CN107058247A - The virus attenuated strain of high-fidelity porcine reproductive and respiratory syndrome and its application - Google Patents

The virus attenuated strain of high-fidelity porcine reproductive and respiratory syndrome and its application Download PDF

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CN107058247A
CN107058247A CN201710500287.8A CN201710500287A CN107058247A CN 107058247 A CN107058247 A CN 107058247A CN 201710500287 A CN201710500287 A CN 201710500287A CN 107058247 A CN107058247 A CN 107058247A
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porcine reproductive
respiratory syndrome
prrsv
plants
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CN107058247B (en
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李鹏成
侯继波
郑其升
张晓燕
陈瑾
于小明
乔绪稳
张元鹏
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Jiangsu Academy of Agricultural Sciences
Yichun University
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Abstract

The present invention provides the virus attenuated strain of high-fidelity porcine reproductive and respiratory syndrome and its application, belongs to biological technical field.The low virulent strain is NJRb plants of porcine reproductive and respiratory syndrome virus PRRSV, and its microbial preservation number is：CGMCC No.13800.The present invention also provides the preparation method of the low virulent strain virus liquid, including：The virus attenuated strain of high-fidelity porcine reproductive and respiratory syndrome is bred using the cells of Marc 145, cell lysis, centrifuging and taking supernatant obtains virus liquid.The present invention also provides porcine reproductive and respiratory syndrome attenuated vaccine, and active component is the virus attenuated strain of the porcine reproductive and respiratory syndrome.Low virulent strain mutation rate of the present invention is low, genetic stability is higher, passes after 100 generations, remains in that original immunogenicity and virulence, the strain has good protection power for high-pathogenicity blue ear disease.The attenuated vaccine of the present invention, has good protection power, security is higher for high-pathogenicity blue ear disease.

Description

The virus attenuated strain of high-fidelity porcine reproductive and respiratory syndrome and its application
Technical field
The invention belongs to biological technical field, and in particular to a kind of virus attenuated strain of high-fidelity porcine reproductive and respiratory syndrome And its application.
Background technology
Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respriratory Syndrome, PRRS blue otopathy) is also known as, is a kind of important strong infection of pig as caused by porcine reproductive and respiratory syndrome viral (PRRSV) Disease, huge economic loss is caused to China's pig industry.PRRS inactivated vaccines are substantially invalid, and PRRS attenuated vaccines have immune The advantages such as protection is fast, effect height, are current China's prevention and control PRRS the only resources.But, PRRS attenuated vaccine strains be easy to variation, Genetic stability is poor, thus cause that immunogenicity is easy to lose, virulence be easy to return it is strong.
The content of the invention
It is an object of the invention to provide a virus attenuated strain of plant height fidelity porcine reproductive and respiratory syndrome, the strain mutation rate Low, genetic stability is higher, passes after 100 generations, remains in that original immunogenicity and virulence, the strain is for high-pathogenicity blue Otopathy has good protection power.
It is a further object of the present invention to provide porcine reproductive and respiratory syndrome virus attenuated vaccine, the attenuated vaccine is for height Pathogenic blue otopathy has good protection power, and security is higher.
The purpose of the present invention adopts the following technical scheme that realization：
A kind of virus attenuated strain of high-fidelity porcine reproductive and respiratory syndrome, is porcine reproductive and respiratory syndrome virus PRRSV- NJRb plants, its microbial preservation number is：CGMCC No.13800.
In the present invention, the nucleotide sequence of the ORF5 genes of the low virulent strain such as SEQ ID NO:Shown in 1.
In the present invention, the nucleotide sequence of the nsp2 genes of the low virulent strain such as SEQ ID NO:Shown in 2.
The present invention also provides the preparation method of the virus attenuated strain virus liquid of the high-fidelity porcine reproductive and respiratory syndrome, bag Include following steps：It is virus attenuated using high-fidelity porcine reproductive and respiratory syndrome described in Marc-145 cells propagation claim 1 Strain, cell lysis, centrifuging and taking supernatant obtains virus liquid.
The present invention also provides porcine reproductive and respiratory syndrome attenuated vaccine, and active component is that pig breeding is integrated with breathing Syndrome virus low virulent strain.
The present invention also provides the weak poison of the porcine reproductive and respiratory syndrome and is preparing the weak malicious epidemic disease of porcine reproductive and respiratory syndrome Application in terms of seedling.
Beneficial effect of the present invention relative to existing low virulent strain and its vaccine：
1. the present invention has screened the virus attenuated strain of high-fidelity porcine reproductive and respiratory syndrome by the pressurization of mutagens, The strain changes NJ plants of tissue tropisms of PRRSV, significantly reduces the preferendum to lung tissue compared with NJ plants of PRRSV；Body Outer fidelity performance improves more than 5 times, and genetic stability is good；Security is higher, continuous passage 5 times in immune swine body, main protection Property antigen gene ORF5 and the key virulence gene nsp2 frequencies of mutation decline 3 times, solve existing PRRSV live vaccines due to heredity The caused immunogenicity of stability difference is easy to lose, virulence is easy to return strong global problem.High-fidelity pig breeding of the present invention is with exhaling Inhale after the syndrome virus attenuated strain kind malicious generation of continuous passage 100, remain in that original immunogenicity and virulence, still can be used for Attenuated vaccine is prepared, production cost is significantly reduced, improves production efficiency.
2. using after attenuated vaccine immunity of the present invention, 100% protection can be provided for inoculation animal.
Brief description of the drawings
Fig. 1 is ORF5 and nsp2 gene PCR amplified production electrophoretograms, and wherein M is DNA Marker, and 1,2,3 is that ORF5 expands Increase fragment；4,5,6 be nsp2 amplified fragments.
Fig. 2 is PRRSV-NJRb plants of inoculation PAM cell CPE observation figures of high-fidelity strain, A：PRRSV-NJRb plants；B： PRRSV-NJ plants；C：Blank control.
Fig. 3 is PRRSV-NJRb plants of piglet in-vivo tissue preferendum qualification figures, and * * represent P ＜ 0.01.
Embodiment
Embodiment NJ plants of Attenuations on Marc-145 cells of 1 PRRSV
1. cell culture
Marc-145 cell growths are in cell culture fluid, in 37 DEG C, 5%CO2Cultivated in incubator.Marc-145 cells Growing up to is used for virus inoculation after individual layer.Cell culture fluid：The calf of (56 DEG C of processing 30min) is inactivated containing 10% (concentration expressed in percentage by volume) Cow's serum and the DMEM cell culture fluids of dual anti-(penicillin, streptomysin, concentration are 100U/mL).
The culture that NJ plants of 2.PRRSV
NJ-a plants of PRRSV be the strong poison of the american type PRRSV that is separated from the acute onset swinery of Nanjing (be disclosed in Zheng its Rise, Li Peng, Cao Ruibing, Hou Jibo, Chen Puyan etc., the GP5 of coexpression PRRSV modification and the recombinant modified bovine vaccine disease of M albumen The structure and its biological characteristics of malicious Ankara strain, bioengineering journal, 2008,24 (5):766-773).In the present invention will It is disclosed in " GP5 of coexpression PRRSV modification and the structure and its biology of the recombinant modified vaccinia virus ankara strain of M albumen NJ-a plants of PRRSV in characteristic " is used as kind of a poison.In addition, in the present invention, PRRSV is abbreviated as into PRRSV NJ NJ-a plants Strain.The malicious continuous passage on the Marc-145 cells for grow up to individual layer is planted by PRRSVNJ plants, when cytopathy reaches 70%~80% When harvest virus, multigelation 3 times, then carry out continuation passage, until the 100th generation.Every 10 generations after the 60th generation, 1 is selected ~2 plaque viruses, are respectively labeled as NJ generations -1, NJ generations -2.Toxicity test is carried out to each generation virus with piglet, specifically Method is as follows：By each 5 piglets of generation virus inoculation, every inoculation 1ml (viral level is shown in Table 1) is observation body temperature and morbidity, dead The piglet quantity (the results are shown in Table 1) died.As a result show：Virulence is remarkably decreased after NJ plants of F80 of PRRSV, is low virulent strain.
Pathogenicity result of the different generations of NJ plants of 1 PRRSV of table to piglet
The high-fidelity PRRSV low virulent strains of embodiment 2 are cultivated and external identification
The poison mutagenesis continuous passage of 1.PRRSV kinds
Mutagenesis continuous passage：Melted using cell culture fluid (in composition be the same as Example 1) culture Marc-145 cells to 70% Close, then add final concentration of 200 μM of mutagens Ribavirin (virazole, purchased from sigma companies) pretreatment 2h, remove Nutrient solution, NJ plants of F80 of PRRSV are added for (NJ plants of PRRSV plants poison and used after the generation of Marc-145 passages 80 according to MOI for 1 The virus of acquisition, the virus is low virulent strain), 1h is infected, PBS is washed twice；Then, it is thin plus containing 200 μM of mutagens Born of the same parents' nutrient solution (in composition be the same as Example 1) continues to cultivate, and finally harvests virus.The viral generation of continuous passage 20 according to the method described above.
Control group is set up simultaneously, is added without mutagens in control group processing, other are with mutagenesis continuous passage method.
2. high-fidelity PRRSV kinds poison plaque purification
Virus after continuously passing for 20 generations through mutagenesis in the present embodiment title 1 carries out clone purification, and specific method is as follows：Will Marc-145 cells are cultivated in six orifice plates to individual layer；Purified virus work is treated with cell culture fluid (in composition be the same as Example 1) Appropriate dilution, cell culture fluid is discarded before inoculation, is rinsed cell monolayer with 5ml PBS or cell culture fluid, is added per hole Replaced in 0.2ml viral dilution liquid, control group with cell culture fluid, put in 37 DEG C of CO2gas incubators and adsorb 1h, per 15min Shake once, to be uniformly distributed virus；PBS washs unadsorbed virus；The cell culture fluid of 2 times of concentrations is taken to add Enter in isometric 2% agar solution (preheating), 1ml is added per hole, the solidification to be cooled of horizontalization platform is after 37 DEG C of carbon dioxide trainings Support and 2~3d of culture is inverted in case；Some plaque harvest viruses are drawn under agar layer with elbow straw, are then inoculated into respectively pre- Cultivated in the blake bottle of the cell monolayer first prepared, then carry out 2 wheel plaque purifications, selecting can give birth in Marc-145 cells The clonal virus grown but can not grown on PAM (porcine alveolar macrophage), it is final to obtain porcine reproductive and respiratory syndrome disease It is malicious PRRSV-NJRb plants.It regard the virus obtained here by plaque purification as kind of a poison.PRRSV-NJRb plants are planted malicious ORF5 bases The nucleotide sequence of cause such as SEQ ID NO:Shown in 1, the nucleotide sequence such as SEQ ID NO of nsp2 genes:Shown in 2.
3. high-fidelity PRRSV strains are identified
Poison is planted using Marc-145 passages to the 50th generation by PRRSV-NJRb plants, obtains PRRSV-NJRb plants of F50 generations, Therefrom 50 clonal virus of picking；By NJ plants of F100 generations of PRRSV, (NJ plants of PRRSV plants the disease that poison passed on for 100 generations and obtained in addition Poison, is abbreviated as non-mutagenized virus) equally it is passaged in the 50th generation, and never mutagenized virus F50 generations and is chosen using Marc-145 cells Take 50 clonal virus.
(1) Trizol methods extract viral RNA
The RNA of above-mentioned 100 clonal virus is extracted respectively.Specific method is as follows：The μ L of plaque virus-4 00 of harvest are added 600 μ L TRIzol reagents (TAKARA), fully cracking；Chlorination imitates 0.2mL, jog 15s；It is stored at room temperature 5min, 4 DEG C, 12 000g centrifuges 15min, takes supernatant colourless aqueous phase (about 500 μ L) into 1.5mL Rnase-free EP pipes, plus 500 μ L isopropanols, The Ep pipes that turn upside down are mixed, and are stored at room temperature 10min；4 DEG C, 12 000g centrifugation 10min, supernatant discarding, using the ethanol of 1mL 75% The aqueous solution (preparation of DEPC water) is washed, 4 DEG C, 12 000g centrifugations 10min；Remove supernatant, drying at room temperature 10min, plus Rnase-free dH2The μ L of O (TAKARA) 20 dissolve, and solution is RNA sample in Ep pipes.
(2) cDNA synthesis
Using PrimeScriptTMAbove-mentioned each viral RNA is carried out reverse transcription and obtained by cDNA synthetic agent box (TAKARA) To cDNA, -70 DEG C save backup.
(3) diversity gene fragment PCR is expanded
Main protection antigen gene ORF5 and key virulence gene nsp2 are the high changes in generally acknowledged PRRSV full-length genomes Area, we separately design the primer of amplification ORF5 and nsp2 fragments, the sequencing of PCR amplifying target genes Hou Song companies.
ORF5 fragment sense primers：5’-ATGAGGTGGGCAACCGTTTTAG-3’；
ORF5 fragment anti-sense primers：5’-GTAATGGAAAACGCCAAAAGCACCT-3’.
Nsp2 fragment sense primers：5’-GGTTTGGCAGACATAAGTGGT-3’；
Nsp2 fragment anti-sense primers：5’-CCTTGAGAAAGCACATAAGCTCC-3’.
PCR reaction systems are：Upstream and downstream primer (10pmol/L) each 1 μ l, dNTPs (2.5mmol/L) 2.0 μ l, 5 × μ l, PrimeSTAR the GXL long-chain high-fidelities enzymes (1.25U/ μ l) 0.5 of 5 μ l, cDNA templates of PrimeSTAR GXL Buffer 2.0 μ l, sterilizing distilled water supplies volume to 25 μ l.PCR response procedures：95 DEG C of pre-degeneration 5min；94 DEG C of reaction of degeneration (RD) 30s, 53 DEG C are moved back Fire reaction 45s, 72 DEG C of extension 45s, carry out 35 circulations, cycle annealing temperature is than a preceding cycle annealing temperature next time Increase by 0.2 DEG C (initial value of annealing temperature is 53 DEG C)；Last 72 DEG C of extensions 10min.In control cDNA is substituted with pure water.Reversion Agents useful for same is originated TAKARA companies in record and PCR reactions.
Pcr amplification product is identified using 1% agarose gel electrophoresis.From Fig. 1, it can be seen that amplified production ORF5 fragments Size be about 725bp, the size of nsp2 fragments is about 2962bp.Company is sent to carry out sequencing analysis PCR primer.It the results are shown in Table The frequency of mutation of 2, PRRSV-NJRb plants of F50 generation (being abbreviated as Rb50) ORF5 genes is substantially less than non-mutagenized virus, the frequency of mutation (i.e. mutation count/103Nt 0.37) is dropped to by 1.86, that is, declines 5 times；Amino acid mutation frequency (i.e. mutation count/103Aa) from 4.88 it is reduced to 0.99.As seen from Table 3, the frequency of mutation of PRRSV-NJRb plants of F50 generation (being abbreviated as Rb50) nsp2 genes also shows Write and be less than non-mutagenized virus, the frequency of mutation drops to 0.2 from 2.06, have dropped 10.3 times；Amino acid mutation frequency drops from 3.22 As little as 0.4, that is, it have dropped 8.05 times.Result above shows, compared with non-mutagenized virus, PRRSV-NJRb pnca gene mutation rates It is remarkably decreased, genetic stability is significantly improved, external fidelity performance pole is significantly improved.Because ORF5 is PRRSV main protectives Antigen gene, nsp2 is PRRSV crucial virulence gene, thus PRRSV-NJRb plants of immunogenicity is not easy to lose, virulence not Yi Fanqiang.Therefore, PRRSV-NJRb plants are the virus attenuated strains of high-fidelity porcine reproductive and respiratory syndrome.
The ORF5 genes of table 2 and the amino acid sequence frequency of mutation comparative result in mutagenized virus and non-mutagenized virus
nt:Nucleotides, aa:Amino acid .*p<0.05,**p<0.01.
The nsp2 genes of table 3 and the amino acid sequence frequency of mutation comparative result in mutagenized virus and non-mutagenized virus
nt:Nucleotides, aa:Amino acid " * " represents p<0.05, " * * " represent p<0.01.
Porcine reproductive and respiratory syndrome virus is planted into poison for PRRSV-NJRb plants, preservation is carried out, preservation information is as follows：
Join the biomaterial (strain) of Ju：PRRSV-NJRb；
Classification And Nomenclature：Porcine reproductive and respiratory syndrome virus Porcine Reproductive and Respriratory Syndrome virus；
Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)；
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；
Preservation date：On May 24th, 2017；
Deposit number：CGMCC No.13800.
The high-fidelity PRRSV low virulent strains tissue tropism of embodiment 3 identifies
Poison inoculation PAM (porcine alveolar macrophage) and Marc-145 cells (MOI=1) respectively are planted by PRRSV-NJRb plants, Continuous passage 5 times, observation change per generation virus CPE (cytopathic effect) separately harvests different generation viruses and carries out titre survey It is fixed.In NJ plants of F80 generations of PRRSV, are used into same procedure passage 5 times simultaneously, as a control group.As a result show：PRRSV-NJRb plants Inoculation PAM cells have no CPE to first generation poison NJRb P1 (referring to that kind of a poison was passed after 1 generation, obtained virus), and allusion quotation occurs in control group Type CPE (Fig. 2), PRRSV-NJRb plants of later each generations are also showed no CPE and (result is not shown) occur.Virus titer measurement result It is shown in Table 4.As a result show：PRRSV-NJRb plants compared with NJ plants of PRRSV, change NJ plants of tissue tropisms of PRRSV, significantly reduce To the preferendum of lung tissue.
The PRRSV-NJRb plants of inoculation PAM of table 4 and Marc-145 cell restrovirus titre comparative results
Note：NJRb P1 represent that planting poison for PRRSV-NJRb plants passed after 1 generation in table 4, obtained virus, other the like； NJ P1 represent that NJ plants of PRRSV plants poison and passed after 1 generation, obtained virus, other the like.(LOG10 represents virus titer in table Take denary logarithm).
Continuous passage genetic stability is evaluated in 4PRRSV-NJRb plants of piglet bodies of embodiment
1. pig is used in experiment
Screen PRRSV serum antigens, the double-negative piglet of antibody.
Serum antigen detection method is as follows：30 age in days piglet blood samples are gathered, serum is separated.Serum is extracted after total serum IgE Using PrimeScriptTM(TAKARA) reverse transcription of cDNA synthetic agent box is into cDNA, using PRRSV ORF5 specific primers (ORF5 upstream and downstream primer) is expanded, and detects PRRSV antigens.
ORF5 sense primers：5’-ATGAGGTGGGCAACCGTTTTAG-3’；
ORF5 anti-sense primers：5’-GTAATGGAAAACGCCAAAAGCACCT-3’.
PRRSV serum antibody application IDEXX kits (Ai Deshi companies of the U.S.) detect that operating procedure is to specifications.
2.PRRSV-NJRb plants in piglet body continuous passage to evaluate genetic stability
Malicious continuous passage 5 times in piglet body are planted by PRRSV-NJRb plants, it is steady to evaluate its heredity by gene sequencing detection It is qualitative.30 ages in days of poison inoculation, PRRSV serum antigens, the double-negative piglet of antibody, every piglet inoculation are planted by PRRSV-NJRb plants 2ml(106.0TCID50/ ml) virus liquid, 20 days after inoculation, from isolated viral in piglet body, 30 ages in days, PRRSV are then reached again The double-negative piglet of serum antigen, antibody, in this way continuous passage 5 times in piglet body.It is daily to see when last time is passed on Examine clinical change, blood separation serum is gathered using RT-PCR methods in 0d, 7d, 14d, 21d, 28d after inoculation (with reference to embodiment 2) Detect viremia virusemia.Cut open within 28 days after inoculation and kill, take lung, bronchial lymph nodes, tonsillotome to carry out tissue virus purification culture.Will Lung, bronchial lymph nodes, tonsil pathological material of disease are milled after smashing to pieces and to be made 1 with sterile PBS buffer：10 tissue suspension, plus Penicillin and streptomycin, puts after 4 DEG C of refrigerator effect 4h, 8000rpm centrifugations 20min；Supernatant is taken to be removed with 0.45 μm of filtering with microporous membrane Bacterium；Take filtrate to change the DMEM culture mediums containing 2%FBS after being inoculated in Marc-145 cell monolayers, 37 DEG C of absorption 1h, continue to cultivate, CPE situations, the harvesting venom when CPE reaches more than 75% are observed daily.After the virus liquid freeze thawing three times, Marc- is inoculated with 145 cells, 50 Plaque Clones of picking (P5 corresponded in table 5,6), expand ORF5 and nsp2 genes using RT-PCR, will expand Increasing fragment send company to be sequenced, and method is with reference to title 3 in embodiment 2.PRRSV is planted in son for NJ plants by poison using above-mentioned same procedure In pig body after continuous passage 5 times, 50 Plaque Clones of picking (comparison virus corresponded in table 5,6) are expanded using RT-PCR Amplified fragments are sent company to be sequenced by ORF5 and nsp2 genes, as a control group.
5, table 6 is the results are shown in Table, poison is planted for PRRSV-NJRb plants and connects discovery after 5 generations of biography in piglet body, ORF5 genes (table 5) are dashed forward Frequency is substantially less than comparison virus, and the frequency of mutation drops to 0.73 by 2.19, declines 3 times, and amino acid mutation frequency is from 5.17 It is reduced to 2.79.Nsp2 genes (table 6) frequency of mutation is also substantially less than comparison virus, and the frequency of mutation drops to 0.39 from 2.48 (have dropped 6.4 times), amino acid mutation drops to 0.53 (have dropped 6.7 times) from 3.54.Result above shows：PRRSV- NJRb plants in piglet body during continuous passage, genetic stability is high, is high-fidelity low virulent strain.
PRRSV-NJRb plants of table 5 ORF5 frequency of mutation comparative results after continuous passage 5 times in piglet body
nt:Nucleotides, aa:Amino acid .* represents p<0.05, * * represents p<0.01.
PRRSV-NJRb plants of table 6 nsp2 frequency of mutation comparative results after continuous passage 5 times in piglet body
nt:Nucleotides, aa:Amino acid .* represents p<0.05, * * represents p<0.01.
The PRRSV-NJRb plants of Immunity identifications of embodiment 5
Poison inoculation Marc-145 cell monolayers are planted by PRRSV-NJRb plants, are harvested when cytopathy reaches 70%~80% Cell culture, multigelation 3 times, centrifuging and taking supernatant is used as PRRSV-NJRb plants of F1 generation virus liquids；By F1 generation virus inoculation Marc-145 cell culture, the harvesting culture equally when cytopathy reaches 70%~80%, multigelation 3 times, from The heart takes supernatant as PRRSV-NJRb plants of F2 for virus liquid；By that analogy, PRRSV-NJRb plants of F100 are harvested for virus liquid.Survey PRRSV-NJRb plants of F100 are determined for virus liquid titre.By the same way, NJ plants of kind poison of PRRSV are used into Marc-145 individual layers Passage obtains NJ plants of F80 of PRRSV for virus liquid to the 80th generation.
Poison and its F100 are planted by PRRSV-NJRb plants, and DMEM is respectively adopted for virus liquid for virus liquid, NJ plants of F80 of PRRSV It is 1.0 × 10 that culture medium, which is diluted to viral level,5.0TCID50/ ml, is used as the vaccine in following inoculation test.
By 4~6 week old PRRSV serum virus antigens, negative antibody piglet 20,4 groups, first group are randomly divided into：Gao Bao True attenuated vaccine group (immune PRRSV-NJRb plants are planted poison)；Second group：High-fidelity attenuated vaccine group is (immune PRRSV-NJRb plants F100 generations)；3rd group：Comparison virus vaccine group (immune NJ plants of F80 generations of PRRSV)；4th group：Blank control group.It is immune to work as My god, first group of PRRSV-NJRb plants of every piglet intramuscular injection 2ml plants poison (1.0 × 105.0TCID50/ ml), second group of every son PRRSV-NJRb plants of F100 of pig muscle injection 2ml are for virus liquid (1.0 × 105.0TCID50/ ml), the 3rd group of every piglet muscle NJ plants of F80 of 2ml PRRSV are injected for virus liquid (1.0 × 105.0TCID50/ ml), the 4th group of every piglet intramuscular injection 2ml DMEM culture mediums.Intramuscular injection in 28 days attacks poison with strong poison PRRSV NJ strain virus liquid (10 after immune6.0TCID50/ ml) 2ml, often Day observation body temperature, record clinical symptoms and morbidity quantity；Respectively at attacking 0 after poison, 7,14,21,28 days collection blood testing virus Mass formed by blood stasis (RT-PCR, with reference to embodiment 2), Continuous Observation 28 days.28 days cut open inspections after poison are attacked, gross anatomy is observed.
Morbid pig criterion：1) at least 3 days body temperature is more than 41 DEG C；Or spiritual loss of appetite, eye conjunctivitis cough The respiratory symptom such as cough, breathe heavily；2) find that sheet consolidation occurs in lung substantially in cut open inspection；3) viremia virusemia, attacks and RT-PCR is used after poison Piglet serum is detected, PRRSV viremia virusemias at least continue 28.Meet the above two, you can be judged to morbidity.
As a result show：After immune, each group piglet is normal, the clinical symptoms such as no fever, apocleisis.Attack after poison, first, second All piglet body temperature of group are normal, and the 3rd group has 1 piglet body temperature to reach more than 41 ° 1 day, first, second, third group of experiment knot Clinical onset symptom is had no after beam, pathology cut open inspection result is shown as 100% protection (5/5).Blank control group in attacking after poison 3 days, The body temperature of all piglets reaches more than 41 DEG C, and more than 41 DEG C at most have reached 10 days；And piglet clinical manifestation have apocleisis, The symptoms such as cough, expiratory dyspnea, attack and start within 10 days after poison dead, pathology cut open inspection symbol all dead to 5 piglets during off-test Close clinical onset standard.The above results are proved：PRRSV-NJRb plants were planted after the malicious generation of continuous passage 100, were remained in that original immune Originality and virulence, still can be used for preparing attenuated vaccine, will significantly reduce production cost, improve production efficiency.
The PRRSV-NJRb plants of piglet in-vivo tissue preferendum identifications of embodiment 6
PRRSV-NJRb strain virus liquid and preparation method thereofs be the same as Example 5.
By 4~6 week old PRRSV serum virus antigens, negative antibody piglet 15,3 groups, first group are randomly divided into：Gao Bao True low virulent strain test group；Second group：The malicious test group of control；3rd group：Blank control group.The inoculation same day, first group of every piglet PRRSV-NJRb plants of F100 virus liquids (1.0 × 10 of intramuscular injection 2ml5.0TCID50/ ml), second group of every piglet intramuscular injection NJ plants of F80 virus liquids (1.0 × 10 of 2ml PRRSV5.0TCID50/ ml), the 3rd group of every piglet intramuscular injection 2ml DMEM culture Base.In 14 days cut open inspections after inoculation, collection lungs, tonsillotome, bronchial lymph nodes, musculature separate PRRSV, Real time PCR quantitative analysis of virus content (T.Opriessnig, N.E.McKeown, K.L.Harmon, et al.Porcine Circovirus Type 2Infection Decreases the Efficacy of a Modified Live Porcine Reproductive and Respiratory Syndrome Virus Vaccine.CLINICAL AND VACCINE IMMUNOLOGY,.2006,13(8),923–929.)。
As a result (Fig. 3) is shown：After PRRSV-NJRb plants of inoculation piglets, virus is in tonsillotome, bronchial lymph nodes, muscle groups Knit good, the no significant difference compared with comparison virus (NJ plants of F80 virus liquids of PRRSV) of middle propagation；And compareed in lung tissue Titre is 10 in viral lung tissue4.3±0.12TCID50/ g, and PRRSV-NJRb plants can not breed substantially, virus titer is in tissue 100.8±0.07TCID50/ g, decrease beyond 1000 times.PRRSV-NJRb plants of tissue tropisms of the result and vitro detection are consistent.
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>The virus attenuated strain of high-fidelity porcine reproductive and respiratory syndrome and its application
<130> 20170626
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 603
<212> DNA
<213>Porcine reproductive and respiratory syndrome virus
<400> 1
atgttgggga agtgcttgac cgcgtgctgt tgctcgcgat tgcttttttt gtggtgtatc 60
gtgccgttct atcttgctgt gctcgtcaac gccagcaaca acaacagctc tcatatccag 120
ttgatttata acttaacgct atgtgagctg aatggcacag attttctggc acaaaaattt 180
gacttggcag tggagacttt tgtcatcttc cccgtgttga ctcacattgt ttcctatgtg 240
gcactcacca ccagccattt ccttgacaca gttggtctgg ccactgtgtc caccgccgga 300
tattatcacg ggcggtatgt cttgagtagc atttacgcag tctgtgctct ggctgcgctg 360
atttgctttg tcattaggct tgcgaagaac tgcatgtcct ggcgctactc ttgtaccaga 420
tataccaact tccttctgga cactaagggc agactctatc gttggcggtc gcccgtcatt 480
gtggagaaaa ggggtaaggt tgaggtcgaa ggtcacctga tcgacctcaa gagagttgtg 540
cttgatggtt ccgcggcaac ccctttaacc agagtttcag cggaactatg gggtcgtccc 600
tag 603
<210> 2
<211> 2850
<212> DNA
<213>Porcine reproductive and respiratory syndrome virus
<400> 2
cggaaagaga gcaaggaaaa cacgctctgg tgcgactact atggtcgctc atcacgcttc 60
gtccgctcat gaaacccggc aggccacgaa gcacgagggt gccggcgcta acaaggctga 120
gcatctcaag cgctactctc cgcctgccga agggaactgt ggttggcact gcatttctgc 180
catcgccaac cggatggtga attccaactt tgagaccacc cttcctgaaa gagtaaggcc 240
ttcagatgac tgggccactg acgaggatct tgtgaacacc atccaaatcc tcaggctccc 300
tgcggccttg gacaggaacg gcgcttgcgg tagcgccaag tacgtgctta aactggaggg 360
tgagcattgg actgtctctg tgatccctgg gatgtcccct actttgctcc cccttgaatg 420
tgttcagggt tgttgtgagc ataagggcgg tcttgtttcc ccggatgcgg tcgaaatttc 480
cggatttgat cctgcctgcc ttgaccgact ggctaaggta atgcacttgc ctagcagtac 540
catcccagcc gctctggccg aatcgaccga cgactccaac cgtccggttt ccccggccgc 600
tactacgtgg actgtttcgc aattctatgc tcgtcataga ggaggagatc atcatgacca 660
ggtgtgcttg gggaaaatca tcagcctttg tcaagttatt gaggattgct gctgccatca 720
gaataaaacc aaccgggcta ctccggaaga ggtcgcggca aagattgatc agtacctccg 780
tggcgcaaca agtcttgagg aatgcttggc caaacttgag agagtttccc cgccgagcgc 840
tgcggacacc tcttttgatt ggaatgttgt gcttcctggg gttgaggcgg cgaatcagac 900
aaccgaccaa cctcacgtca actcatgctg caccctggtc cctcccgtga ctcaagagcc 960
tttgggcaag gactcggtcc ctctgaccgc cttctcactg tccaattgct attaccctgc 1020
acaaggtgac gaggttcatc accgtgagag gttaaattcc gtactctcta agttggaaga 1080
ggttgtcctg gaagaatatg ggctcatgtc cactggactt ggcccgcgac ccgtgctgcc 1140
gagcgggctc gacgagctta aagaccagat ggaggaggat ctgctagaac taaccaacac 1200
ccaggcgact tcagaaatga tggcctgggc ggctgagcag gtcgatttaa aagcttgggt 1260
caaaagctac ccgcggtgga caccaccacc ccctccacca agagttcaac ctagaagaac 1320
aaagtctgtc aaaagcttgc cagagggcaa gcctgtccct gctccgcgca ggaaggtcag 1380
atccgattgc ggcagcccgg ttttgatggg cgacaatgtc cctaacggtt cggaagaaac 1440
tgtcggtggt cccctcaatt ttccgacacc atccgagccg atgacaccta tgagtgagcc 1500
cgtacttgtg cccgcgtcgc gacgtgtccc caagctgatg acacctttga gtgggtcggc 1560
accagttcct gcaccgcgta gaactgtgac aacaacgctg acgcaccagg atgagcctct 1620
ggatttgtct gtgtcctcac agacggaata tgaggctttc cccctagcac catcgcagaa 1680
catgggcatc ctggaggcga gggggcaaga agttgaggga gtcctgagtg aaatctcgga 1740
tatactaaat gacaccaacc ctgcacctgt gtcatcaagc agctccctgt caagtgttaa 1800
gatcacacgc ccaaaatact cagctcaagc catcatcgac tctggcgggc cttgcagtgg 1860
gcatctccaa aaggaaaaag aagcatgcct cagcatcatg cgtgaggctt gtgatgcgtc 1920
caagcttggt gatcctgcta cgcaggagtg gctctctcgc atgtgggata gggttgacat 1980
gctgacttgg cgcaacacgt ctgcttacca ggcgtttcgc atcttaaatg gcatgtttga 2040
gtttctccca aagatgattc tcgagacacc gccgccccac ccgtgcgggt ttgtgatgtt 2100
acctcgcacg cctgcacctt ccgtgagtgc agagagtgac ctcaccattg gttcagtggc 2160
caccgaggat gttccacgca tcctcgggaa aataggagac actgacgagc tgcttgaccg 2220
gggtccctcg gctccctcca agggagaacc ggtctgtgac caacctgcca aagatccccg 2280
gatgtcgcca cgggagtctg acgagagcat gatagctccg cccgcagata caggtggtgt 2340
cggctcattc actgatttgc cgtcttcaga tggtgtggat gtggacgggg gggggccgtt 2400
aagaacggta aaaacaaaag caggaaggct cttagaccaa ctgagctgcc aggtttttag 2460
cctcgtttcc catctcccta ttttcttctc acacctcttc aaatctgaca gtggttattc 2520
tccgggtgat tggggttttg cagctttcac tctattttgc ctctttctat gttacagtta 2580
cccattcttc ggttttgctc ccctcttggg tgtattttct gggtcttctc ggcgtgtgcg 2640
aatgggggtt tttggctgct ggttggcttt tgctgttggt ctgttcaagc ctgtgtccga 2700
cccagtcggc actgcttgtg agtttgactc gccagagtgt aggaacgtcc ttcattcttt 2760
tgagcttctc aaaccttggg accctgtccg cagccttgtt gtgggccccg tcggtctcgg 2820
ccttgccatt cttggcaggt tactgggcgg 2850

Claims (6)

1. a kind of virus attenuated strain of high-fidelity porcine reproductive and respiratory syndrome, is porcine reproductive and respiratory syndrome virus PRRSV- NJRb plants, its microbial preservation number is：CGMCC No.13800.
2. the virus attenuated strain of high-fidelity porcine reproductive and respiratory syndrome according to claim 1, it is characterised in that：Its ORF5 base The nucleotide sequence of cause such as SEQ ID NO:Shown in 1.
3. the virus attenuated strain of high-fidelity porcine reproductive and respiratory syndrome according to claim 1, it is characterised in that：Its nsp2 base The nucleotide sequence of cause such as SEQ ID NO:Shown in 2.
4. the preparation method of the virus attenuated strain virus liquid of high-fidelity porcine reproductive and respiratory syndrome described in claim 1, including such as Lower step：Using the virus attenuated strain of high-fidelity porcine reproductive and respiratory syndrome described in Marc-145 cells propagation claim 1, split Cell is solved, centrifuging and taking supernatant obtains virus liquid.
5. porcine reproductive and respiratory syndrome attenuated vaccine, it is characterised in that active component is pig breeding described in claim 1 with exhaling Inhale syndrome virus attenuated strain.
6. the weak poison of porcine reproductive and respiratory syndrome described in claim 1 is in terms of porcine reproductive and respiratory syndrome attenuated vaccine is prepared Application.
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CN111849924A (en) * 2020-06-23 2020-10-30 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Similar NADC30 porcine reproductive and respiratory syndrome virulent strain, attenuated strain and application thereof
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