CN101089177A - Type II genetic marker strain of porcine circovirus and its application - Google Patents
Type II genetic marker strain of porcine circovirus and its application Download PDFInfo
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Abstract
The present invention discloses Porcine circovirus type 2 strain PCV2/PE with genetic marker and in the preservation number of CGMCC No.1617 and its application. The new Porcine circovirus strain is obtained with clinically separated Porcine circovirus type 2 material, and through PCR amplification of the virus genome, cis-connecting two genomes and inserting into plasmid vector to constitute infectious molecular cloning, designing the substituting base with the primer, inserting SalI restrictive endonuclease site as genetic marker into the virus genome, and recombining the plasmid transfected cell. The Porcine circovirus strain has obviously raised propagation capacity, capacity of meeting the basic requirement on vaccine virus seed and special genetic marker for identification from wild viruses, and possesses important applications.
Description
Technical field
The present invention relates to a kind of new strain, relate in particular to a kind of porcine circovirus 2 type genetic marker strain and application thereof, belong to biological technical field.
Background technology
Porcine circovirus 2 type (Porcine circovirus type 2, PCV2) mainly cause the weanling pig multisystemic wasting syndrome (Postweaning multisystemic wasting syndrome, PMWS).This disease confirms first in Canada that in 1997 all there are this pathogenetic report in Europe, the United States, the many countries of Ya Gezhou in succession.China swinery also generally has this disease popular, pig industry is constituted significant damage (Lang Hongwu, Zhang Guangchuan, Wu Faquan etc. wean pig multisystemic exhaustion syndrome Serum Antibody Detection. Chinese animal doctor's science and technology, 2000,30 (3): 3-5.), except that PMWS, pigskin scorching with nephritic syndrome, hyperplasia necrotizing pneumonia, porcine respiratory syndrome, breeding difficulty, congenitally tremble, diseases such as fetus myocarditis and enteritis also confirm relevant with the PCV2 infection.PCV2 is a kind of circular small-particle virus, and diameter 17nm is the icosahedron symmetry, no cyst membrane, and genome is made up of sub-thread minus strand closed-circular DNA, contains 1767 or 1768 bases.Genome comprises 2 big open reading frames (ORFs), ORF1 genes encoding rdrp virus (Replicase), ORF2 genes encoding viral capsid proteins (Capsid).Pathogenic main and the body immune system of virus infection interacts relevant, can cause the necrosis of general lymphocyte, causes that lymphocyte consume and scavenger cell reduce, and causes the immunity of organism inhibition, increases the susceptibility to various pathogenic agent.
At present, PCV2 infects the PMWS and the relative disease that cause 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.Adopting vaccine immunity to become the key of control PCV2 relative disease, is the effective ways that reduce financial loss.Because this virus is low at vitro culture poison valency, causes certain difficulty for the development of vaccine.
For making up the PCV2 infective molecule cloning, mainly take two kinds of strategies at present: the one, viral genome is cloned, carry out dual-gene group of cis again and connect, on recombinant plasmid, form a complete viral genome regulation and control unit, obtain infectious virus through the plasmid transfection cell; Another kind is that viral single-gene group is cloned on the plasmid, cuts and the gel electrophoresis recovery through enzyme, carries out self-cyclisation and forms simulating nature viral genome structure, and transfectional cell obtains virus.Studies show that above-mentioned two kinds of methods all can successfully obtain infectious virus.Comparatively speaking, the former is workable, good reproducibility; The latter is wayward because of links such as gel-purified and self-cyclisation, makes the infective molecule cloning transfection efficiency not high, and is repeatable bad.
Recently, (fenaux M such as Fenaux, Opriessing T, Halbur PG, et al.Immunogenicity andpathogenicity of chimeric infectious DNA clones of pathogenic porcine circovirus type 2 (PCV2) and nonepathogenic PCV1 in weanling pigs[J] .J Virol, 2003,77 (20): 11232-11243.Fenaux M, Opriessing T, Halbur PG, et al.A chimeric porcine circovirus (PCV) with the immunogenic PCV type 2 (PCV2) cloned into the genomic backbone of thenonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs[J] .JVirol, 2004,78 (12): 6297-6303; Fenaux M; Opriessnig T; Halbur PG; et al.Two amino acidmutations in the capsid protein of type 2 porcine circovirus (PCV2) enhanced PCV2replication in vitro and attenuated the virus in vivo[J] .J Virol.2004; 78 (24): 13440-6) reported that utilizing PCV1 and PCV2 genome ORF 2 coding capsid protein districts to replace has made up the mosaic type molecular cloning; the inoculation test of pig body proves its pathogenic weakening; and can induce body to produce PCV2 capsid protein antibody; can produce immune protective effect, but virus is not seen below continuous report as yet in the situation of vitro culture.The inventor also once carried out similar research, did not succeed yet the mosaic type virus that makes up goes down to posterity in vitro culture, and this may be relevant with the function of two kinds of virogene coding regions replacement back change rdrp virus.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, and a kind of new strain of porcine circovirus 2 type that has genetic marker is provided.
Technical problem to be solved by this invention realizes by following technological approaches:
A kind of porcine circovirus 2 type (Porcine circovirus type 2) strain that has genetic marker, called after: PCV2/PE; Preserving number is: CGMCC No.1617; The preservation place is: China Committee for Culture Collection of Microorganisms common micro-organisms center; The preservation address is: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date is: on February 9th, 2006.
The present invention is a material with the isolating PCV2 wild-type of the sick pig of clinical PMWS strain, adopt genetic engineering technique to make up the viral genome infective molecule cloning, replace the mode of base by design of primers, replace base by design of primers, in viral genome, insert SalI system property restriction enzyme site as genetic marker, obtain to have the new strain of genetic marker through recombinant plasmid transfected cell.
The present invention is when making up the viral infection molecular cloning, and 2 bases of terminal replacement form a SalI restriction enzyme and cut sequence in genome ORF 1 coding region.Do not change genes encoding reading frame and aminoacid sequence in the test design, thereby guaranteed the function of recombinant virus replicative enzyme and the infectivity of pair cell.Owing to insert a SalI restriction enzyme site in the new strain genome of the present invention, adopt PCR strain of the present invention and wild-type virus can be distinguished mutually with the restriction fragment length polymorphism analytical method.
The PCV2/PE strain that the present invention is constructed, the passage initial stage does not produce CPE, and is basic similar to wild-type virus cells infected result.Along with increasing progressively of cell cultures passage number, viral adaptability strengthens, and the fecundity of PCV2/PE strain significantly promotes.New strain passed for 60 generations continuously through cell, the stable performance of vitro culture propagation, and malicious valency can reach 10
6.6TCID
50/ ml possesses the basic demand of vaccine kind poison.The 60th generation viral cultures can reach more than 50% with 5% inoculum size cells infected positive rate.When viral proliferation reached certain level, cells infected hypertrophy occurred, becomes phenomenons such as circle, refractivity change.Cultivate adaptability by virus at cell in vitro and go down to posterity, can significantly improve the viral proliferation ability, a large amount of viruses are accumulated in cell and are made host cell that corresponding pathology take place.
Adopt the test of immunoperoxidase monolayer cell, immuno-electron microscope, nucleic acid sequence analysis to show that detect virus specific antigen in virus infected cell of the present invention, its antigenicity, morphology of virus and gene order are consistent with parent's strain.Get viral cultures of the present invention and inoculate 4 of 35 age in days negative antibody piglets through vein and collunarium approach, show fervescence, progressive emaciation, the coarse and body surface swollen lymph node symptom of quilt hair after the inoculation, all can detect virus antigen and nucleic acid in the multiple internal organs after compeling to kill.
Seed culture of viruses is made in PCV2/PE strain of the present invention,, obtained viral cultures through cell cultures technology; viral cultures is through formalin-inactivated; promptly get inactivated vaccine with adjuvant emulsion, the vaccine immunity potency test is the result show, prepared inactivated vaccine has stronger immune protection effectiveness.
In addition, utilize the viral cultures of PCV2/PE strain of the present invention to adopt immunoperoxidase monolayer cell test (IPMA) method can detect PCV2 antibody in the sample serum accurately, easily, for the evaluation of porcine circovirus 2 type epidemiology survey and immune effect of vaccine provides a kind of effective technology means.
In a word, the new strain of the present invention is that a new way has been opened up in the research such as pathogenic, pathogenesis, vaccine immunity and molecular diagnosis of porcine circovirus 2 type.
Description of drawings
Fig. 1: the reaction of recombinant clone plasmid enzyme restriction is identified.
Swimming lane 1: single-gene group+carrier; Swimming lane 2: dual-gene group+carrier; Swimming lane 3: empty carrier; M is the DNA standard specimen.
Fig. 2: the IPMA of virus antigen detects in the PCV2 infective molecule cloning transfectional cell.
A: virus infected cell; B: non-infected cells contrast.
Recombinant type and wild-type PCV2 are differentiated in Fig. 3: PCR and restriction fragment length polymorphism analysis.
Swimming lane 1: recombinant type virus PCR product S alI enzyme is cut and is produced 870bp and 186bp two bands; Swimming lane 2: wild-type virus PCR product (1057bp) SalI enzyme is cut and is negative; M is the DMA standard specimen.
Fig. 4: PCV2/PE strain virus particle immunocomplex electron microscopic observation.
Fig. 5: the immunohistochemical methods method detects virus antigen in the experimental infection pig lymphoglandula.
Embodiment
Further describe the present invention by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit the scope of the invention.
The structure of [embodiment 1] strain of the present invention
1 material and method
1.1 virus, cell, antibody, bacterial strain and carrier PCV2 wild-type strain system are that the sick pig of PMWS separates acquisition (Liu Changming from clinical manifestation, Zhang Chaofan, the gorgeous force etc. of endangering. the porcine circovirus 2 type cell cultures adapts to the cultivation and the evaluation of strain. Chinese animal and veterinary association's the 6th scientific seminar's collection of thesis of animal and veterinary biotechnology association (Jinan), 2005, p196-199.) the positive porcine blood serum (it is 3200 times that IPMA tires) of PCV2 is used for virus antigen and detects; Used e. coli strains (Top10), carrier (pUC19) and the restriction enzyme enzyme of gene clone is available from Japanese Bao Bio-Engineering Company (Takara); The pig kidney continuous cell line (PK15) that no pig circular ring virus 1 type (PCV1) pollutes is by external introduction, and preserve this chamber.
1.2 design of primers and pcr amplification primer are according to GenBank (AF201311) sequences Design.PCV2-F:5 '-
GTCGACTGTTCTGTAGCATTCTTCCA-3 ', corresponding sequence is 920～946; PCV2-R:5 '-
GTCGACGGAGGAAGGGGGCCAGTT-3 ', corresponding sequence is 925～901.Underscore place tilted letter is represented the base of replacing, and constitutes the SalI restriction enzyme site.Viral DNA extracts and carries out (Liu CM according to a conventional method, Ihara T, Nunoya T, et al.Development of an ELISA based on the baculovirus-expressedcapsid protein of porcine circovirus type 2 as antigen[J] .J Vet Med Sci, 2004,66 (3): 237-242.), TaKaRa Ex Taq PCR test kit carries out the viral genome amplification, specific procedure is: pre-94 ℃ of 2min of sex change, 35 circulation [94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1.5min], 72 ℃ of 7min of extension.The PCR product is through 1% agarose gel electrophoresis Analysis and Identification.
1.3 the viral genome clone cuts through the SalI enzyme with order-checking PCR product and handles and the gel electrophoresis purifying, be inserted into the plasmid vector pUC19 SalI restriction enzyme site of same treatment, method is seen document (Sa nurse Brooker J, Russell's work DW (Huang Peitang etc. translate). molecular cloning experiment guide [M]. the 3rd plate, Science Press, 2002.).373A type dna sequence dna instrument order-checking (Perkin-Elmer test kit), the DNAsis software analysis.
Test-results: pcr amplification product is 1.7kb, cuts processing through the SalI enzyme, is cloned into the pUC19 carrier.Get 3 positive colonies and carry out sequencing, unanimity is not found base mispairing and disappearance as a result.1768 based compositions of genome total length, contain ORF1 and ORF2 coding region, AF166528, NC002173, AF264843, AF027217, AF118897, AF364094 and the AF201897 sequence of sequence and GenBank login are relatively, ORF1 district homology is 97.6%～99.8%, the ORF2 district is 93.7%～98.6%, and full genome is 96.2%～99.3%.
1.4 the structure full-length gene group recombinant plasmid of infective molecule cloning, ScaI complete degestion (single endonuclease digestion site on the pU19 carrier) carries out SalI partially digested (inserting genome both sides restriction enzyme site) more in advance.Long-armed (3506bp) contain the single-gene group reclaimed in gel electrophoresis and another contains single-gene group galianconism (2689bp), and gel-purified contains two of viral full-length gene group long-armed and galianconism with carrier swims and be with, and uses T
4Ligase enzyme connects 2 viral genome of cis connection on carrier of acquisition, both is connected the back obtain to contain the recombinant plasmid (pUC/PCV2 that dual-gene group of cis arranged
+), enzyme is cut qualification result and is seen Fig. 1.
1.5 the recombinant plasmid cell transfecting test infective molecule cloning plasmid transfection PK15 cell of purifying, transfection reagent is Lipofectamine2000 (Invitrogen), is undertaken by producer's explanation.Cell after the transfection is put 37 ℃ of 5%CO
2Gather in the crops viral cultures after cultivating 72h, join synchronously by 10% inoculum size in the cell suspension of new digestion, 37 ℃ leave standstill cultivation 24h formation individual layer, carry out D-glucosamine by the Tischer method and handle (Tischer I, Peters D, Rasch R, et al.Replication of porcine circovirus:induction by glucosamine andcell cycle dependence[J] .Arch Virol, 1987,96:39-57.), virus culture is through 3 freeze thawing follow-up generations.
1.6 the rescue of labeled virus is with containing dual-gene group of recombinant plasmid (pUC/PCV2
+) transfection PK15 cell, IPMA detects virus antigen.The positive cell of virus infection is dyed red-brown (Fig. 2 a), the uninfecting virus cell contrast reaction (Fig. 2 b) that is negative.Positive cell is point type and distributes, main poly-in nuclear area and tenuigenin district.The result shows, successfully obtains new strain (name PCV2/PE) with recombinant plasmid transfected cell, and it has identical antigenicity with street strain.
1.7 immunoperoxidase monolayer cell test (IPMA) is adopted in the evaluation of recombinant virus, carrying out virus infected cell antigen and viral level measures, immunoelectron microscopic method identifying virus morphology, concrete grammar is seen document (Liu Changming, Zhang Chaofan, the gorgeous force etc. of endangering. the porcine circovirus 2 type cell cultures adapts to the cultivation and the evaluation of strain. Chinese animal and veterinary association's the 6th scientific seminar's collection of thesis of animal and veterinary biotechnology association (Jinan), 2005, p196-199; Liu Changming, Lu Yuehua, Zhang Chaofan etc. the expression of porcine circovirus 2 type recombinant C ap albumen in insect baculovirus. Chinese animal and veterinary association's the 6th scientific seminar's collection of thesis of animal and veterinary biotechnology association (Jinan), 2005, p200-202.).
1.8 genetic marker strain and wild-type virus nucleic acid telling test adopt primer P1 and P2 respectively artificial strain (PCV2/PE) and wild-type strain (PCV2/LG) genome that makes up of the present invention to be carried out pcr amplification, design of primers is positioned at SalI restriction enzyme site both sides, P1:5 '-CAGCAAGAAGAATGGAAGAAGCGGA-3; P2:5 '-CCAGGACTACAATATCCGTGTAACT-3.The amplifying target genes segment is long to be 1057bp, and the PCR reaction conditions is with 1.2.Product is cut through the SalI enzyme can obtain 870bp and two segments of 189bp; The wild-type virus genome does not have the SalI restriction enzyme site, and the PCR product is constant, and wild-type virus pcr amplification product endonuclease reaction is negative, PCR product constant (Fig. 3).This shows, adopt PCR method genetic marker strain of the present invention can be differentiated mutually with wild-type virus.
Embodiment 2 usefulness strain of the present invention prepares inactivated vaccine
Make seed culture of viruses with the PCV2/PE strain, through cell cultures technology, obtain viral cultures, viral level reaches 1 * 10
6.0TCID
50More than/the ml.Viral cultures is prepared into inactivated vaccine through formalin-inactivated with the high-quality adjuvant emulsion.Carried out the check of work in-process and finished product vaccine by the requirement of veterinary drug biological products rules, goods carry out following clinical trial with being up to the standards.
The preparation and the using method thereof of embodiment 3 PCV2-IPMA antibody diagnosing reagent kits.
Get the PK15 cell suspension of new digestion, inoculate 2.5%PCV2/PE virus liquid synchronously, be inoculated in 1,3,5,7,9,11 odd columns (100 μ L/ hole) of 96 porocyte culture plates, even column (2,4,6,8,10,12) is the normal healthy controls cell hole of not virus inoculation, and culture plate is put 37 ℃ of 50mL/L CO
2Cultivate 48h～72h under the condition and form individual layer, take out the back and embathe cell hole 1 time with PBS, with the liquid-solid 20min of deciding of 33% acetone-PBS, dry rearmounted-20 ℃ of preservations are standby after drying up.
PCV2-IPMA diagnostic kit schedule of operation: the detection reaction plate of getting method for preparing is put the room temperature preheating, embathe 1 time with PBS, with 0.1% gelatin-PBS liquid by 2 times of serial dilution porcine blood serum to be checked such as 1: 25,1: 50,1: 100, do reference with the PCV2 positive and negative serum, the serum of every part of dilution joins 1 antigen hole and 1 cell control well interior (100uL/ hole) respectively, puts 37 ℃ of wet boxes and hatches 1.5h; PBS washs 3 times, adds the SPA-HRP liquid (Zymed product) of 1: 3000 times of dilution, and every hole 50uL puts 37 ℃ of wet boxes and hatches 1h; PBS washing 3 times adds 50uL/ hole substrate reactions liquid [3-amino-9-ethyl imidazol(e) (AEC) is the Sigma product], puts color development at room temperature 30min, gets rid of except that using distilled water wash 1 time behind the substrate solution, dry back observation by light microscope result of determination.PCV2 positive serum and the dyeing of virus infected cell hole are red-brown, answer the non-coloring reactor to be judged to positive reaction with the healthy cell control wells; The equal non-coloring reaction of PCV2 negative serum and virus infection hole and cell control well is judged to feminine gender, and result of determination was effective when the positive and negative control serum was all set up.
Test example 1 virus strain infection of the present invention cell characteristics and morphology of virus are learned and are identified
PK15 cell (the external introduction, the Harbin veterinary institute is preserved), successive cell subculture 60 times are infected in the PCV2/PE strain that the present invention makes up.In preceding 50 generations, do not produce cytopathy (CPE) substantially, but along with the lifting of viral passage number, as 60 generation viral cultures inoculating cell and control group contrast, can be observed slight CPE, show as the cell hypertrophy, become circle, refractivity variation etc.Increase with generation, virus titer progressively raises.5th, the viral cultures poison valency survey of 10,20,30,45 and 60 generations is respectively 10
2.5, 10
2.7, 10
3.5, 10
4.7, 10
5.5, 10
6.6TCID
50/ ml.
Get viral cultures of the present invention and carry out the immuno-electron microscope evaluation, observe the mixture (Fig. 4) of the virus-like particle formation of form homogeneous, rounded particle, diameter is about 17nm, with wild-type virus form basically identical, finds no exogenous virus and pollutes.
Test example 2 virus strain infection of the present invention animal experiments
One, test method
Get the 60th generation viral cultures of the present invention (cultivating according to ordinary method), inoculate 4 of 35 age in days negative antibody weanling pigs through vein and each 1ml of collunarium approach, inoculation contrasts 2 of pigs with establishing not.Isolated rearing, body weight, body temperature variation are measured in blood sampling regularly, observe the clinical response situation.Test pig 35d behind virus inoculation compels to kill, and carries out pathologic finding and PCR and measures.
Two, test-results
With 4 of PCV2/PE strain inoculation test pigs, have 2 tangible clinical responses to occur in inoculation back, show as by hair coarse, the body surface lymphadenectasis, poor growth, skin rashes etc., body temperature reaches 41.3 ℃ for the moment; Other test pig reactions are slightly light.Inoculation back 35d compels to dissect extremely finding, the multiple Lymphoid tissue performance of test group pig enlargement and hyperemia in various degree, and interstitial pneumonia, the greyish white focus of kidney point-like, valvula caeca filter palpebral edema expands.Immunohistochemical methods detects and shows, lymphoglandula filter born of the same parents see that a large amount of virus antigen positive cell (see figure 5)s are arranged in the district.PCR detects multiple internal organs and all is positive, and more with pouring under intestines pouring, groin pouring, the jaw, tonsilla and spleen viral level, the heart, liver, lung, kidney and testis take second place.The 1st week of inoculation back plays PCR and detects the serum-virus mass formed by blood stasis, and the 2nd week played IPMA and detects serum antibody.Control group pig duration of test no abnormality seen, various detections are negative.
The security of test example 3 inactivated vaccines of the present invention and immune protection effectiveness test
Safety testing: with the prepared PCV2/PE inactivated vaccine of embodiment 2, by each 3 of various dose (0.5ml/ head, 1ml/ head, 2ml/ head, 4ml/ head) intramuscular inoculation 25～30 ages in days test piglets, clinical observation was not seen no abnormal reaction in 28 days, proved that vaccine product is safe to this animal.
Vaccine immunity potency test: with the prepared PCV2/PE inactivated vaccine of embodiment 2, adopt 0.5ml/ head, 1ml/ head, 2ml/ three kinds of dosage immunity 25～30 age in days age in days piglets, 3 every group, establish 3 of not vaccination contrast pigs.Sun is all changeed in antibody test in 21 days behind all vaccine immunities, and with detecting viral toxicaemia behind the strong virus attack, calculating test protection ratio result is that 0.5ml, 1ml and 2ml immune group all obtain 100% protection, control group 100% morbidity.
Test example 4 PCV2 antibody diagnosing reagent kits detect serum sample PCV2 antibody test
PCV2/PE viral cultures diluent is inoculated in the PK15 cell suspension of new digestion (2 * 10
4), in 96 porocyte culture plates, do not inoculate control wells by 100 μ L/ hole dispensings with establishing virus, put 37 ℃ of 50mL/L CO
2Cultivate under the condition and form individual layer, liquid-solid fixed with acetone-PBS, dry rearmounted-20 ℃ of preservations.
IPMA schedule of operation: PBS embathes once, with 0.1% gelatin-PBS liquid porcine blood serum to be checked is pressed 2 times of serial dilutions such as 1: 25 grade, compare with PCV2 positive or negative serum, the serum of every part of dilution adds 1 antigen hole and 1 cell control well (100uL/ hole) respectively, put 37 ℃ of wet boxes and hatch 1.5h, PBS washing 3 times, the SPA-HRP liquid (50uL/ hole) that adds dilution, put 37 ℃ of wet boxes and hatch 1h, PBS washing 3 times adds 50uL/ hole substrate reactions liquid (AEC), put the existing look 30min of room temperature, get rid of and remove substrate solution, use distilled water wash 1 time, use the observation by light microscope result of determination.
PCV2 positive serum and the dyeing of virus infected cell hole are red-brown, and are judged to positive reaction with healthy cell control wells non-coloring reactor; The equal non-coloring reaction of PCV2 negative serum and virus infection hole and cell control well is judged to feminine gender.
The result shows that detecting artificial challenge's porcine blood serum the 1st all antibody recall rates with PCV2-IPMA test kit of the present invention is that 50%, the 2～5 all antibody recall rates are 100%, detects all negative with establishing the control group pig anteserum sample.With test kit 520 parts of serum in 13 pig farms, ground such as Heilungkiang, Jilin, Liaoning, Hebei, Shanghai have been carried out the PCV2 antibody test, 8/13 pig farm antibody positive rate is greater than 50%, and antibody positive rate is distributed in 57.1%～100%.In sum, PCV2-IPMA test kit of the present invention provides a kind of new technique means for the evaluation of epidemiology survey and immune effect of vaccine.
Claims (4)
1. a porcine circovirus 2 type (Porcine circovirus type 2) strain PCV2/PE, its preserving number is CGMCC No.1617.
2. the application of the PCV2/PE strain of claim 1 in setting up weanling pig multisystemic wasting syndrome animal model comprises: with described PCV2/PE strain artificial challenge test pig.
3. the application of the PCV2/PE strain of claim 1 in preparation prevention porcine circovirus 2 type vaccine.
4. diagnostic kit that detects porcine circovirus 2 type antibody is characterized in that containing the viral cultures of the PCV2/PE strain of claim 1.
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| CN102120987A (en) * | 2010-12-10 | 2011-07-13 | 中国农业科学院哈尔滨兽医研究所 | Construction of four different sub-genotypes of porcine circovirus type 2 molecular cloning strains and application thereof |
| CN103421748A (en) * | 2013-09-06 | 2013-12-04 | 青岛易邦生物工程有限公司 | Porcine circovivus2 strain and application thereof |
| CN106701794A (en) * | 2015-11-13 | 2017-05-24 | 广东永顺生物制药股份有限公司 | Virus rescue method of porcine circovirus 2 dual-copy infectious clone |
| CN106701821A (en) * | 2015-11-13 | 2017-05-24 | 广东永顺生物制药股份有限公司 | Application of porcine circovirus type-2 dual-copy infectious clone in indirect immunofluorescence experiment |
| CN106701793A (en) * | 2015-11-13 | 2017-05-24 | 广东永顺生物制药股份有限公司 | Application of porcine circovirus type 2 double-copy infectious clone to indirect ELISA detection method |
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| US6391314B1 (en) * | 1997-10-03 | 2002-05-21 | Merial | Porcine circoviruses vaccines diagnostic reagents |
| US6497883B1 (en) * | 1999-06-10 | 2002-12-24 | Merial | Porcine circovirus recombinant poxvirus vaccine |
| CN1579553A (en) * | 2004-05-18 | 2005-02-16 | 浙江大学 | Method for preparing II-type pig's ring-virus nucleic vaccine and the use thereof |
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| CN102120987A (en) * | 2010-12-10 | 2011-07-13 | 中国农业科学院哈尔滨兽医研究所 | Construction of four different sub-genotypes of porcine circovirus type 2 molecular cloning strains and application thereof |
| CN103421748A (en) * | 2013-09-06 | 2013-12-04 | 青岛易邦生物工程有限公司 | Porcine circovivus2 strain and application thereof |
| CN106701794A (en) * | 2015-11-13 | 2017-05-24 | 广东永顺生物制药股份有限公司 | Virus rescue method of porcine circovirus 2 dual-copy infectious clone |
| CN106701821A (en) * | 2015-11-13 | 2017-05-24 | 广东永顺生物制药股份有限公司 | Application of porcine circovirus type-2 dual-copy infectious clone in indirect immunofluorescence experiment |
| CN106701793A (en) * | 2015-11-13 | 2017-05-24 | 广东永顺生物制药股份有限公司 | Application of porcine circovirus type 2 double-copy infectious clone to indirect ELISA detection method |
| CN117467698A (en) * | 2023-09-20 | 2024-01-30 | 南京农业大学 | Porcine circovirus type 3 recombinant plasmid and application thereof |
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| CN100489092C (en) | 2009-05-20 |
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Application publication date: 20071219 Assignee: Harbin Weike Biotechnology Development Co., Ltd. Assignor: Harbin Veterinary Medicine Inst., China Academy of Agriculture Contract record no.: 2014230000506 Denomination of invention: Type II genetic marker strain of porcine circovirus and its application Granted publication date: 20090520 License type: Exclusive License Record date: 20140911 |
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