CN102787100B - High-prolificacy porcine circovirus type-2 strain and application thereof - Google Patents

High-prolificacy porcine circovirus type-2 strain and application thereof Download PDF

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CN102787100B
CN102787100B CN 201210312682 CN201210312682A CN102787100B CN 102787100 B CN102787100 B CN 102787100B CN 201210312682 CN201210312682 CN 201210312682 CN 201210312682 A CN201210312682 A CN 201210312682A CN 102787100 B CN102787100 B CN 102787100B
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CN102787100A (en
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周继勇
金玉兰
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Zhejiang University ZJU
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ZHEJIANG TONGDIAN BIOLOGICAL TECHNOLOGY CO LTD
Zhejiang University ZJU
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Abstract

The invention discloses a high-prolificacy porcine circovirus type-2 strain and application thereof. The porcine circovirus type-2 strain is named as PCV2-ZJ/H strain, the preservation number of the strain is CGMCC No.6391, the gene sequence of the strain is shown as SEQ ID NO:1, and nucleotide at a 1376 locus in the nucleotide sequence of a genome of the strain is absent. The porcine circovirus type-2 strain has high prolificacy, the virus titer (TCID50) in PK-15 cells reaches 107.6/mL and is more than 10 times higher than that (106.3/mL) of a parent virus, and a vaccine prepared with the strain has high immune protection capacity.

Description

A kind of high fecundity porcine circovirus 2 type strain and application thereof
Technical field
The invention belongs to field of virology, be specifically related to a kind of new strain of porcine circovirus 2 type and application in the preparation inactivated vaccine thereof that utilizes the high fecundity of virological molecular genetics technological transformation acquisition.
Background technology
(Porcine circovirus, pathogenic, antigenicity PCV) and nucleotide sequence can be divided into it two genotype, i.e. pig circular ring virus 1 type (PCV1) and porcine circovirus 2 type (PCV2) according to pig circular ring virus.
PCV2 is nearly ten years emerging a kind of pig circular ring virus 2 viral disease (Porcine circovirus virus-associated disease, virus PCVAD) of causing.The pathogenic of the pig of PCV2 is feature with immune damage, make infected pigs be in serious immunosuppressive condition, mainly show as clinically become thin, pathology such as lymphadenectasis, also can cause multisystem depletion, expiratory dyspnea, breeding difficulty etc. with multiple cause of disease polyinfection.
Pig circular ring virus belongs to PCV-II section PCV-II and belongs to.Under Electronic Speculum, PCV2 be a kind of do not have a cyst membrane be icosahedron symmetrical circular small-particle virus, diameter 17nm, the DNA genome with sub-thread ring-type.Insensitive to organic solvents such as chloroform, the tincture of iodine, alcohol, can be 70 ℃ of stable survivals 15 minutes.But reagent such as Pyrogentisinic Acid, quaternary ammonium compounds, sodium hydroxide and oxygenant are responsive.
Found at present the PCV2 strain of the different genes group size that 1768,1767 Nucleotide are formed, the nucleotide homology of the two is more than 90%.The PCV2 genome research can be divided into PCV2 PCV2a, PCV2b and three gene hypotypes of PCV2c, and genome nucleotide and amino acid identity are all about 95% between three types.
The PCV2 genome contains 11 reads frames, i.e. ORF1～ORF11, and each is read the frame size and differs greatly.Wherein 5 '-3 ' direction of ORF1, ORF5, ORF7, ORF10 is identical, ORF2, ORF3, ORF4, ORF6, ORF8, ORF9 are identical with 5 ' of ORF11-3 ' direction, it is overlapping that these reading frames show as Gene Partial, can take full advantage of the limited genetic material of PCV-II.
Recently in year, PCV becomes the research focus of Chinese scholars, and a lot of scholars have made up the infections clone of PCV1 and PCV2 in different ways, and the recombinant virus that it is saved out is applied in the correlative study.Granted publication number discloses a kind of pig circular ring virus 1 type infections clone and the virus of saving thereof for the Chinese invention patent of CN101423836B and has used, this invention is inserted into structure acquisition infective molecule cloning in the pUC19 carrier with 2 genome cis connections of pig circular ring virus 1 type, and in this infections clone, insert Sal I restriction enzyme site as molecular target, the recombinant virus of being saved out has molecular target, available PCR differentiates itself and parental virus mutually in conjunction with RFLP, and will save virus for the preparation of in the reagent or medicine that detect pig circular ring virus 1 type antibody.
At present, PCV2 infects the pig circular ring virus 2 viral disease that causes and brings about great losses for global pig industry, and the immunosuppression that virus infection produces can cause existing vaccine immunity failure, causes multiple cause of disease to mix sense, and makes pestilence more serious.Adopting vaccine immunity to become the key of control PCV2 relative disease, is the effective ways that reduce financial loss.Virus titer is low during the PCV2 vitro culture, is the high basic reason of vaccine price, also is the biggest obstacle of current vaccine development.
Summary of the invention
The invention provides a kind of new strain of porcine circovirus 2 type with high fecundity (high titre), it is poor to have solved existing PCV2 strain fecundity, is applied to develop the high problem of vaccine cost.
A kind of porcine circovirus 2 type strain, called after porcine circovirus 2 type ZJ/H strain, be abbreviated as the PCV2-ZJ/H strain, this strain is preserved in the China Committee for Culture Collection of Microorganisms common micro-organisms center that is positioned at No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on July 17th, 2012, and preserving number is CGMCC No.6391.
Described porcine circovirus 2 type strain makes by method:
(1) genomic dna with PCV2-HZ0201 virus is template, be primer amplification PCV2 complete genome sequence with FSAC and RSAC, gel-purified reclaims the test kit purified pcr product then, is connected to plasmid pMD18-T, transform the Top10 competent cell, obtain recombinant plasmid pMD-HZ0201.
FSAC：GAA CCGCGGGCTGGCTGAACTTTTGAAAGT
RSAC：GCA CCGCGGAAATTTCTGACAAACGTTACA
(2) be template with the pMD-HZ0201 plasmid that builds, utilize the nucleotide deletion Auele Specific Primer to carry out the amplification of Dpn I site-directed mutagenesis, gel-purified reclaims the test kit purified pcr product then, is connected to plasmid pMD18-T, transform the Top10 competent cell, obtain recombinant plasmid pMD-ZJ/H.
(3) through the correct pMD-ZJ/H recombinant plasmid of sequencing, cut with restriction enzyme Sac II enzyme, with the cyclisation of T4DNA ligase enzyme, again with cyclisation plasmid lipofectamine TM2000 transfection PK-15 cells, the cell culture of results virus particle.
(4) the viral PCV2ZJ/H that saves by indirect immunofluorescence assay (IFA) method validation then.Virus to rescue goes down to posterity, and measures the viral median infective dose (TCID of different generations with indirect immunofluorescence method 50), relatively rescue virus and the fecundity of parental virus on the PK-15 cell.Cultivate and IFA mensuration the virus titer (TCID of the PCV2-ZJ/H virus particle of saving out in the PK-15 cell through passage 50) reach 10 7.6/ mL is than parental virus (10 6.3/ mL) malicious valency exceeds more than 10 times.
The present invention also provides a kind of gene of described porcine circovirus 2 type PCV2-ZJ/H strain, the base sequence of described gene shown in SEQ ID NO.1, with respect to the wild-type strain, the nucleotide deletion in 1376 sites in this strain genome nucleotide sequence.
The present invention provides a kind of recombinant vectors that comprises described gene again, and the initial carrier of described carrier can be pMD18-T.
The present invention also provides the application of described porcine circovirus 2 type ZJ/H strain in the preparation inactivated vaccine, and described inactivated vaccine is mainly used in preventing zoogenetic infection pig annulus 2 C-type virus Cs, and described animal can be pig.
The invention provides a kind of method of utilizing described porcine circovirus 2 type ZJ/H strain to prepare inactivated vaccine, comprising:
Be that seed culture of viruses prepares viral liquid with porcine circovirus 2 type ZJ/H strain, viral liquid mixes with adjuvant after deactivation, dilution, namely makes inactivated vaccine.
Tiring to greater than 10 of described viral liquid 7.0TCID 50/ mL.
It is beta-propiolactone that reagent is adopted in described deactivation, and its final concentration in viral liquid is generally 0.05～1.0%.
Described immunological adjuvant is generally selected mineral oils adjuvant, natural drug class adjuvant or aluminium glue adjuvant for use, and the tired adjuvant of described mineral oil can be selected ISA206VG for use, and the viral liquid volume ratio after adjuvant and the dilution was generally 5: 5～9: 1.
The invention provides the inactivated vaccine that is equipped with that utilizes described porcine circovirus 2 type ZJ/H strain system.
The present invention has manually made the new strain of porcine circovirus 2 type that a strain has high fecundity by nucleotide deletion, gene frameshit technology and infections clone technology, for the vaccine development that promotes PCV2 provides new seed culture of viruses resource.
Virus of the present invention has high fecundity, the virus titer (TCID in the PK-15 cell 50) reach 10 7.6/ mL is than parental virus (10 6.3/ mL) malicious valency exceeds more than 10 times, and the vaccine that utilizes it to make has good immune protection power.
Description of drawings
Fig. 1 cuts evaluation figure (M swimming lane: DNAMarker for the SacII enzyme of mutant and HZ0201 normal gene; Swimming lane A-D: the mutant clone enzyme is cut;
Fig. 2 is that the PCV2/Cap protein monoclonal antibody after the 1376 site deletant rescues detects;
Fig. 3 is that PCV2-ZJ/H strain fecundity is measured figure.
Embodiment
Embodiment 1 nucleotide site 1376 deletion mutantion strain infections clone make up and identify
1.1 virus, cell and plasmid
PCV2-HZ0201 strain (Genbank NO.AY188355), coli strain TG1, the negative PK-15 clone of carrier pMD18-T, PCV, PCV2/Cap protein monoclonal antibody, FITC sheep anti-mouse igg are test materials, no specificity requirement.
Wherein, the preparation of PCV2/Cap protein monoclonal antibody, FITC sheep anti-mouse igg can reference (Shang SB et al.Fine mapping of antigenic epitopes on capsid proteins of porcine circovirus and antigenic phenotype of porcine circovirus Type 2.Molecular Immunology.2009.46:327-334), and all the other are the commercially available prod.
1.2 design of primers is with synthetic
According to the PCV2-HZ0201 strain sequence that provides among the GenBank, at the continuous mononucleotide disappearance of 1376～1379bp design.Use the Prime5.0 biosoftware, primers F SAC and the RSAC of the synthetic 1 pair of amplification PCV2 genome total length of design, the line part is the SacII restriction enzyme site in the table; Primers F ORF2 and the RORF2 of 1 couple of amplification ORF2; 4 pairs of primers that constitute the mononucleotide disappearance, F1376/R1376 cause 1376 sites disappearance, amplified production called after A; F1377/R1377 causes 1377 sites disappearance, amplified production called after B; F1378/R1378 causes 1378 sites disappearance, amplified production called after C; F1379/R1379 causes 1379 sites disappearance, amplified production called after D.
Concrete primer sequence is as shown in table 1, and it is synthetic that primer is given birth to worker bio-engineering corporation by Shanghai, and with before being dissolved in the ultrapure water of sterilizing to final concentration 10 μ mol/L ,-20 ℃ of preservations are standby.
Table 1 pcr amplification the primer
The primer title Primer sequence (5 ' → 3 ')
F1376 GAATAACAGCACTGAGCCCACTCCCCT
R1376 AGTGGGCTCAGTGCTGTTATTCT
F1377 CTAGAATAACAGCACTGGGCCCACTCC
R1377 AGTGGGCTCCAGTGCTGTTATTCTA
F1378 CTAGAATAACAGCACTGGACCCACTC
R1378 GAGTGGGTCCAGTGCTGTTATTCTA
F1379 CTAGAATAACAGCACTGGAGCCACT
R1379 GAGTGGCTCCAGTGCTGTTATTCTA
FSAC GAA CCGCGGGCTGGCTGAACTTTTGAAAGT
RSAC GCA CCGCGGAAATTTCTGACAAACGTTACA
FORF2 GAGGATCCATGACGTATCCAAGGAGG
RORF2 GCCCTCGAGTTAAGGGTTAAGTGGG
1.3PCV2 whole genome amplification and purifying
With PCV2-HZ0201 strain (10 5.75TCID 50/ mL) suspended with 1: 10 and be inoculated in the PK15 cell, infect back 72h and receive poison, cell is placed-70 ℃ of refrigerator multigelations three times, the collecting cell nutrient solution, the centrifugal 5min of 12,000r/min gets viral supernatant liquid extracting viral DNA.PCV2-HZ0201 strain virus DNA with above-mentioned extracting is template, utilizes Auele Specific Primer FSAC and RSAC amplification PCV2 genome total length.
The PCR reaction system is: 2.5 μ L, 10 * PCR buffer, and each 0.5 μ L of FSAC and RSAC primer, 0.5 μ LdNTP, 0.12 μ l Probest DNA polymerase, plasmid template≤200ng, deionized water complement to 25 μ L.Fully behind the mixing, carry out PCR.
The PCR response procedures is: 95 ℃ of pre-sex change 5min, 35 circulations [72 ℃ are extended 2min for 94 ℃ of sex change 15s, 55 ℃ of annealing 45s], 72 ℃ of total elongation 10min.
The PCR product that obtains is identified with 1% agarose gel electrophoresis.Gel-purified reclaims test kit (AXYGEN AP-GX-250) purified pcr product then, is connected with carrier pMD18-T to make up the pMD-HZ0201 plasmid template.
1.4 the mononucleotide deletion mutant makes up
Be template with the pMD-HZ0201 plasmid that builds, the primer that utilizes 4 couple in the table 1 to constitute the mononucleotide disappearance carries out Dpn I site-directed mutagenesis respectively and obtains different loci mononucleotide missing gene sequence.
PCR reaction system: 2.5 μ L, 10 * PCR buffer, each 0.5 μ L of upstream and downstream primer, 0.5 μ LdNTP, 0.12 μ l Probest DNA polymerase high-fidelity enzyme, plasmid template≤200ng, ddH 2O complements to 25 μ L.
The PCR program is: 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ are extended 5min, 15 circulations; 72 ℃ of total elongation 10min.
The PCR product that obtains is identified with 1% agarose gel electrophoresis; PCR product (different loci mononucleotide missing gene sequence) is connected the evaluation of checking order of pMD18-T plasmid respectively, obtain different mononucleotides disappearance recombinant plasmids.
1.5 mononucleotide disappearance virus rescue
The mononucleotide of extracting disappearance recombinant plasmid is carried out the SacII enzyme to be cut, enzyme is cut qualification result as shown in Figure 1, gene after the recovery point disappearance, the goal gene of recovery is the DNA that has SacII restriction enzyme site end, adds the T4 ligase enzyme and connect cyclisation virus in the EP of sterilization pipe.
Linked system is: T4DNA ligase enzyme 1 μ L, the goal gene that enzyme cuts back to close 〉=1 μ g connects Buffer 1 μ L, behind the mixing, spends the night in 16 ℃ of connections.
Connect product liposome 2000 (invitrogen) transfection PK-15 cell; Cell after the transfection is drawn nutrient solution; Clean 3 times with PBS.Every hole adds an amount of methanol/acetone (1: 1) and covers cell surface, and-20 ℃ of fixing 20min discard stationary liquid, dry in the ventilation.The skimmed milk 2mL of adding 5%, sealing 40～60min.Discard skimmed milk, will resist the PCV2/Cap protein monoclonal antibody in skimmed milk, to be diluted to suitable proportion respectively, add in the hand-hole, hatch 60min.Discard mixed solution, clean 3 times with PBS, each 3min.
The FITC sheep anti-mouse igg is diluted to suitable proportion in skimmed milk, adds in the hand-hole, hatch 60～90min again.Discard mixed solution, clean 3 times with PBS, each 3min.Cleaned, every hole adds the anti-quencher of 100 μ L, places under the fluorescent microscope and observes.The result as shown in Figure 2, in 1376～1379 disappearances P0 generations behind the virus transfection, gone down to posterity P1 after generation, 1376 sites disappearance virus can continue to go down to posterity, and each generation all can detect fluorescence, illustrate that virus rescue is successful.
Virus stain called after pig circular ring virus (Porcine circovirus) 2 type ZJ/H strains with 1376 sites disappearance in the genome, this strain is preserved in the China Committee for Culture Collection of Microorganisms common micro-organisms center that is positioned at No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on July 17th, 2012, and preserving number is CGMCC No.6391.
The replication in vitro ability of embodiment 2 unit point nucleotide deletion strains
2.1 test method
Detection by to former generation and the viral indirect immunofluorescence (IFA) that goes down to posterity obtains saving successful mutated viruses.Mutated viruses P0 for infecting the PK-15 cell, behind 37 ℃ of cultivation 72h, is placed-70 ℃, and multigelation three times is with collecting viral liquid after the filter filtration sterilization.With 1: 10 inoculation PK-15 cell that suspends, carry out the cultivation of going down to posterity of virus.Cultivating 8 generations to malicious valency continuously in the PK-15 cell stablizes.
To save successful PCV2-ZJ/H virus to infect cultivations of going down to posterity of PK-15 cell at 1: 10 2.2 fecundity is measured, in this process, the virulence of virus strengthens gradually, continuous passage cultivate 8 generations extremely malicious valency stablize, each virus is carried out replication mensuration.In each generation, all surveyed the TCID of the virus of cultivating 50
The viral liquid of getting PCV2-ZJ/H respectively dilutes with MEM, from 10 -1To 10 -10, totally 10 gradients join (every hole 100 μ l) in 96 orifice plates with dilution viral liquid well, and each sample is established 4 repeating holes; In 5%CO 2With cultivate 72h under 37 ℃ of conditions; Give a baby a bath on the third day after its birth time with PBS, stationary liquid is in-20 ℃ of fixing 20min; 37 ℃ of sealings of 5% skimmed milk 2h; Add the PCV2/Cap protein monoclonal antibody; With PBST flushing 3 times, add the goat-anti pig IgG of FITC mark, 37 ℃ of effect 1h; Wash 3 times with PBST, whether fluorescence microscope produces green fluorescence; Calculate the malicious TCID that recuperates that whenever is commissioned to train by the Reed-Muench method 50
The result as shown in Figure 3.PCV2-ZJ/H strain (1376 sites disappearance virus) TCID of virus after the 8th generation of going down to posterity 50Reached 10 7.6/ mL, the TCID of wild strain PCV2-HZ0201 50Be 10 6.3/ mL, the PCV2-ZJ/H virus of rescue is higher more than 10 times than wild-type strain.
The inactivated vaccine of embodiment 3 strain preparations of the present invention is to the protection test of mouse
Using cleaning level Balb/C mouse measures the immunogenicity of PCV2-ZJ/H strain.
3.1 prepare inactivated vaccine with strain of the present invention
To tire greater than 10 7.0TCID 50The PCV2-ZJ/H strain virus liquid final concentration of/ml is that 0.05% beta-propiolactone was 4 ℃ of deactivations 48 hours, 37 ℃ of water-baths 2 hours, the venom of deactivation is suitably diluted with the MEM nutrient solution, in 3: 1 (v/v) ratios, with venom and ISA206VG adjuvant (France's match Bick) mix, emulsification, be prepared into oil-in-water-type PCV2-ZJ/H strain inactivated vaccine.
3.2 experimental animal and testing program
Clean level BALB/C mice random packet, 10 every group 8 ages in week of PCV2 antigen and antibody test feminine gender.Attack appetite, healthy state that mouse is observed in the poison back.Before attacking poison docking blood sampling and attack poison after cut open to kill and take a blood sample separation of serum.Immunity back the 21st day is attacked with the PCV2-ZJ/H strain, and every abdominal injection 0.2ml attacks poison and cuts open in the time of 21 days and get spleen tissue extremely, and tissue is used for the virus separation.
3.3 the mouse immune effect evaluation of vaccine
Mouse tissue is weighed, adds the MEM serum free medium, grinds 3min with 25 times/s of tissue grinder concussion in 1: 20 ratio, grind the freeze thawing 3 times of finishing after, the centrifuging and taking supernatant, filtration sterilization is collected.The PK15 cell monolayer washes with PBS, 0.05% pancreatin is put 37 ℃ of digestion 5～8 minutes, be mixed with the cell suspension that concentration is 1 * 106/ml with the MEM substratum, cell suspension grinds to consider with mouse tissue in 10: 1 ratio inoculates 12 porocyte plates after liquid mixes, put 37 ℃ of incubators and cultivate after 72 hours harvested cell freeze thawing 3 times.Continuous blind passage three generations.
The cell cultures individual layer in 1～3 generation of blind passage is abandoned cell conditioned medium, and cell monolayer is fixed 30 minutes with 1: 1 cold acetone-methanol solution in-20 ℃.Under 37 ℃, seal in 5% milk powder solution; PBST cleans; The PCV2/Cap protein monoclonal antibody of PBST dilution is hatched under 37 ℃; PBST cleans; The sheep anti-mouse igg that adds the FITC mark is hatched; PBST cleans; Directly under inverted fluorescence microscope, observe.The result shows that not immune viral separation rate of attacking malicious mouse spleen is 80%; 0.2ml the virus separation positive rate that the dosage group was attacked poison in back 21 days in immunity is 20%.
The inactivated vaccine of embodiment 4 strain preparations of the present invention is to the protection test of piglet
Use the healthy piglet of PCV2 antibody and antigen negative, the immunogenicity of PCV2-ZJ/H strain is measured.
4.1 the deactivation of strain of the present invention and emulsification
To tire to greater than 10 7.0TCID 50The PCV2-ZJ/H strain virus liquid final concentration of/ml be 0.05% beta-propiolactone 4 ℃ of deactivations 48 hours, 37 ℃ of water-baths 2 hours.The venom of deactivation is suitably diluted, in 3: 1 (v/v) ratios, with venom and ISA206VG adjuvant (France's match Bick) mix, emulsification, be prepared into oil-in-water-type PCV2-ZJ/H strain inactivated vaccine.
4.2 experimental animal and testing program
12 of the healthy piglets of 14～18 ages in days of selection PCV2 antibody and antigen negative are divided into 2 groups, 5 every group at random.The 1st intramuscular injection PCV2-ZJ/H strain inactivated vaccine, the 2ml/ head; The 2nd group of blank group of vaccinate not.Every pig collunarium 1ml, intramuscular injection 2ml are attacked with the PCV2-ZJ/H strain in immunity back the 21st day.The blood sampling in the 7th, 14 day of immunity back.Attack the poison back and cutd open each treated animal extremely on the 14th day, gather lungs, spleen, hilar lymph node, lymphoodi mandibulares, shoulder ALN, inguinal lymph nodes, mesenteric lymph nodes, iliac lymph nodes, carry out PCV2 virus and separate.
4.3 the piglet immunological effect evaluation of vaccine
Tissue homogenate (1: 3) with 0.22 μ m membrane filtration degerming, is inoculated the PK-15 cell of fresh digestion in 1: 10 ratio, and behind the harvested cell culture after cultivating 72h under 37 ℃, 5%CO2 condition, multigelation three times, inoculation is of future generation.
Continuously the cell monolayer in blind passage 1-3 generation is abandoned cell conditioned medium.Cell monolayer is fixed 30 minutes with 1: 1 cold acetone-methanol solution in-20 ℃.Cell monolayer is fixed 30 minutes with 1: 1 cold acetone-methanol solution in-20 ℃.Under 37 ℃, seal in 5% milk powder solution; PBST cleans; The PCV2/Cap protein monoclonal antibody of PBST dilution is hatched under 37 ℃; PBST cleans; The sheep anti-mouse igg that adds the FITC mark is hatched; PBST cleans; Directly under inverted fluorescence microscope, observe.
Experimental result is as shown in table 2, and vaccine immunity group pathological material of disease is behind PK-15 passage 3 times, and except 1 pig lungs is separated to PCV2 virus, its hetero-organization all is not separated to virus, and protection ratio is 80%.The blank group pathological material of disease of not using vaccine immunity all can be separated to virus PK-15 passage 2 times, and it is 100% (5/5) that virus is separated positive rate.The viral separating resulting of different tissues shows (table 2), attack poison back different tissues virus separation positive rate very big-difference is arranged, separates positive rate for the highest with inguinal lymph nodes and lungs virus, and iliac lymph nodes, mesenteric lymph nodes, the viral separation of shoulder ALN positive rate are minimum.
Table 2 piglet different tissues PCV2 is separating resulting again
Figure BDA00002074252000091
Annotate: the * molecule is that virus is separated positive size of animal, and denominator is immune animal quantity.
Figure IDA00002074253000011
Figure IDA00002074253000021
Figure IDA00002074253000031
Figure IDA00002074253000041
Figure IDA00002074253000051
Figure IDA00002074253000061

Claims (10)

1. a porcine circovirus 2 type strain is characterized in that, called after pig circular ring virus (Porcine circovirus) 2 type ZJ/H strains, and preserving number is CGMCC No.6391.
2. as the gene of claim 1 porcine circovirus 2 type strain, it is characterized in that the base sequence of described gene is shown in SEQ ID NO.1.
3. recombinant vectors that comprises the described gene of claim 2.
4. recombinant vectors as claimed in claim 3 is characterized in that, initial carrier is plasmid pMD18-T.
5. the application of porcine circovirus 2 type strain in the preparation inactivated vaccine according to claim 1.
6. method of utilizing porcine circovirus 2 type strain according to claim 1 to prepare inactivated vaccine comprises:
Be that seed culture of viruses prepares viral liquid with porcine circovirus 2 type ZJ/H strain, viral liquid mixes with adjuvant after deactivation, dilution, namely makes inactivated vaccine.
7. method as claimed in claim 6 is characterized in that, reagent that described deactivation is adopted is beta-propiolactone.
8. method as claimed in claim 6 is characterized in that, the tiring greater than 10 of described viral liquid 7.0TCID 50/ mL.
9. method as claimed in claim 6 is characterized in that, described adjuvant is ISA206VG.
10. the inactivated vaccine of method preparation as described in arbitrary as claim 6～9.
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