 106 0TCID50/ml的猪伪狂犬病病毒株 HN1201株或其培养物灭活疫苗。
10、 根据权利要求 8所述的疫苗组合物， 其中， 所述疫苗组合物包 括猪伪狂犬病病毒株 HN1201株或其培养物的 25〜10(^g/剂量 gD蛋白抗 原。
11、 根据权利要求 7〜10任一项所述的疫苗组合物， 其中， 所述疫 苗组合物进一步包括介质、 佐剂、 赋形剂。
12、 一种制备权利要求 8所述疫苗组合物的方法， 所述制备方法包 括增殖培养所述猪伪狂犬病病毒株 HN1201株或其培养物， 灭活， 加入 佐剂， 搅拌均匀。
13、 一种制备权利要求 8所述疫苗组合物的方法， 所述方法包括： ( 1 )克隆所述猪伪狂犬病病毒 gD蛋白重组基因；
( 2 )表达所述猪伪狂犬病病毒 gD重组蛋白；
( 3 )将所述猪伪狂犬病病毒 gD蛋白抗原与佐剂按比例混合，乳化。
14、根据权利要求 7〜10任一项所述的疫苗组合物在制备预防和治疗 猪伪狂犬病病毒相关疾病的药物中的应用。
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CN106267182A (zh) * 2015-06-29 2017-01-04 普莱柯生物工程股份有限公司 一种猪伪狂犬病病毒亚单位疫苗的制备方法及疫苗组合物和应用
CN106267182B (zh) * 2015-06-29 2020-02-07 普莱柯生物工程股份有限公司 一种猪伪狂犬病病毒亚单位疫苗的制备方法及疫苗组合物和应用
CN113151190A (zh) * 2021-01-26 2021-07-23 铜仁职业技术学院 一种猪伪狂犬病病毒强毒株
CN113151190B (zh) * 2021-01-26 2023-10-31 铜仁职业技术学院 一种猪伪狂犬病病毒强毒株
CN119823234A (zh) * 2024-11-12 2025-04-15 天康制药股份有限公司 一种猪伪狂犬病毒抗原及其制备方法和应用
CN119823234B (zh) * 2024-11-12 2026-04-24 天康制药股份有限公司 一种猪伪狂犬病毒抗原及其制备方法和应用

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