CN108704128A - A kind of canine distemper parvovirus bigeminy subunit vaccine - Google Patents

A kind of canine distemper parvovirus bigeminy subunit vaccine Download PDF

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CN108704128A
CN108704128A CN201810463652.7A CN201810463652A CN108704128A CN 108704128 A CN108704128 A CN 108704128A CN 201810463652 A CN201810463652 A CN 201810463652A CN 108704128 A CN108704128 A CN 108704128A
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CN108704128B (en
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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
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Qingdao Agricultural University
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Abstract

The present invention provides a kind of canine distemper parvovirus bigeminy subunit vaccine, includes vaccine adjuvant and antigen, and the antigen is that amino acid sequence is SEQ ID NO:2 H protein and vp2 albumen.The canine distemper parvovirus bigeminy subunit vaccine of the present invention has stable biological characteristics; and there is good immunogenicity; securely and reliably; there is extraordinary protecting effect to the canine distemper poison of attacking that GN plants of poison attacks QN plants of poison and canine parvovirus by force; prevalence and the propagation of canine distemper and parvovirus can effectively be prevented; economic loss caused by reducing both diseases, has broad application prospects.

Description

A kind of canine distemper parvovirus bigeminy subunit vaccine
Technical field
The invention belongs to veterinary biological product preparing technical fields, and in particular to a kind of canine distemper parvovirus bigeminy is sub- The preparation method and application of subunit vaccine.
Background technology
As people's material life is become better and better, raising pets increases therewith, especially dog cat as companion animals, Quantity drastically increases, and thus also inadvertently also increases the probability to contact with each other between each pet.And CDV (Canine Distemper virus, CDV) and CPV (Canine parvovirus, CPV) it is highest as incidence in dog and lethality Infectious disease pathogens, it is very harmful to canine, and by the continuous evolution of decades, pathogenic continuous increase.
Canine distemper (Canine distemper, CD) is the canine distemper virus (Canine by Paramyxoviridae Distemper virus, CDV) caused by Canidae, the acute, hot of Mustelidae and part Procyonidae animal, high degree in contact pass It catches an illness.It is mainly characterized by biphasic or bipolar type fever, the mucosal inflammations such as inflammation, nose, alimentary canal and catarrhal pneumonia, skin eczema and Nervous symptoms.Have been reported that confirmation has detected canine distemper virus nucleic acid in the tissue of patient for suffering from Pagets diseases.It can be seen that The host range of canine distemper expands constantly, and pattern of infection is increasing.In recent years, with China army dog, police dog, experiment With increasing considerably for dog and pet dog breeding amount and increasing for strange land exchange, incidence of the canine distemper in China dog and Lethality has raised trend, and clinical manifestation also with it is previous different.The disease is at present to China's canine farming, hair Skin animal farming industry and conservation of wildlife industry endanger one of maximum epidemic disease, therefore in widespread attention.
Canine parvovirus (Canine parvovirus, CPV) is to cause Canidae or other Carnivora parvovirus The cause of disease of sick (Canine parvovirus infection).From 1978 for the first time find since, quickly with canine distemper virus (Canine Distemper virus, CDV) becomes two kinds of most important cause of diseases of dog, often causes dog seriously hemorrhagic stomach The symptoms such as enteritis, vomiting and dehydration.Because virus surface is covered without cyst membrane so that CPV has very strong defence external interference Ability.It is resistant to zero degree low temperature, and insensitive to general disinfectants, and illness dog can discharge a large amount of to ambient enviroment Virion, for non-immune dog, the infective dose that is averaged is 1000 virion.Every ounce of excrement of sick dog is about 3500000 virion are 35000 times of average infective dose.Canine parvovirus disease be one kind popular between all kind of dog often See, acute strong contagious disease, the health of canine is caused greatly to threaten.Clinically mainly with hemorrhagic enteritis, Based on vomiting is acutely, total white blood cells decline, are dehydrated, body temperature increases.
Hundstaupe pyreticosis does not have the drug of special efficacy to be treated, so early prevention and diagnosis just seem increasingly important.CDV The shortcomings of time that inactivated vaccine is very fast because generated antibody declines, antigenicity is poor, induction body fluid is immune is short.With weak poison Vaccine is widely used, it has been found that there is many defects, it can cause immunosupress and nervous system to a certain extent Damage, further, since the interference of maternal antibody and factors, the immune dog such as mutation of viral gene also fail to be protected completely There are the report of immune population outburst canine distemper in shield, many countries.It is imperative to study the strong vaccine of new immunity.Dog is thin Though small virus is from appearance to current only more than 30 years history, the virus evolution speed is fast, and serotype also continuously emerges newly Variation, disease caused by the virus is not still controlled effectively.The commercialized vaccine strain being commonly used is CPV-2 hypotypes, and domestic main popular strain is New CPV-2a hypotypes, the difference of this prevalence strain and vaccine strain May be to cause one of vaccine immunity failure cause.Research and develop the novel epidemic disease for animals based on major antigen gene nucleotide Seedling is the dominant direction in this century.
Invention content
The object of the present invention is to provide a kind of canine distemper parvovirus bigeminy subunit vaccine and preparation method thereof and answer With having the characteristics that safe and good immunogenicity, achieve the purpose that prevent canine distemper and dog tiny, improve and be immunized Effect reduces vaccine cost.It holds time short, the problems such as cost is higher to solve inactivated vaccine immune effect.
Canine distemper parvovirus bigeminy subunit vaccine provided by the present invention, includes vaccine adjuvant and antigen, described Antigen be amino acid sequence be SEQ ID NO:2 H protein and vp2 albumen；
The vp2 albumen, amino acid sequence are SEQ ID NO:4；
The gene of H protein is encoded, one kind nucleotide sequence is SEQ ID NO:1；
The gene of vp2 albumen is encoded, one kind nucleotide sequence is SEQ ID NO:3；
Further, the H protein is expressed by baculoviral with VP2 albumen；
Preferably, the H protein and the concentration of VP2 albumen are not less than 8 μ g/mL；
Vaccine provided by the present invention, wherein vaccine adjuvant are allyl sucrose crosslinked acrylic acid polymer solution and third The composite adjuvant of three alcoholic solution mixed preparings.
The canine distemper parvovirus bigeminy subunit vaccine of the present invention has stable biological characteristics, and with good Immunogenicity, securely and reliably, to canine distemper by force GN plants of poison attack QN plant of poison and canine parvovirus attack poison with extraordinary Protecting effect can effectively prevent prevalence and the propagation of canine distemper and parvovirus, reduce economic damage caused by both diseases It loses, has broad application prospects.
Description of the drawings
Fig. 1：QN-1 plants of CDV and CDV3 plants of H gene sequence alignment result figures；
Fig. 2：QN-1 plants of CDV and type strain H gene consecutive nucleotides comparison result figure；
Fig. 3：QN-1 plants of H gene sequence alignment result figures of CDV.
Specific implementation mode
The present invention is described in detail with reference to embodiment.
Embodiment 1：The clonal expression of canine distemper virus H gene
The required strain of the present embodiment, bacterial strain, cell and plasmid are as follows：
Vero cells and pFastBac1 plasmids are preserved by this laboratory；E. coli DH5a, it is purchased from precious life Object engineering (Dalian) Co., Ltd；Sf9 insect cells have China Animal Health and Epidemiology Center's present.
H gene used in the present invention is obtained from QN-1 plants of amplifications of CDV, and QN-1 plants are supported in Shandong Province Zhucheng Grow the mink of field, about 60 ages in days or so；There is doubtful canine distemper symptom in not immune mink canine distemper vaccine product, mink group, Eye, nose have apparent secretion, anorexia, asoscope drying, but do not occur mink death, suspect by mink canine distemper The infection of virus attenuated strain.Viral screening is carried out to the sample obtained from the farm, is finally obtained one plant of hundstaupe pyreticosis Malicious CDV QN-1 plants of Caninedistempervirus CDV QN-1 were deposited in military positioned at China on April 11st, 2018 The China typical culture collection center of Chinese Wuhan Universitys, deposit number：CCTCC NO:V201814；
1, the clone of H gene and VP2 genes
Molecular biology identification primer sequence is shown in Table 1.Primer is synthesized by Beijing SBS Genetech bio-engineering corporation, uses ddH2O Final concentration of 20pmol/ μ L are diluted to, -20 DEG C of preservations are set.
Table 1：VP2 genes and H gene primer sequence
aPosition of the sequence in full-length genome.
H gene and VP2 gene PCRs amplified production DNA plastic recovery kits purify recycling, by operational manual progress. Target gene fragment after recycling is identified is connect according to 10 μ L coupled reaction systems with pMD18-T carriers, and kit is pressed in operation Specification carries out.It is converted at once after connection, the operating procedure of transformation experiment is with reference to molecular biology big experiment.Using E.Z.N.A.TMPlasmid Mini Kit I (small amount plasmid extraction kit I types) extract plasmid, by kit specification into Row operation.
The PCR of recombinant plasmid is identified：It takes 1 μ L of recombinant plasmid as template, PCR amplification is carried out using amplimer.Reaction After, take 5 μ L PCR products to carry out detected through gel electrophoresis.Recombinant plasmid is subjected to PCR identifications, size is obtained and is expected greatly Small consistent target fragment.
The digestion of recombinant plasmid is identified：Double digestion identification is carried out using restriction enzyme BamH I and Hind III, will be weighed Group plasmid with BamH I and III digestions of Hind obtain two segments with expection size it is consistent.
3) sequencing of H gene and VP2 genes
It is that positive recombinant plasmid send Beijing six directions Hua Da Gene science share to have to take bacterium solution PCR and double digestion to identify Limit company is sequenced, and sequencing result carries out Blast on NCBI and compares the correctness for confirming and row being sequenced.
Sequencing result show the H gene of CDV QN-1 Strain amino acid sequence (amino acid sequence be SEQ ID NO: 2, the nucleotides sequence of encoding gene is classified as SEQ ID NO:1) there is larger difference with the separation strains reported both at home and abroad, with CDV3 phases It is made a variation at 14 than having, 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, P583- A583, it is shown that viral variation.The two mutational sites wherein Y525-S525 and D526-V526 are happened at host cell knot The receptor key area of conjunction, can influence the combination of CDV and host cell, this is likely to lead to the reduction of CDV QN-1 virulence Reason.
The amino acid sequence of VP2 albumen is SEQ ID NO:4, nucleotides sequence is classified as SEQ ID NO:3.
2, the structure of recombinant expression carrier pFastBac1-H and pFastBac1-VP2
1) 2 pairs of expression primers are designed, is held in primer 5 ' and introduces BamHI and XhoI restriction enzyme sites and protectiveness base, it is contemplated that Clip size is respectively for 1921bp and 1755bp, PCR amplification H gene and VP2 genes, target fragment and expression vector The double digestion of pFastBac1 connects the genetic fragment that glue recycles with pFastBac1 carriers in ice bath, and linked system is 10 μ L after linked system mixing, place it in 16 DEG C overnight.It is converted at once after connection, the operating procedure ginseng of transformation experiment According to molecular biology big experiment.Using E.Z.N.A.TMPlasmid Mini Kit I (small amount plasmid extraction kit I types) are carried Plasmid is taken, is operated by kit specification.
2) identification of recombinant expression carrier
Random picking single bacterium colony, carries out the recombination dientification of bacteria on transformed bacteria culture plate.
After recombinant bacterium culture and extracting plasmid, plasmid makees 10 times of dilutions, carries out PCR identifications.PCR product is through 1% fine jade Sepharose electroresis appraisal.
By the recombinant plasmid of extraction, after evenly mixing with BamH I and XhoI double digestions, in 37 DEG C of water-bath 4h, 1% agar Sugared gel electrophoresis observes digestion as a result, using empty plasmid carrier as negative control simultaneously.
By recombined pronucleus expression plasmid pFastBac1-H and pFastBac1-VP2 after BamH I, XhoI double digestions, 1% There are 2 specific bands in agarose gel electrophoresis, and gained stripe size is respectively 4775bp and 1921bp, 4775bp and 1755bp is consistent with expected size, will be accredited as positive recombinant plasmid and Hua Da Gene science limited liability company is sent to be sequenced. Tetraploid rice is carried out with the sequence that GenBank has been delivered, sequencing, which is determined as positive plasmid, to be saved backup.
3,1) expression and identification of the pFastBac1-H and pFastBac1-VP2 in Sf9 insect cells recombinates shuttle bar Grain pFastBac1-H and pFastBac1-VP2 distinguishes transfection Sf 9 insect cell
The good insect cell of growth conditions is taken, carries out viable count, calculating its survival rate, (cell survival rate is higher than 95%).It is about 2 × 10 to take the good insect cell of 2mL growth conditions, cell count6It is added in six orifice plates, cell is allowed to paste Wall grows at least 1h, and sets up negative control.Prepare the 1.5mL centrifuge tubes of two sterilizings, a centrifuge tube is added 100 μ L's The restructuring rod granule (about 1-2ng) of serum free medium and 2 μ L, and by its mixing.100 μ L are added in another centrifuge tube without blood 8 μ L Cellfectin Reagent lipofectamines of clear culture medium sum, gently by its mixing.On two centrifuge tubes It marks respectively, in being stored at room temperature 15-20min.Mixed liquor in two centrifuge tubes is mixed, and is gently mixed Even, in incubation at room temperature 30min, the 800 μ L of serum free medium that preheating is then added in above-mentioned mixed liquor are uniformly mixed, so Gently the dropping on adherent Sf9 insect cells in advance drop by drop by mixed liquor afterwards, this process is had to slowly, postposition Culture 5h is protected from light in 27 DEG C of incubators.After cultivating 5h, the culture medium in six orifice plates is sopped up, the fresh Sf- of preheating is added 900TM III SFM (1 ×) serum free medium is subsequently placed in 27 DEG C of incubators and is protected from light culture 3-5d, until disease occurs in cell Until change.
After identification correctly recombination shuttle rod granule transfection Sf 9 insect cell, transfects successful cell and detail occur Cytopathy：The cell rupture etc. that cell becomes larger, is rounded, having.And the cell growth for being transferred to empty carrier is good.
2) harvest of recombinant baculovirus
After cell culture 3-5d, if transfected successfully, it can be appreciated that apparent cytopathy, cell dia will increase, carefully Karyon is full of entire cell, and some cells will appear rupture, and finally, cell is split away off from tablet.It can gently blow at this time The cell in six orifice plates is made a call to, cell suspension is collected, is transferred in sterile 10mL centrifuge tubes in 4 DEG C of preservations, if to protect for a long time It deposits, can place it in -80 DEG C of refrigerators, this poison is P1 generation poison.
3) amplification of recombinant baculovirus
The P1 generation poison same day is collected, well-grown Sf9 insect cells 2mL, cell count about 2x10 are taken6A cell, inoculation Into six porocyte culture plates, cell adherent growth 3h is allowed, and set up negative control, take P1 generation poison about 30uL to be inoculated into above-mentioned It in six porocyte culture plates, is placed in 27 DEG C of cell incubators and cultivates 3-5d, during which pay attention to the metamorphosis for observing cell, when Cells showed cytopathic and when split away off from tablet, carries out receiving poison according to the method for collecting P1 generation poison, this When the baculoviral that receives be P2 generation poison, inoculate cell according to the method, you can obtain the P3 generation poison of high virus titer.
4) Western blot detect the identification of pFastBac1-H protein expressions
Electric transfer is carried out after recombinant protein is carried out SDS-PAGE electrophoretic separation, then with rabbit-anti CDV positive serums for one Anti-, it is secondary antibody that HRP, which marks goat anti-rabbit igg, carries out Western-blot identifications, recombinant protein Head Domain average molecular matter About 50.8ku is measured, is consistent with expected size.Show that recombinant protein can be specifically bound with positive serum, has good anti- Originality.
5) Western blot detect the identification of pFastBac1-VP2 protein expressions
Electric transfer is carried out after recombinant protein is carried out SDS-PAGE electrophoretic separation, then with rabbit-anti CPV positive serums for one Anti-, it is secondary antibody that HRP, which marks goat anti-rabbit igg, carries out Western-blot identifications, recombinant protein VP2 relative molecular masses are about 64.5ku is consistent with expected size.Show that recombinant protein can be specifically bound with positive serum, there is good antigenicity
4, virus titer measures
Recombinant baculovirus titre detection is carried out to original seed culture of viruses, using indirect immunofluorescence (IFA) method, using Reed- Muench methods carry out titre calculating, and virus titer is not less than 2 × 107IFU/ml。
Embodiment 3：Canine distemper virus H gene is manufactured experimently with dog tiny VP2 subunit vaccines laboratory and product quality research
Virus and cell
The seed culture of viruses of manufacture this product is CDV and CPV recombinant baculovirus, by Qingdao Agricultural University's structure, keeping and is supplied It answers, virus titer >=107IFU/ml；The 6th generation of Sf9 Cells for production, by Qingdao Agricultural University's identification, keeping and supply.
1, virus inoculation and culture
By seed culture of viruses by 0.01% inoculum concentration inoculating cell density up to 1 × 106The Sf9 cells of cells/ml, 27 DEG C of conditions Lower continuously culture 72 hours, harvest vial supernatant, and 12000rpm is centrifuged 20 minutes, collect supernatant, and as production is malicious, and -20 It DEG C saves backup.
When in bioreactor Sf9 cell densities up to 1 × 106When cells/ml, by production with seed culture of viruses press 1% inoculation Amount connects poison, connects and continues culture 120 hours, rotating speed 80rpm, oxygen dissolving value 60%, pH value 6.2 for 27 DEG C after poison.After culture 120 hours, Virus-culturing fluid is harvested, marked product title prepares date and quantity.The virus liquid of harvest is in 2~8 DEG C of preservations.By being synchronised It is rapid to prepare the tiny recombinant virus semi-finished product of dog, the virus liquid of harvest is sampled, indirect immunofluorescence (IFA) is carried out and detects virus Titre.
The production viral disease poison assay result of table 2
2, vaccine formulation
1) virus liquid of harvest is passed through 1.0 μm of filter membrane systems and 0.22 μm of filter membrane system filtering, filtrate point by filtering successively Loaded on sterile chamber, the harvest date is indicated, 2~8 DEG C of preservations are placed in.
2) 0.2M inactivators binary ethylenimine (BEI) is added with certain proportion in filtered virus liquid for inactivation, dense eventually Degree is 5mM, and 37 DEG C continuously stir inactivation 72 hours, are added after reaction terminating in the sodium thiosulfate of final concentration of 5mM and extra BEI, preserve under the conditions of 2~8 DEG C.
3) inactivation of viruses liquid is examined
Inactivation examine takes inactivation of viruses liquid be added Sf9 cells, 27 DEG C culture 72 hours after blind passage, 3 generation of continuous blind passage, often In generation, carries out indirect immunofluorescence (IFA) and detects.The equal redgreen specificity fluorescent of product often is passed on for inactivation of viruses liquid, it is positive right Correlate the green specificity fluorescent of appearance.
Steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, and asepsis growth is answered.
Destination protein content detection takes inactivation of viruses liquid to carry out destination protein content detection, every milliliter of destination protein content ≥16μg/ml。
4) main ingredient that composite adjuvant prepares composite adjuvant solution is molten by allyl sucrose crosslinked acrylic acid polymer Liquid A and glycerin solution B presses 5:1 ratio mixing, is finally added appropriate axenic purification water mixing preparation group according to adjuvant dosage At.
5) vaccine preparation
Mainly the recombinant baculovirus of H protein containing canine distemper inactivates liquid, the tiny VP2 eggs of dog to vaccine formulation scheme vaccine composition White recombinant baculovirus inactivation liquid, composite adjuvant ingredient solution A and solution B.Vaccine formulation contains dog by every milliliter of vaccine final concentration The tiny VP2 albumen of the hot H protein of pest, dog is not less than 8ug, and amount of antigen presses 1 with adjuvant:1 ratio is prepared.
Vaccine formulation process will examine qualified inactivation antigen liquid be adjusted to destination protein content >=16ug/ml is placed in It is sterile to match in seedling tank, blender stirring at low speed is started, by 1:1 ratio weighs composite adjuvant solution and is slowly added to match in seedling tank, And be stirred 20 minutes with 200rpm, it is uniformly mixed.
6) it dispenses
After vaccine is sufficiently mixed, 20ml/ bottles of quantitative separatings seal, labeling.
3, product inspection
1) vaccine is sampled and observes vaccine color under conditions of room temperature natural light abundance by character, should be colourless or micro- Huang Color contamination suspension.
2) loading quantity inspection is by existing《Chinese veterinary pharmacopoeia》Annex is checked, regulation should be met.
3) steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, and asepsis growth is answered.
4) safety verification takes the susceptible beasle dog of health (canine distemper is not higher than 1 with the tiny neutralizing antibody of dog:4) 20, with Machine is divided into 4 groups.1~3 group be vaccine immunity group, every group 5,4 groups be control group, 5.1 group subcutaneous point 5 points of injection canine distempers Viral H gene and the tiny VP2 subunit vaccines of dog (pFastBac1-H+pFastBac1-VP2 plants), 1 part of every injection (1ml), 2 groups of subcutaneous point of 10 points of injection canine distemper virus H genes and the tiny VP2 subunit vaccines (pFastBac1-H+ of dog PFastBac1-VP2 plants), 2 parts (1ml) of every injection, 3 groups of subcutaneous point of 15 points of injection canine distemper virus H genes and dog are tiny VP2 subunit vaccines (pFastBac1-H+pFastBac1-VP2 plants), 5 parts (1ml) of every injection, to experiment after being immunized Dog and control dog are observed continuously 14, whether observe and record body temperature, the state of mind, appetite, excrement situation of change and injection site There are adverse reaction, first day on-test to test last bu also known as amount experiment dog weight, dog upgrowth situation is evaluated, in experiment Last day, every group is cutd open and kills 3 dogs, checks that whether there is or not pathological changes for injection site.Within the observation period, three groups of canine distemper virus H Gene is with the tiny VP2 subunit vaccines of dog (pFastBac1-H+pFastBac1-VP2 plants) immune group and control group dog in body Temperature, the state of mind, appetite, excrement are normal, and injection site is without the adverse reactions such as swelling and inflammation, injection site dissect inspection Do not occur anomalous variation, vaccine inoculation group dog growing state and control group difference is not notable.
5) efficacy test
Taking healthy susceptible canine, (canine distemper is not higher than 1 with the tiny neutralizing antibody of dog:4) 30,3 groups are randomly divided into.1 group For vaccine immunity group, 10, homemade bigeminy subunit vaccine, subcutaneous point of 5 points of injection canine distemper virus H genes and dog is immunized Tiny VP2 subunit vaccines (pFastBac1-H+pFastBac1-VP2 plants), 1 part (1ml) of every injection.2 groups are hundstaupe Heat-tiny bigeminy vaccine immune group, 10,3 groups of control groups, every group 10.The same day on the 21st after immune, blood sampling measure serum dog Pest heat and the tiny neutralize antibody titers of dog.Canine distemper antibody titer is measured by neutralization test, and the tiny antibody titer of dog passes through blood Solidifying/hemagglutination-inhibition test measures.The results show that canine distemper virus H gene and the tiny VP2 subunit vaccines of dog (pFastBac1-H+pFastBac1-VP2 plants) group neutralize antibody titers are above control vaccine group.After blood sampling, every group It is random to take 5 every (to contain 100 ID with 1ml50) GN plants of canine distemper virus (internal organs poison) attack, be observed continuously 21, observe Record experiment dog and the Temperature changing and clinical symptoms for compareing dog.Every group takes other 5 every (to contain 100 ID with 1ml50) QN plants of attacks of canine parvovirus, are observed continuously 21, observe and record experiment dog and compare the Temperature changing and clinical symptoms of dog.
3 antibody test result of table and protest test result
The results show that three batches of canine distemper virus H genes and the tiny VP2 subunit vaccines (pFastBac1-H+ of dog PFastBac1-VP2 plants) inoculation test dog is 5/ with QN plants of attacks of canine parvovirus to GN plants of canine distemper virus (internal organs poison) 5 protections, control group 5/5 are fallen ill.Concrete outcome is shown in Table 3.
Sequence table
<110>Qingdao Agricultural University
<120>A kind of canine distemper parvovirus bigeminy subunit vaccine
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1824
<212> DNA
<213>Canine distemper virus (canine distemper virus)
<400> 1
atgctctcct accaagacaa ggtgggtccc ttctacaagg acaatgcaag acccaattca 60
tccaagctgt ccccagtgac agaacagcat gggggcagga gaccacctta tttgttgttt 120
gtccttctca tcctattggt tggaatcctg gccctgcttg ctatcactgg agttcgattt 180
caccaagtat caactagcaa tatggaattt agcagattgc tgaaagagga tatggagaaa 240
tcagaggccg tacatcatca agtcatagat gtcttgacac cgctcttcaa gattattggg 300
gatgagattg ggttacggtt gccacaaaag ctaaacgaga tcaaacaatt tatccttcaa 360
aagacaaatt tcttcaatcc gaacagagaa ttcgatttcc gcgatctcca ctggtgcatt 420
aacccgccta gtaaggtcaa ggtgaatttt acaaattact gtgagacaat tgggatcaga 480
aaatctattg catcggcagc aaatcccatc cttttatcag ccctctctgg gggcaggagt 540
gacatattcc caccatacag atgcagtgga gctactactt cagtaggcaa agttttcccc 600
ctatcagtct cgttatccat gtctttgatc tcaagaacct cacagataat caatatgctg 660
accgctacct cagacggcgt gtatggcaaa acttacttgc tagtgcctga tgatatagaa 720
cgggagttcg acactcaaga gattcgagtc tttgaaatag gcttcattaa aaggtggctg 780
aatgacatgc cattactcca agcaaccaac tatatggtcc tcccggagaa ttccaaagcc 840
aaggtatgta ccatagcagt gggtgagttg acactggctt cctcgtgtgt agaagagagc 900
actgtattat tataccatga cagcaggggt tcacaagatg gtattctagt agtgacactg 960
gggatatttg gggcaacacc tatggatcat attgaggaag tgatacctgt cgctcaccca 1020
tcaatggaga aaatacatat aacaaaccac cgtggtttta taaaagattc aattgcaacc 1080
tggatggtgc ctgccctggc ctctgagaaa caagaagaac aaaaaggttg gctggagtca 1140
gcttgtcaaa gaaaaaccta ccccatgtgc aaccaaacgt catgggaacc cttcggagga 1200
ggacagttgc catcttatgg gcggttgaca ttacctctag atgcaagtgt tgaccttcaa 1260
cttaacatat cgttcacata cggtccggtt atactgaatg gagatgctat ggattattat 1320
caaagcccac ttttgaactc cggatggctt accattcctc ctaaaaacgc aacaatcctt 1380
ggattgataa acaaagcaag tagaggagac cagttcactg tgatacccca agtattaaca 1440
tttgcgccca gggaatcatg tggaaattgt tatttaccta ttcaaacatc tcaaattata 1500
gatagagatg tcctcatcga gtccaatgta gtggtgttgc ctacacagag ttttagatat 1560
gtcatagcaa cgtctgtcat atcacgaaat gatcatgcga ttgtttatta tgtttatcac 1620
ccaatccgga ccatttctta tacgcaccca tttagactaa ctaccaaggg tagacctgat 1680
ttcctaagga ttgaatgttt tgtgtgggat gataatttgt ggtgtcacca attttacaga 1740
tacgagccta acatcgccaa ctctacaacc agtgttgaga atttagtccg tataagattc 1800
tcatgtaacc gttcaaatcc ctga 1824
<210> 2
<211> 607
<212> PRT
<213>Canine distemper virus (canine distemper virus)
<400> 2
Met Leu Ser Tyr Gln Asp Lys Val Gly Pro Phe Tyr Lys Asp Asn Ala
1 5 10 15
Arg Pro Asn Ser Ser Lys Leu Ser Pro Val Thr Glu Gln His Gly Gly
20 25 30
Arg Arg Pro Pro Tyr Leu Leu Phe Val Leu Leu Ile Leu Leu Val Gly
35 40 45
Ile Leu Ala Leu Leu Ala Ile Thr Gly Val Arg Phe His Gln Val Ser
50 55 60
Thr Ser Asn Met Glu Phe Ser Arg Leu Leu Lys Glu Asp Met Glu Lys
65 70 75 80
Ser Glu Ala Val His His Gln Val Ile Asp Val Leu Thr Pro Leu Phe
85 90 95
Lys Ile Ile Gly Asp Glu Ile Gly Leu Arg Leu Pro Gln Lys Leu Asn
100 105 110
Glu Ile Lys Gln Phe Ile Leu Gln Lys Thr Asn Phe Phe Asn Pro Asn
115 120 125
Arg Glu Phe Asp Phe Arg Asp Leu His Trp Cys Ile Asn Pro Pro Ser
130 135 140
Lys Val Lys Val Asn Phe Thr Asn Tyr Cys Glu Thr Ile Gly Ile Arg
145 150 155 160
Lys Ser Ile Ala Ser Ala Ala Asn Pro Ile Leu Leu Ser Ala Leu Ser
165 170 175
Gly Gly Arg Ser Asp Ile Phe Pro Pro Tyr Arg Cys Ser Gly Ala Thr
180 185 190
Thr Ser Val Gly Lys Val Phe Pro Leu Ser Val Ser Leu Ser Met Ser
195 200 205
Leu Ile Ser Arg Thr Ser Gln Ile Ile Asn Met Leu Thr Ala Thr Ser
210 215 220
Asp Gly Val Tyr Gly Lys Thr Tyr Leu Leu Val Pro Asp Asp Ile Glu
225 230 235 240
Arg Glu Phe Asp Thr Gln Glu Ile Arg Val Phe Glu Ile Gly Phe Ile
245 250 255
Lys Arg Trp Leu Asn Asp Met Pro Leu Leu Gln Ala Thr Asn Tyr Met
260 265 270
Val Leu Pro Glu Asn Ser Lys Ala Lys Val Cys Thr Ile Ala Val Gly
275 280 285
Glu Leu Thr Leu Ala Ser Ser Cys Val Glu Glu Ser Thr Val Leu Leu
290 295 300
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 (7)

1. a kind of canine distemper parvovirus bigeminy subunit vaccine, which is characterized in that the bigeminy subunit vaccine includes Vaccine adjuvant and antigen, the antigen are that amino acid sequence is SEQ ID NO:2 H protein and vp2 albumen.
2. bigeminy subunit vaccine as described in claim 1, which is characterized in that the vp2 albumen, amino acid sequence are SEQ ID NO:4。
3. bigeminy subunit vaccine as described in claim 1, which is characterized in that the H protein, the nucleosides of encoding gene Acid sequence is SEQ ID NO:1.
4. bigeminy subunit vaccine as claimed in claim 1 or 2, which is characterized in that the vp2 albumen, encoding gene Nucleotides sequence is classified as SEQ ID NO:3.
5. bigeminy subunit vaccine as described in claim 1, which is characterized in that the H protein passes through rod-shaped with VP2 albumen Prepared by expressing viral.
6. bigeminy subunit vaccine as described in claim 1, which is characterized in that the concentration of the H protein and VP2 albumen is not Less than 8 μ g/mL.
7. bigeminy subunit vaccine as described in claim 1, which is characterized in that the vaccine adjuvant is handed over for allyl sucrose The composite adjuvant of acrylic acid polymer solution and glycerin solution mixed preparing.
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