CN108704128B

CN108704128B - Canine distemper parvovirus bigeminal subunit vaccine

Info

Publication number: CN108704128B
Application number: CN201810463652.7A
Authority: CN
Inventors: 单虎; 张洪亮; 盖春云; 秦志华; 张传美; 宋晓明; 周保琨; 孙强; 孙有德; 朱丰龙
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2018-05-15
Filing date: 2018-05-15
Publication date: 2022-06-21
Anticipated expiration: 2038-05-15
Also published as: CN108704128A

Abstract

The invention provides a canine distemper parvovirus bigeminy subunit vaccine which comprises a vaccine adjuvant and an antigen, wherein the antigen is H protein and vp2 protein of which the amino acid sequences are SEQ ID NO. 2. The canine distemper parvovirus bivalent subunit vaccine has stable biological characteristics, good immunogenicity, safety and reliability, has a very good protection effect on virus attack of a canine distemper virulent GN strain and virus attack of a canine parvovirus QN strain, can effectively prevent the prevalence and spread of canine distemper and parvovirus, reduces economic loss caused by the two diseases, and has a wide application prospect.

Description

Canine distemper parvovirus bigeminal subunit vaccine

Technical Field

The invention belongs to the technical field of veterinary biological product preparation, and particularly relates to a preparation method and application of a canine distemper parvovirus bivalent subunit vaccine.

Background

With the better physical life of people, the pet breeding is increased, particularly, the number of the pets, such as dogs and cats, is increased sharply as companion animals, and therefore, the probability of mutual contact among the pets is increased inadvertently. CDV (Canine distemper virus, CDV) and CPV (Canine parovus, CPV) are used as infectious disease pathogens with the highest morbidity and mortality in dogs, have great harm to dogs, and have continuously increased pathogenicity after decades of continuous evolution.

Canine Distemper (CD) is an acute, febrile, highly contagious disease of canines, ferrets and some raccoons caused by the Canine Distemper Virus (CDV) of the paramyxoviridae family. It is mainly characterized by bipolar fever, inflammation of mucous membrane such as nose, digestive tract and the like, catarrhal pneumonia, skin eczema and nerve symptoms. Canine distemper virus nucleic acid has been reported to be detected in tissues of patients suffering from Pagets disease. The host range of canine distemper is continuously expanded, and the infection spectrum is increasingly increased. In recent years, with the great increase of the feeding amount of military dogs, police dogs, experimental dogs and pet dogs and the increase of allopatric communication in China, the incidence rate and the fatality rate of canine distemper in dogs in China tend to increase, and the clinical manifestations are different from the prior art. The disease is one of the most harmful epidemic diseases in the canine raising industry, fur animal breeding industry and wild animal protection industry in China at present, so the disease is widely regarded.

Canine Parvovirus (CPV) is the causative agent of Canine or other carnivora parvovirus diseases (Canine parvovirus infection). Since the first discovery in 1978, the Canine Distemper Virus (CDV) rapidly becomes two most important pathogens of dogs and often causes severe hemorrhagic gastroenteritis, vomiting, dehydration and the like of dogs. CPV has strong capability of defending external interference because the surface of the virus is not covered by a capsular membrane. It can tolerate low temperature of zero and is not sensitive to common disinfectants, sick dogs can release a large amount of virus particles to the surrounding environment, and the average infection dose of the dogs is 1000 virus particles for non-immunized dogs. The sick dog had approximately 350 million virus particles per ounce of feces, which was 35000 times the average infectious dose. Canine parvovirus is a common, acute, virulent, contact infectious disease that is prevalent among all canine species and poses a significant threat to canine health. Clinically, hemorrhagic enteritis, severe vomiting, total leukocyte decrease, dehydration and body temperature increase are mainly taken as main reasons.

The canine distemper has no specific medicine for treatment, so the early prevention and diagnosis are particularly important. The CDV inactivated vaccine has the defects of rapid reduction of the generated antibody, poor antigenicity, short time for inducing humoral immunity and the like. With the wide use of attenuated vaccines, a plurality of defects are found, which can cause immunosuppression and damage to a certain degree of nervous system, in addition, because of factors such as interference of maternal antibodies, mutation of virus genes and the like, the immunized dog cannot be completely protected, and the outbreak of canine distemper in immune groups is reported in many countries. Research on new vaccines with strong immunity is imperative. Although the canine parvovirus has only a history of more than thirty years from appearance to the present, the virus has a fast evolution speed, new variations of serotypes continuously appear, and diseases caused by the virus are still not effectively controlled. At present, a commonly used commercial vaccine strain is a CPV-2 subtype, while a domestic main epidemic strain is a New CPV-2a subtype, and the difference between the epidemic strain and a vaccine strain can be one of the reasons for vaccine immunity failure. Research and development of novel veterinary vaccines mainly based on main antigen gene nucleotide are the leading direction of the century.

Disclosure of Invention

The invention aims to provide a canine distemper parvovirus bigeminy subunit vaccine as well as a preparation method and an application thereof, which have the characteristics of high safety and good immunogenicity, achieve the purpose of preventing canine distemper and canine parvovirus, improve the immune efficacy and reduce the vaccine cost. Thereby solving the problems of short maintenance time of the immune effect of the inactivated vaccine, higher cost and the like.

The canine distemper parvovirus bivalent subunit vaccine provided by the invention comprises a vaccine adjuvant and antigens, wherein the antigens are H protein and VP2 protein with amino acid sequences of SEQ ID NO. 2;

the amino acid sequence of the VP2 protein is SEQ ID NO. 4;

a gene for coding H protein, wherein one nucleotide sequence of the gene is SEQ ID NO. 1;

a gene for coding VP2 protein, one nucleotide sequence of which is SEQ ID NO. 3;

further, the H protein and the VP2 protein are expressed by baculovirus;

preferably, the concentration of the H protein and the concentration of the VP2 protein are not lower than 8 mug/mL;

the vaccine adjuvant is a composite adjuvant prepared by mixing allyl sucrose cross-linked acrylic acid polymer solution and glycerol solution.

The canine distemper parvovirus bivalent subunit vaccine has stable biological characteristics, good immunogenicity, safety and reliability, has a very good protection effect on virus attack of a canine distemper virulent GN strain and virus attack of a canine parvovirus QN strain, can effectively prevent the prevalence and spread of canine distemper and parvovirus, reduces economic loss caused by the two diseases, and has a wide application prospect.

Drawings

FIG. 1: a CDV QN-1 strain and CDV3 strain H gene sequence alignment result chart;

FIG. 2: a CDV QN-1 strain and a standard strain H gene sequence nucleotide comparison result chart;

FIG. 3: CDV QN-1 strain H gene sequence alignment result chart.

Detailed Description

The present invention will be described in detail with reference to examples.

Example 1: cloning expression of H gene of canine distemper virus

Strains, cells and plasmids required for this example were as follows:

vero cells and pFastBac1 plasmid were both stored in this laboratory; coli DH5a, available from precious bioengineering (Dalian) limited; sf9 insect cells were given by the Chinese centers for animal hygiene and epidemiology.

The H gene used by the invention is obtained by amplifying CDV QN-1 strain, wherein the QN-1 strain is a mink in a certain farm in cities in Shandong province, and is about 60 days old; the product of the non-immunized mink canine distemper vaccine has the suspected symptoms of canine distemper in a mink group, obvious secretion in eyes and nose, anorexia and nasoscope dryness, but no mink death case occurs, and the mink canine distemper vaccine is suspected to be infected by a mink canine distemper virus attenuated strain. Screening viruses of a sample obtained from the farm to finally obtain a canine distemper virus CDV QN-1 Caninesterpervirus CDV QN-1, wherein the canine distemper virus CDV QN-1 is preserved in the China center for type culture Collection at Wuhan university, Wuhan, 2018, 4 and 11 days, and the preservation number is as follows: CCTCC NO: V201814;

1. cloning of H Gene and VP2 Gene

The sequences of the primers identified in molecular biology are shown in Table 1. The primers were synthesized by the Biotech company Baisheng Beijing using ddH₂O was diluted to a final concentration of 20 pmol/. mu.L and stored at-20 ℃.

Table 1: VP2 gene and H gene primer sequence

^aThe position of the sequence in the whole genome.

The PCR amplification products of the H gene and the VP2 gene are purified and recovered by a DNA gel recovery kit according to the operation instruction. And connecting the recovered and identified target gene fragment with a pMD18-T vector according to a 10-L ligation reaction system, and performing the operation according to the kit instructions. The transformation was carried out immediately after ligation, and the procedures of the transformation experiments were referred to large experiments in molecular biology. E.z.n.a was used.^TMPlasmid Mini Kit I (Plasmid miniprep Kit type I) extracts plasmids and operates according to the Kit instructions.

PCR identification of recombinant plasmids: taking 1 mu L of recombinant plasmid as a template, and carrying out PCR amplification by using an amplification primer. After the reaction is finished, 5 mu L of PCR product is taken for gel electrophoresis detection. And carrying out PCR identification on the recombinant plasmid to obtain a target fragment with the size consistent with the expected size.

Enzyme digestion identification of the recombinant plasmid: restriction enzymes BamH I and Hind III are used for double enzyme digestion identification, and the recombinant plasmid is digested by BamH I and Hind III to obtain two fragments with the same size as expected.

3) Sequencing of H Gene and VP2 Gene

And (3) taking the recombinant plasmid with positive bacteria liquid PCR and double enzyme digestion identification, sending the recombinant plasmid to Beijing Liuhe Hua Dagen science and technology Co., Ltd for sequencing, and performing Blast comparison on a sequencing result on NCBI (national center for Biotechnology) to confirm the correctness of the sequence to be tested.

Sequencing results show that the amino acid sequence (the amino acid sequence is SEQ ID NO:2, and the nucleotide sequence of the coding gene is SEQ ID NO:1) of the H gene of the CDV QN-1 virus strain is greatly different from isolates reported at home and abroad, and has 14 mutations compared with CDV3, such as P10-A10, P18-A18, Q29-E29, Q215-E215, A269-T269, S295-L295, A436-G436, Q441-E441, A457-G457, Y525-S525, D526-V526, H540-D540, P550-S550 and P583-A583, which show the virus changes. The two mutation sites of Y525-S525 and D526-V526 occur in the key region of the host cell binding receptor, which can affect the binding of CDV to the host cell, and is probably the reason of reducing the virulence of CDV QN-1.

The amino acid sequence of the VP2 protein is SEQ ID NO. 4, and the nucleotide sequence is SEQ ID NO. 3.

2. Construction of recombinant expression vectors pFastBac1-H and pFastBac1-VP2

1) Designing 2 pairs of expression primers, introducing BamHI and XhoI enzyme cutting sites and protective bases at the 5' end of the primers, wherein the sizes of the expected fragments are 1921bp and 1755bp respectively, carrying out PCR amplification on H genes and VP2 genes, carrying out double enzyme cutting on a target fragment and an expression vector pFastBac1, connecting the gene fragment recovered from the gel and a pFastBac1 vector in ice bath, wherein the connecting system is 10 mu L, and placing the mixture at 16 ℃ overnight after uniformly mixing the connecting system. The transformation was carried out immediately after ligation, and the procedures of the transformation experiments were referred to large experiments in molecular biology. E.z.n.a was used.^TM Plasmid Mini Kit I (plasmid miniprep Kit type I) extracts plasmids, and the procedures were performed according to the Kit instructions.

2) Identification of recombinant expression vectors

Randomly picking single bacterial colony on the transformed bacteria culture plate, and carrying out recombinant bacteria identification.

After the recombinant bacteria are cultured and plasmids are extracted, the plasmids are diluted by 10 times for PCR identification. The PCR product was identified by electrophoresis on a 1% agarose gel.

The extracted recombinant plasmid is evenly mixed by double enzyme digestion of BamHI and XhoI, and then is treated by water bath at 37 ℃ for 4h, 1% agarose gel electrophoresis is carried out to observe the digestion result, and meanwhile, a blank plasmid vector is used as a negative control.

After the recombinant prokaryotic expression plasmid pFastBac1-H and pFastBac1-VP2 are subjected to BamH I and XhoI double enzyme digestion, 2 specific bands appear by 1% agarose gel electrophoresis, the sizes of the obtained bands are 4775bp and 1921bp, and 4775bp and 1755bp respectively, the sizes are consistent with the expected sizes, and the recombinant plasmid which is identified as positive is sent to Huada science and technology corporation for sequencing. And carrying out homology comparison with published sequences of GenBank, and storing plasmids which are determined to be positive by sequencing for later use.

3. Expression and characterization of pFastBac1-H and pFastBac1-VP2 in Sf9 insect cells 1) recombinant shuttle plasmids pFastBac1-H and pFastBac1-VP2 transfect Sf9 insect cells, respectively

And (4) taking insect cells with good growth state, counting the living cells, and calculating the survival rate (the survival rate of the cells is higher than 95%). 2mL of insect cells with good growth conditions were collected, and the cell count was about 2X10⁶Cells were allowed to grow adherently for at least 1h by addition to six-well plates and a negative control was set up. Two sterilized 1.5mL centrifuge tubes were prepared, and one added 100. mu.L of serum-free medium and 2. mu.L of recombinant bacmid (ca. 1-2ng) and mixed well. mu.L of serum-free medium and 8. mu.L of Cellffectin Reagent lipofectin were added to another centrifuge tube and gently mixed. Marking on two centrifuge tubes respectively, and standing at room temperature for 15-20 min. Mixing the mixed solution in the two centrifuge tubes, gently mixing, incubating at room temperature for 30min, and adding into the above mixed solutionAdding 800 mu L of preheated serum-free culture medium, uniformly mixing, then gently dripping the mixed solution one drop by one drop on Sf9 insect cells adhered to the wall in advance, wherein the process is slow, and then placing the insect cells in an incubator at 27 ℃ for 5 hours in a dark place. After 5h of incubation, the medium in the six well plates was aspirated off, preheated fresh Sf-900TM III SFM (1X) serum free medium was added, and the cells were incubated in a 27 ℃ incubator protected from light for 3-5 days until they were diseased.

After Sf9 insect cells were transfected with recombinant shuttle rods that identified the correct, the cells that were successfully transfected developed detailed cytopathic effects: cell enlargement, rounding, some cell rupture, etc. While cells transferred into the empty vector grew well.

2) Harvesting of recombinant baculovirus

After 3-5 days of cell culture, if transfection is successful, obvious cytopathic effect is seen, the cell diameter is increased, the cell nucleus is filled with the whole cell, some cells are broken, and finally, the cells are separated from the plate. At this time, the cells in the six-well plate can be blown gently, the cell suspension is collected and transferred into a sterile 10mL centrifuge tube to be stored at 4 ℃, if the cell suspension needs to be stored for a long time, the cell suspension can be placed in a refrigerator at minus 80 ℃, and the virus is P1 substitute.

3) Amplification of recombinant baculovirus

On the day of collecting P1 toxin generation, 2mL of well-grown Sf9 insect cells were collected, and the number of the cells was about 2X10⁶Inoculating the cells into a six-well cell culture plate, allowing the cells to grow for 3h in an adherent manner, setting a negative control, inoculating 30uL of P1 generation virus into the six-well cell culture plate, placing the six-well cell culture plate in a 27 ℃ cell culture box for culture for 3-5d, observing morphological change of the cells, collecting the virus according to a method for collecting P1 generation virus when the cells have cytopathic effect and fall off from the plate, wherein the baculovirus received is the P2 generation virus, and inoculating the cells according to the method to obtain the P3 generation virus with high virus titer.

4) Identification of pFastBac1-H protein expression by Western blot detection

Carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic separation on the recombinant protein, then carrying out electric transfer printing, and then carrying out Western-blot identification by using rabbit anti-CDV positive serum as a primary antibody and using HRP (horse radish virus) labeled goat anti-rabbit IgG as a secondary antibody, wherein the relative molecular mass of the recombinant protein Head Domain is about 50.8ku and is consistent with the expected size. The recombinant protein can be specifically combined with positive serum, and has good antigenicity.

5) Identification of pFastBac1-VP2 protein expression by Western blot detection

Carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic separation on the recombinant protein, then carrying out electric transfer printing, and then carrying out Western-blot identification by using rabbit anti-CPV positive serum as a primary antibody and HRP (horse radish peroxidase) labeled goat anti-rabbit IgG as a secondary antibody, wherein the relative molecular mass of the recombinant protein VP2 is about 64.5ku and is consistent with the expected size. Shows that the recombinant protein can be specifically combined with positive serum and has good antigenicity

4. Viral titer assay

Performing recombinant baculovirus titer detection on the original virus seeds, performing titer calculation by adopting an indirect Immunofluorescence (IFA) method and a Reed-Muench method, wherein the virus titer is not less than 2 multiplied by 10⁷IFU/ml。

Example 3: laboratory trial production of canine distemper virus H gene and canine parvoVP 2 subunit vaccine and product quality research

Viruses and cells

The virus seeds for preparing the product are CDV and CPV recombinant baculovirus, which are constructed, stored and supplied by Qingdao agricultural university, and the virus titer is more than or equal to 10⁷IFU/ml; sf9 production cell generation 6, identified, stored and supplied by Qingdao agricultural university.

1. Virus inoculation and culture

Inoculating the virus seeds with 0.01% of inoculum size to reach the cell density of 1 × 10⁶cells/ml Sf9 cells, at 27 ℃ for 72 hours, harvest virus supernatant, 12000rpm centrifugal 20 minutes, collect the supernatant, is production virus, at-20 ℃ for storage.

When the Sf9 cell density in the bioreactor reaches 1X 10⁶Inoculating 1% of the virus seeds for production at cell/ml, culturing at 27 deg.C for 120 hr, rotating at 80rpm, dissolved oxygen value of 60%, and pH value of 6.2. After 120 hours of culture, the virus culture fluid was harvested and labeled with product name, preparation date and quantity. Disease of harvestAnd preserving the venom at 2-8 ℃. Preparing a canine parvovirus semi-finished product according to a phase synchronization step, sampling the harvested virus liquid, and detecting the virus titer by indirect Immunofluorescence (IFA).

TABLE 2 determination of the virus content in production

2. Vaccine formulation

1) Filtering, namely filtering the harvested virus liquid by a 1.0-micron filter membrane system and a 0.22-micron filter membrane system in sequence, subpackaging the filtrate in sterile containers, indicating the harvesting date, and storing at 2-8 ℃.

2) Inactivating, namely adding 0.2M of inactivating agent diethylene imine (BEI) into the filtered virus solution according to a certain proportion, continuously stirring and inactivating for 72 hours at 37 ℃, adding 5mM of sodium thiosulfate to neutralize redundant BEI after the reaction is ended, and storing at 2-8 ℃.

3) Inactivated virus fluid test

Inactivation test inactivated virus solution is added into Sf9 cells, blind transmission is carried out after culture for 72 hours at 27 ℃, 3 continuous blind transmission generations are carried out, and indirect Immunofluorescence (IFA) detection is carried out on each generation. The passage products of each generation of inactivated virus liquid have no green specific fluorescence, and the positive control should have green specific fluorescence.

The sterility test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the growth is carried out aseptically.

And (3) detecting the content of the target protein by taking the inactivated virus liquid, wherein the content of the target protein per milliliter is more than or equal to 16 mu g/ml.

4) The main component of the compound adjuvant solution prepared by the compound adjuvant is prepared by mixing allyl sucrose cross-linked acrylic acid polymer solution A and glycerol solution B according to the proportion of 5:1, and finally adding a proper amount of sterile purified water according to the dosage of the adjuvant and uniformly mixing.

5) Vaccine preparation

The vaccine preparation scheme mainly comprises canine distemper H protein recombinant baculovirus inactivated liquid, canine parvoVP 2 protein recombinant baculovirus inactivated liquid, compound adjuvant component solution A and solution B. The vaccine is prepared according to the final concentration of each milliliter of vaccine, the content of canine distemper H protein and the content of canine fine VP2 protein are not less than 8ug, and the antigen amount and the adjuvant are prepared according to the proportion of 1: 1.

In the vaccine preparation process, the qualified inactivated antigen solution is adjusted to have the target protein content of more than or equal to 16ug/ml and is placed in an aseptic vaccine preparation tank, a stirrer is started to stir at a low speed, the composite adjuvant solution is weighed according to the proportion of 1:1 and is slowly added into the vaccine preparation tank, and the mixture is stirred at 200rpm for 20 minutes and is uniformly mixed.

6) Dispensing

After the vaccines are fully mixed, quantitatively subpackaging 20 ml/bottle, covering and sealing, and labeling.

3. Inspection of finished product

1) Character the vaccine is sampled to observe the color of the vaccine under the condition of sufficient natural light at room temperature, and the color is colorless or yellowish suspension.

2) The loading inspection is carried out according to the appendix of the current Chinese animal pharmacopoeia and is in line with the regulations.

3) The sterility test is carried out according to the appendix of the traditional Chinese veterinary pharmacopoeia, and the bacteria can grow aseptically.

4)20 healthy and susceptible beagle dogs (canine distemper and canine parvoneutralizing antibody are not higher than 1:4) are taken for safety inspection and randomly divided into 4 groups. Groups 1-3 were vaccine immunization groups, 5 per group, 4 control groups, and 5. 1 subcutaneous 5-point injection of H gene of canine distemper virus and canine fine VP2 subunit vaccine (pFastBac1-H + pFastBac1-VP2 strain), 1 head (1ml) per injection, 2 subcutaneous 10-point injection of H gene of canine distemper virus and canine fine VP2 subunit vaccine (pFastBac1-H + pFastBac1-VP2 strain), 2 head (1ml) per injection, 3 subcutaneous 15-point injection of H gene of canine distemper virus and canine fine VP2 subunit vaccine (pFastBac1-H + pFastBac1-VP2 strain), 5 head (1ml) per injection, 14 days after immunization for test dogs and control dogs, observation and recording of body temperature, mental state, appetite, feces change condition and local adverse reaction, weighing test initial day, test body weight on the last day, evaluating growth condition of dogs, and testing dogs, each group was dissected and killed 3 dogs, and injection sites were examined for pathological changes. In an observation period, dogs in an immune group and a control group of three groups of canine distemper virus H genes and a canine tiny VP2 subunit vaccine (pFastBac1-H + pFastBac1-VP2 strain) are normal in body temperature, mental state, appetite and excrement, adverse reactions such as swelling and inflammation do not exist in the injection part, abnormal changes do not occur in the injection part autopsy examination, and the difference between the growth condition of the vaccinated group dogs and the growth condition of the control group dogs is not obvious.

5) Efficacy test

30 healthy susceptible dogs (canine distemper and canine parvoneutralizing antibody are not higher than 1:4) are randomly divided into 3 groups. Group 1 was the vaccine immunization group, 10 were immunized against homemade bivalent subunit vaccine, and the canine distemper virus H gene and canine parvoVP 2 subunit vaccine (pFastBac1-H + pFastBac1-VP2 strain) were injected subcutaneously at 5 spots, with 1 head (1ml) per injection. The 2 groups are canine distemper-parvo bivalent vaccine immune groups, 10, 3 control groups, and 10 in each group. Blood is collected 21 days after immunization, and the titer of the canine distemper and canine parvoneutralizing antibody in serum is determined. Canine distemper antibody titers were determined by the neutralization assay, and canine parvoantibody titers were determined by the hemagglutination/hemagglutination inhibition assay. The results show that the neutralizing antibody titer of the canine parvovirus H gene and canine parvoVP 2 subunit vaccine (pFastBac1-H + pFastBac1-VP2 strain) group is higher than that of the control vaccine group. After the blood collection was completed, 5 random samples of each blood sample were collected and administered in 1ml (containing 100 IDs)₅₀) The test dogs and the control dogs were observed and recorded for 21 days after challenge with the GN strain (organ virus) of canine distemper virus. Each group was divided into another 5 individuals each with 1ml (containing 100 IDs)₅₀) The dog parvovirus QN strain of the test dog and the control dog are continuously observed for 21 days, and the body temperature change and clinical symptoms of the test dog and the control dog are observed and recorded.

TABLE 3 antibody test results and challenge protection test results

The results show that three batches of canine distemper virus H genes and canine parvoVP 2 subunit vaccines (pFastBac1-H + pFastBac1-VP2 strains) inoculated in test dogs have 5/5 protection against the canine distemper virus GN strain (visceral virus) and the canine parvovirus QN strain, and the control group has 5/5 diseases. The specific results are shown in Table 3.

Sequence listing

<110> Qingdao agricultural university

<120> canine distemper parvovirus bivalent subunit vaccine

<160> 4

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<210> 2

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Met Leu Ser Tyr Gln Asp Lys Val Gly Pro Phe Tyr Lys Asp Asn Ala

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Lys Val Lys Val Asn Phe Thr Asn Tyr Cys Glu Thr Ile Gly Ile Arg

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Tyr His Asp Ser Arg Gly Ser Gln Asp Gly Ile Leu Val Val Thr Leu

305 310 315 320

Gly Ile Phe Gly Ala Thr Pro Met Asp His Ile Glu Glu Val Ile Pro

325 330 335

Val Ala His Pro Ser Met Glu Lys Ile His Ile Thr Asn His Arg Gly

340 345 350

Phe Ile Lys Asp Ser Ile Ala Thr Trp Met Val Pro Ala Leu Ala Ser

355 360 365

Glu Lys Gln Glu Glu Gln Lys Gly Trp Leu Glu Ser Ala Cys Gln Arg

370 375 380

Lys Thr Tyr Pro Met Cys Asn Gln Thr Ser Trp Glu Pro Phe Gly Gly

385 390 395 400

Gly Gln Leu Pro Ser Tyr Gly Arg Leu Thr Leu Pro Leu Asp Ala Ser

405 410 415

Val Asp Leu Gln Leu Asn Ile Ser Phe Thr Tyr Gly Pro Val Ile Leu

420 425 430

Asn Gly Asp Ala Met Asp Tyr Tyr Gln Ser Pro Leu Leu Asn Ser Gly

435 440 445

Trp Leu Thr Ile Pro Pro Lys Asn Ala Thr Ile Leu Gly Leu Ile Asn

450 455 460

Lys Ala Ser Arg Gly Asp Gln Phe Thr Val Ile Pro Gln Val Leu Thr

465 470 475 480

Phe Ala Pro Arg Glu Ser Cys Gly Asn Cys Tyr Leu Pro Ile Gln Thr

485 490 495

Ser Gln Ile Ile Asp Arg Asp Val Leu Ile Glu Ser Asn Val Val Val

500 505 510

Leu Pro Thr Gln Ser Phe Arg Tyr Val Ile Ala Thr Ser Val Ile Ser

515 520 525

Arg Asn Asp His Ala Ile Val Tyr Tyr Val Tyr His Pro Ile Arg Thr

530 535 540

Ile Ser Tyr Thr His Pro Phe Arg Leu Thr Thr Lys Gly Arg Pro Asp

545 550 555 560

Phe Leu Arg Ile Glu Cys Phe Val Trp Asp Asp Asn Leu Trp Cys His

565 570 575

Gln Phe Tyr Arg Tyr Glu Pro Asn Ile Ala Asn Ser Thr Thr Ser Val

580 585 590

Glu Asn Leu Val Arg Ile Arg Phe Ser Cys Asn Arg Ser Asn Pro

595 600 605

<210> 3

<211> 1755

<212> DNA

<213> Canine distemper virus (canine distemper virus)

<400> 3

atgagtgatg gagcagttca accagacggt ggtcagcctg ctgtcagaaa tgaaagagct 60

acaggatctg ggaacgggtc tggaggcggg ggtggtggtg gttctggggg tgtggggatt 120

tctacgggta ctttcaataa tcagacggaa tttaaatttt tggaaaacgg atgggtggaa 180

atcacagcaa actcaagcag acttgtacat ttaaatatgc cagaaagtga aaattataga 240

agagtggttg taaataattt ggataaaact gcagttaacg gaaacatggc tttagatgat 300

acccatgcac aaattgtaac accttggtca ttggttgatg caaatgcttg gggagtttgg 360

tttaatccag gagattggca actaattgtt aatactatga gtgagttgca cttagttagt 420

tttgaacaag aaatttttaa tgttgtttta aagactgttt cagaatctgc tactcagcca 480

ccaactaaag tttataataa tgatttaact gcatcattga tggttgcatt agatagtaat 540

aatactatgc catttactcc agcagctatg agatctgaga cattgggttt ttatccatgg 600

aaaccaacca taccaactcc atggagatat tattttcaat gggatagaac attaatacca 660

tctcatactg gaactagtgg cacaccaaca aatatatacc atggtacaga tccagatgat 720

gttcaatttt atactattga aaattctgtg ccagtacact tactaagaac aggcgatgaa 780

tttgctacag gaacattttt ttttgattgt aaaccatgca gactaacaca tacatggcaa 840

acaaatagag cattgggctt accaccattt ctaaattctt tgcctcaagc tgaaggaggt 900

actaactttg gttatatagg agttcaacaa gataaaaggc gtggtgtaac tcaaatggga 960

aatacaaaca ttattactga agctactatt atgagaccag ctgaggttgg ttatagtgca 1020

ccatattatt cttttgaggc gtctacacaa gggccattta aaacacctat tgcagcagga 1080

cgggggggag cgcaaacaga tgaaaatcaa gcagcagatg gtgatccaag atatgcattt 1140

ggtagacaac atggtcagaa aactaccaca acaggagaaa cacctgagag atttacatat 1200

atagcacatc aagatacagg aagatatcca gaaggagatt ggattcaaaa tattaacttt 1260

aaccttcctg taacaaatga taatgtattg ctaccaacag atccaattgg aggtaaaaca 1320

ggaattaact atactaatat atttaatact tatggtcctt taactgcatt aaataatgta 1380

ccaccagttt atccaaatgg tcaaatttgg gataaagaat ttgatactga cttaaaacca 1440

agacttcatg taaatgcacc atttgtttgt caaaataatt gtcccggtca attatttgta 1500

aaagttgcgc ctaatttaac aaatgaatat gatcctgatg catctgctaa tatgtcaaga 1560

attgtaactt actcagattt ttggtggaaa ggtaaattag tatttaaagc taaactaaga 1620

gcctctcata cttggaatcc aattcaacaa atgagtatta atgtagataa ccaatttaac 1680

tatgtaccaa gtaacattgg aggtatgaaa attgtatatg agaaatctca actagcacct 1740

agaaaattat attaa 1755

<210> 4

<211> 584

<212> PRT

<213> Canine distemper virus (canine distemper virus)

<400> 4

Met Ser Asp Gly Ala Val Gln Pro Asp Gly Gly Gln Pro Ala Val Arg

1 5 10 15

Asn Glu Arg Ala Thr Gly Ser Gly Asn Gly Ser Gly Gly Gly Gly Gly

20 25 30

Gly Gly Ser Gly Gly Val Gly Ile Ser Thr Gly Thr Phe Asn Asn Gln

35 40 45

Thr Glu Phe Lys Phe Leu Glu Asn Gly Trp Val Glu Ile Thr Ala Asn

50 55 60

Ser Ser Arg Leu Val His Leu Asn Met Pro Glu Ser Glu Asn Tyr Arg

65 70 75 80

Arg Val Val Val Asn Asn Leu Asp Lys Thr Ala Val Asn Gly Asn Met

85 90 95

Ala Leu Asp Asp Thr His Ala Gln Ile Val Thr Pro Trp Ser Leu Val

100 105 110

Asp Ala Asn Ala Trp Gly Val Trp Phe Asn Pro Gly Asp Trp Gln Leu

115 120 125

Ile Val Asn Thr Met Ser Glu Leu His Leu Val Ser Phe Glu Gln Glu

130 135 140

Ile Phe Asn Val Val Leu Lys Thr Val Ser Glu Ser Ala Thr Gln Pro

145 150 155 160

Pro Thr Lys Val Tyr Asn Asn Asp Leu Thr Ala Ser Leu Met Val Ala

165 170 175

Leu Asp Ser Asn Asn Thr Met Pro Phe Thr Pro Ala Ala Met Arg Ser

180 185 190

Glu Thr Leu Gly Phe Tyr Pro Trp Lys Pro Thr Ile Pro Thr Pro Trp

195 200 205

Arg Tyr Tyr Phe Gln Trp Asp Arg Thr Leu Ile Pro Ser His Thr Gly

210 215 220

Thr Ser Gly Thr Pro Thr Asn Ile Tyr His Gly Thr Asp Pro Asp Asp

225 230 235 240

Val Gln Phe Tyr Thr Ile Glu Asn Ser Val Pro Val His Leu Leu Arg

245 250 255

Thr Gly Asp Glu Phe Ala Thr Gly Thr Phe Phe Phe Asp Cys Lys Pro

260 265 270

Cys Arg Leu Thr His Thr Trp Gln Thr Asn Arg Ala Leu Gly Leu Pro

275 280 285

Pro Phe Leu Asn Ser Leu Pro Gln Ala Glu Gly Gly Thr Asn Phe Gly

290 295 300

Tyr Ile Gly Val Gln Gln Asp Lys Arg Arg Gly Val Thr Gln Met Gly

305 310 315 320

Asn Thr Asn Ile Ile Thr Glu Ala Thr Ile Met Arg Pro Ala Glu Val

325 330 335

Gly Tyr Ser Ala Pro Tyr Tyr Ser Phe Glu Ala Ser Thr Gln Gly Pro

340 345 350

Phe Lys Thr Pro Ile Ala Ala Gly Arg Gly Gly Ala Gln Thr Asp Glu

355 360 365

Asn Gln Ala Ala Asp Gly Asp Pro Arg Tyr Ala Phe Gly Arg Gln His

370 375 380

Gly Gln Lys Thr Thr Thr Thr Gly Glu Thr Pro Glu Arg Phe Thr Tyr

385 390 395 400

Ile Ala His Gln Asp Thr Gly Arg Tyr Pro Glu Gly Asp Trp Ile Gln

405 410 415

Asn Ile Asn Phe Asn Leu Pro Val Thr Asn Asp Asn Val Leu Leu Pro

420 425 430

Thr Asp Pro Ile Gly Gly Lys Thr Gly Ile Asn Tyr Thr Asn Ile Phe

435 440 445

Asn Thr Tyr Gly Pro Leu Thr Ala Leu Asn Asn Val Pro Pro Val Tyr

450 455 460

Pro Asn Gly Gln Ile Trp Asp Lys Glu Phe Asp Thr Asp Leu Lys Pro

465 470 475 480

Arg Leu His Val Asn Ala Pro Phe Val Cys Gln Asn Asn Cys Pro Gly

485 490 495

Gln Leu Phe Val Lys Val Ala Pro Asn Leu Thr Asn Glu Tyr Asp Pro

500 505 510

Asp Ala Ser Ala Asn Met Ser Arg Ile Val Thr Tyr Ser Asp Phe Trp

515 520 525

Trp Lys Gly Lys Leu Val Phe Lys Ala Lys Leu Arg Ala Ser His Thr

530 535 540

Trp Asn Pro Ile Gln Gln Met Ser Ile Asn Val Asp Asn Gln Phe Asn

545 550 555 560

Tyr Val Pro Ser Asn Ile Gly Gly Met Lys Ile Val Tyr Glu Lys Ser

565 570 575

Gln Leu Ala Pro Arg Lys Leu Tyr

580

Claims

1. The canine distemper parvovirus bigeminy subunit vaccine is characterized by comprising a vaccine adjuvant and an antigen, wherein the antigen is H protein and VP2 protein of which the amino acid sequences are SEQ ID NO. 2; the amino acid sequence of the VP2 protein is SEQ ID NO. 4.

2. The bivalent subunit vaccine according to claim 1, wherein the H protein and VP2 protein are produced by baculovirus expression.

3. The bivalent subunit vaccine according to claim 1, wherein the concentration of H protein and VP2 protein is not less than 8 μ g/mL.

4. The bivalent subunit vaccine according to claim 1, wherein the vaccine adjuvant is a composite adjuvant prepared by mixing allyl sucrose cross-linked acrylic acid polymer solution and glycerol solution.