CN101603035B - Porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain and method for producing living vaccine with same - Google Patents
Porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain and method for producing living vaccine with same Download PDFInfo
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Abstract
The invention relates to a porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain and a method for producing living vaccine with same; latter half part of GP2 gene and GP3-GP5 gene of the porcine reproductive and respiratory syndrome virus (PRRSV) classic virus PTK strain, M ferritin, N ferritin and spacer sequences thereof and all nucleotide sequences of 3' non-coding region are all deleted by using reverse genetic manipulation and gene combination technology, then replaced with corresponding gene of Chinese highly pathogenic porcine reproductive and respiratory syndrome variant strain and are recombined to be the chimeric recombinant vaccine strain (PC strain) with the PRRSV classic strain and variant strain genes simultaneously, the chimeric recombinant vaccine PC strain is used to produce the living vaccine which has low toxicity, ensures no returning of strong toxicity within 5 generations of returning swine and the heritability is stable; the invention has good immunogenicity, and can prevent the porcine reproductive and respiratory syndrome virus classic strain and highly pathogenic variant strain from being infected simultaneously.
Description
Technical field
The invention belongs to the veterinary biologics technical field, be specifically related to a kind of method that makes up the attenuated live vaccines strain and produce the porcine reproductive and respiratory syndrome living vaccine with this strain with gene recombination technology.
Background technology
Pig blue-ear disease is to be generated heat, miscarry with sow by what porcine reproductive and respiratory syndrome virus (PRRSV) caused, and the Pre-and Post-Weaning Piglets mortality ratio raises, and different ages pig dyspnoea etc. is the disease of Clinical symptoms.1991, people such as Dutch scholar Terpstra were separated to Europe class PRRSV, called after Lelystad virus (Lv) in infected pigs's body; In the same year, people such as American scholar Benfield are separated to american type PRRSV, called after VR-2332.In recent years, Chinese scholar in succession isolation identification the strains such as CH-1a, S1, HB-1 and HB-2 of american type PRRSV.
Pig " hyperpyrexia disease " is the great animal epidemic that takes place in China recently, does not all have the correlative study report before this at home and abroad.The experiment confirm that processes such as Ning Yibao are a large amount of PRRS virus to infect be the major cause that causes hyperpyrexia disease.And report at first at home.
Diagnosis and vaccine immunity Prevention Technique at traditional pig blue-ear disease has lot of research both at home and abroad, but these prior aries can not satisfy China's emerging highly pathogenic PRRS prevention and control needs, must study special vaccine product and differential diagnosis technology at highly pathogenic PRRS targetedly.
Great deal of experiment data proves: the pig blue-ear disease living vaccine that manually goes down to posterity a little less than causing has advantage such as immunizing power, duration of immunity length preferably, but blue otopathy problem occurred after using on some pig farms, and its safety problem has caused people's common concern.When using attenuated vaccine, for the consideration of vaccine to the pig security reason, general just suggestion is used in the zone that pig blue-ear disease took place, and does not advise using in the plant that pig blue-ear disease did not take place.Why forbidding preventing pig blue-ear disease with living vaccine in European Union, also is because once broken out blue otopathy after attenuated live vaccines is used on some pig farms.The U.S. is equally very careful in the use of blue otopathy living vaccine.Do not cause weak pig blue-ear disease living vaccine though some other country in America and Asia various regions and states do not oppose to go down to posterity the popular regional end user worker of pig blue-ear disease, do not advocate on the pig farm that pig blue-ear disease is not taking place and use attenuated vaccine.There is not the problem of the poison that looses in inactivated vaccine because cause of disease has been inactivated, relative living vaccine, and security is much better, but compares with living vaccine, and immune effect is poor, and duration of immunity is also short.The result who uses at China's highly pathogenic PRRS inactivated vaccine proves: the pig blue-ear disease area takes place, because pig body itself is infected pigs's reproductive and respiratory syndrome virus, not only do not have the effect of diseases prevention after the inactivated vaccine immunity, can cause certain side reaction on the contrary.On vaccine use object, say that different with living vaccine is that inactivated vaccine preferably uses in the area that pig blue-ear disease is not taking place.
Different with the pig blue-ear disease of classics, highly pathogenic PRRS is caused by height variation pig blue-ear disease strain, no matter is classical living vaccine, or inactivated vaccine, all can not prevent the generation of this disease.Therefore, for effective prevention and control highly pathogenic PRRS, vaccine research is the task of top priority.Experience and lessons according to classical strain vaccine research are necessary to carry out the research of novel gene engineered vaccine very much.
1, pig blue-ear disease conventional vaccine:
Along with the discovery of pig blue-ear disease, the vaccine research of pig blue-ear disease is also just carried out thereupon, although everybody differs to the immune effect evaluation that has pig blue-ear disease vaccine now.Commercial both at home and abroad at present pig blue-ear disease vaccine has inactivated vaccine and attenuated live vaccines two classes.The trend of develop actively has also appearred in recombinant vaccine research.
1.1 pig blue-ear disease attenuated live vaccines:
Aspect classical pig blue-ear disease attenuated live vaccines, U.S. Boehringer Ingleheim company releases commercialization attenuated vaccine Resp PRRS/ReproTM first in nineteen ninety-five, got permission to be used for 3～18 age in week pig preventive vaccination.A plurality of subsequently units have all released the pig blue-ear disease attenuated vaccine.Spain, Holland, the U.S. and China have developed pig blue-ear disease attenuated live vaccines and commercialization at present.Can for each in stage swinery use, but mainly use for the piglet inoculation in 3～18 ages in week, be used for the anti-dyspnoea of making pig blue-ear disease.It is reported that used the homology strong virus attack on the 110th day behind the inoculation pig blue-ear disease attenuated vaccine, animal can be protected fully, and to the protection of strong virus attack or even lifelong.The feeder pig inoculation just excited protective immune response after 7 days, and more than sustainable 16 weeks, compared with nonvaccinated contrast pig, and the inoculation pig is attacked poison back toxin expelling time decreased, and pulmonary lesion alleviates, and it is good to grow.Yet most reports think that there is safety problem in the pig blue-ear disease attenuated live vaccines.Reports such as Botner, in Denmark, the negative pig of 1000 bull pig blue-ear disease serology has been used attenuated vaccine, pig blue-ear disease has taken place in swinery in the near future, and from aborted fetus and stillborn foetus, be separated to vaccine virus, but find vaccine virus diaplacental infection fetus, and propagate that some swinery then shows acute pig blue-ear disease sample syndrome to nonvaccinated sow.The investigator is also arranged with this vaccine inoculation boar,, find to have degradation phenomenon under seminal fluid toxin expelling and the semen quality subsequently with strong virus attack.
The Opriessnig report takes place to be separated to a strain PRRSV 98-38803 behind the pig blue-ear disease from a pig farm of repeatedly inoculating Ingelvac PRRS MLV, and confirms that this strain comes from Ingelvac PRRS MLV.So returning strong possibility, the virulence of pig blue-ear disease attenuated vaccine exists.From above result, it is fast that the pig blue-ear disease attenuated vaccine immunity produces the phase, controlling on the pig blue-ear disease clinical symptom significantly better than the pig blue-ear disease inactivated vaccine.But the strong poison of pig blue-ear disease is consistent with weak poison in the intravital reaction process of pig, so the safety problem that life-time service pig blue-ear disease attenuated vaccine is brought is still dispute maximum in the present PRRSV immune.Although weak malicious seedling immunity can make the pig body obtain certain immunizing power, the insecurity of attenuated vaccine especially virulence to return the strong variation possibility that waits be to have limited that it is widely-used.
Aspect the pathogenic blue otopathy attenuated live vaccine of height, at present, though China has the mode of how tame unit a little less than causing by passage to study the high blue otopathy attenuated live vaccine that causes a disease, and obtains good progress, their security need be done further observation.
1.2 pig blue-ear disease inactivated vaccine:
Aspect classical pig blue-ear disease inactivated vaccine, Bayer company released inactivated vaccine PRRomiSet M in 1997, and after this Spain, Holland, Canada and China have all developed pig blue-ear disease inactivated vaccine and commercialization.Be mainly used in prevention replacement gilt and multiparity sow and infect the breeding difficulty that pig blue-ear disease caused.The advantage of inactivated vaccine is that security is good, but mainly stimulates the pig body to produce humoral immune reaction, and is powerless to removing the PRRS virus that infects in the scavenger cell, therefore only depends on the deactivation vaccine immunity can not make pig produce firm immunizing power.Shortcoming after the inactivated vaccine immunity is that immunizing dose is big, immune time is many, the immunizing power generation phase is longer, thereby is not suitable for piglet immunological.It is reported, attack poison after the inactivated vaccine immunity and can not protect the infection of piglet that the titre and the non-immune group of the time length of viremia and virus do not have marked difference behind the virus infection to PRRS virus.Piglet and feeder pig band poison is the most important reason that the pig farm PRRS virus continues existence, therefore, is difficult to control and eradicate PRRS virus in the existence on pig farm and popular by inactivated vaccine.
Aspect the pathogenic blue otopathy inactivated vaccine of height, since summer in 2006, pig high-pathogenicity blue ear disease epidemic situation takes place in China some areas successively, the Ministry of Agriculture has organized units concerned such as China Animal Disease Control And Prevention Center, China Veterinery Drug Inspection Office's joint research and development, carried out the research of high-pathogenicity blue ear disease inactivated vaccine, this vaccine was put into serial production in May, 2007.This vaccine utilizes the popular high-pathogenicity blue ear disease NVDC-JXA1 of variation strain strain as kind of a poison, this vaccine is the interim authentication code of present unique acquisition and as the inactivated vaccine of the urgent prevention of government bid and purchase usefulness, but uses the result to show: this vaccine exists and the same problem of other inactivated vaccine.
At present, China also has how tame unit at the high blue otopathy living vaccine that causes a disease of research, but they do not put use on market all among studying as yet.
2 porcine reproductive and respiratory syndrome recombinant vaccines
Just the pig blue-ear disease new generation vaccine kind at laboratory development is a lot of at present, mainly contains subunit vaccine, genetically engineered reorganization/chimeric and dna vaccination, though obtained progress is gratifying, they substantially are in conceptual phase.
2.1 subunit vaccine
More clearly to each structural protein Studies on Immunogenicity of PRRS virus at present; behind the PRRS virus infected pigs body; the immunoglobulin (Ig) of anti-PRRS virus mainly is at N albumen and M albumen in the serum, though GP3 can not stimulate the generation of neutralizing antibody, can provide protection to piglet.GP4, GP5 and M albumen can produce neutralizing antibody, and GP5 is stronger than the ability that GP4 produces neutralizing antibody.The research spininess of subunit vaccine and other new generation vaccines is to these albumen.
2.2 genetically engineered recombiant vaccine
Gagnon etc. are recombinant expressed by genes such as GP5 and M albumen have been carried out, and make up the research of genetically engineered recombiant vaccine, for frontier has been opened up in the research of pig blue-ear disease new generation vaccine.Work with PRRS virus artificial preparation recombinant virus also launches; Verheije etc. made up PRRS virus LV strain infectious CDNA clones, obtain the PRRS virus of artificial mutation, and carried out the mutant strain security and immune protection effectiveness detects.The genetically engineered recombiant vaccine provides certain protection can for the pig body at present, but effect is incomplete.
2.3DNA vaccine
Kwang, Pirzadeh, the Yang Han of China Agricultural University spring etc. have prepared expresses the dna vaccination of PRRS virus ORFK4, ORF5, ORF6 and ORF7 respectively and is used for animal experiment, shows that dna vaccination can induce the generation of humoral immunization and cellular immunization.
Although at present of a great variety, can produce ideal safeguard protection effect to the pig body without any a kind of vaccine at the vaccine research of PRRS.
The research and development of porcine reproductive and respiratory syndrome (or pig blue-ear disease) vaccine are global problems, the strong partially problem of living vaccine ubiquity virulence, and the subject matter that inactivated vaccine exists is that immune efficacy is not good enough.At present, still there is not a kind of vaccine of satisfaction to be used for the prevention of this disease in the world.And the highly pathogenic PRRS that occurred in China in 2006; because the sudden change of pathogenic genes; classical pig blue-ear disease vaccine does not have immune protective efficiency basically to it; the inactivated vaccine of producing with the high-pathogenicity porcine reproductive and respiratory syndrome virus strain isolated can not produce effectively protection to street strain equally, and the weakening strain that goes down to posterity is produced the problem that there is virulence equally in attenuated vaccine.Therefore, press at present work out a kind of safe and effective, the vaccine that immunity spectrum is wide.
Summary of the invention
The present invention is in order to overcome problem and the shortcoming that above-mentioned prior art exists, the method that a kind of porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain is provided and produces living vaccine, the present invention utilizes reverse genetic manipulation and gene recombination technology that cause a disease porcine reproductive and respiratory syndrome virus pnca gene and classical porcine reproductive and respiratory syndrome low virulent strain gene of height is chimeric, reassemble into a new chimeric attenuated vaccine strain and produce vaccine, solve the problems referred to above that this type of vaccine exists effectively with this strain.
Technical solution of the present invention is:
The porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain, utilize reverse genetic manipulation and gene recombination technology latter half and GP3～GP5 gene with the GP2 gene of the classical malicious PTK strain of porcine reproductive and respiratory syndrome virus (PRRSV), the M protein gene, behind all nucleotide sequence Delete Alls of N protein gene and intervening sequence thereof and 3 ' non-coding region, replace to the corresponding gene of the high pig blue-ear disease variation strain of causing a disease of China, reassemble into a chimeric recombinant vaccine strain (PC strain) that contains classical strain of porcine reproductive and respiratory syndrome and variant gene simultaneously, the gene complete sequence total length of chimeric recombinant vaccine strain (PC strain) is 15562bp, the aminoacid sequence of chimeric recombinant vaccine strain (PC strain) gene.This chimeric recombinant strain toxicity is low, to pig safety, good immunogenicity is arranged, and produces the infection that vaccine can prevent classical strain of porcine reproductive and respiratory syndrome and variant with the PC strain.
The high corresponding gene that causes a disease pig blue-ear disease variation strain of described China is latter half, GP3 gene, GP4 gene, GP5 gene, M protein gene, N protein gene and the intervening sequence thereof of the Chinese highly pathogenic PRRS GP2 of variation strain gene and all nucleotide sequences of 3 ' non-coding region, and the purpose nucleotide sequence total length that embeds is 2421bp, the structural protein of the PRRSV that mainly encodes and 3 ' non-coding region.Wherein the latter half of GP2 gene, GP3 gene, GP4 gene, GP5 gene, M protein gene, N protein gene are the structural protein of virus, and the nucleotide sequence of 3 ' non-coding region is relevant with activity with duplicating of virus.Can test by the amplification of specificity RT-PCR method.The aminoacid sequence of the corresponding gene of the high pig blue-ear disease variation strain of causing a disease of the China that embeds.
Described porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain is produced the method for living vaccine, carries out according to the following steps:
(1) seedling going down to posterity and cultivating with cell: Marc145 or MA104 clone through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, are continued to cultivate with cell growth medium, when forming fine and close individual layer, standby; Be used to continue to go down to posterity or virus inoculation;
(2) breeding of cell seed culture of viruses: the cell bottle that PC strain virus liquid has been grown into good cell monolayer by 1/10 volume access, adsorb after 1 hour, add cell maintenance medium in the clone individual layer, continue to cultivate, after appearring in the 70-80% cell, pathology gathers in the crops, 2-3 rearmounted preservation below-15 ℃ of the venom freeze thawing of results takes a morsel and does the inspection of semifinished product;
(3) join seedling, packing and freeze-drying: the virus-culturing fluid that is up to the standards is mixed by 1: 1 volume ratio with conventional stablizer, fully shake up, quantitatively packing in a container; Every part contains the cell venom and is no less than 10
5.0TCID
50, carry out lyophilize after the packing rapidly and get product.
Porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain PC of the present invention strain is that the ORF1-ORF2 gene fragment first half of the classical malicious PTK strain of porcine reproductive and respiratory syndrome virus (PRRSV) is made carrier, insert the high porcine reproductive and respiratory syndrome China isolated strain ORF2 gene fragment latter half ORF3-ORF7 gene fragment of causing a disease, be built into the chimeric PC strain of reorganization, container name is the PB13117 plasmid.The nucleotide sequence of carrier comprises: PBluescript SK complete sequence, CMV promotor, T7 promotor and pig breed in the genome sequence of breath syndrome virus (PRRSV) PTK strain, and the size of the CMV promotor T7 promotor of PBS13117 plasmid is respectively 565bp and 19bp.Only use the T7 promotor among the present invention, terminator is the t1t2 polyphone transcription terminator of rrnB rRNA operon, size is 402bp, the CMV promotor comes from cytomegalovirus, the T7 promotor derives from phage, the rrnB terminator derives from intestinal bacteria, insert Chinese high-pathogenicity blue ear disease variant purpose nucleotide sequence after, the gene total order of chimeric poison PC strain is classified 15562bp as.The technical process that the present invention makes up chimeric reorganization PC strain as shown in Figure 1.
Marker gene is ampicillin resistance gene (Ampr), and gene ORF size is 861bp, and its function has the ability of tolerance finite concentration penbritin for making the bacterium that contains this gene, derives from intestinal bacteria.
The genetic manipulation method:
Genetic manipulation method used in the present invention comprises that conventional RT-PCR, enzyme are cut, connected, conversion, in-vitro transcription, cell transfecting equimolecular biological method.
The detection method of the transgenic microorganism of porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain PC strain adopts the RT-PCR method to detect.
The present invention has the following advantages:
Chimeric reorganization PC strain is in propagation on the cell and genetic stability: chimeric reorganization PC of the present invention strain is after the virus rescue success, can express and bring into play function at Marc145 or MA104 cell inner stablity ground, the chimeric strain that makes up can be stablized propagation on Marc145 or MA104 cell, continuous passage above its various characteristics of 20 generations does not change.Returned for 5 generations in this animal body, virulence is not returned by force, and heritability is stable.
Production of vaccine and vaccine characteristic: chimeric reorganization PC of the present invention strain has good safety and immunogenicity to this animal, thus produce vaccine with it, in order to the infection of prevention porcine reproductive and respiratory syndrome classical strains and highly pathogenic mutant strain.
The chimeric reorganization PC strain that the present invention makes up can be on Marc145 or MA104 cell high efficiently multiplying, make its generation pathology.This strain virulence is low, and passback pig 5 generation virulence is not returned by force, and heritability is stable; Has good immunogenicity; because this chimeric reorganization PC strain has kept the gene structure of classical strain of porcine reproductive and respiratory syndrome and highly pathogenic mutant strain simultaneously; vaccine immunity with its manufacturing is renderd a service height, can protect the infection of pig blue-ear disease classical strains and highly pathogenic mutant strain simultaneously.
The vaccine that the present invention produces has following several big advantage:
1, because genetic background is clear, and vaccine virulence can not occur and return by force, this can overcome the problem of the attenuated vaccine security of manually going down to posterity.
2, because it is a living vaccine, can stimulate humoral immunization, again can the irritation cell immunity, and also gene is not subjected to the mutagenesis influence of going down to posterity for a long time, and gene structure is more approaching with prime strain.Should have good immune effect.
3, owing to both contained the portion gene of classical strain in the vaccine, contain the portion gene of variant again, theoretically, it is a bivalent vaccine, both can be used for preventing classical pig blue-ear disease, can prevent highly pathogenic PRRS again.
4, since gene constructed be manually to insert the expression gene, thereby, can make a differential diagnosis to this vaccine.This is to the immune efficacy of estimating vaccine and carry out disease and eradicate very favourablely, and this point is that traditional vaccine is difficult to accomplish.
5, a little less than long-time relatively virus went down to posterity in cell and causes, the time that the recombinant vaccine building process is spent was wanted much shorter, and getting is not that we have obtained under the prerequisite of safety barrier.This is badly in need of China seeming meaningful especially under the situation of highly pathogenic PRRS vaccine.
Description of drawings
The process flow sheet of Fig. 1 chimeric reorganization PC strain for the present invention makes up.
Embodiment
Porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain of the present invention, utilize reverse genetic manipulation and gene recombination technology latter half and GP3～GP5 gene with the GP2 gene of the classical malicious PTK strain of porcine reproductive and respiratory syndrome virus (PRRSV), the M protein gene, behind all nucleotide sequence Delete Alls of N protein gene and intervening sequence thereof and 3 ' non-coding region, replace to the corresponding gene of the high pig blue-ear disease variation strain of causing a disease of China, reassemble into a chimeric recombinant vaccine strain (PC strain) that contains classical strain of porcine reproductive and respiratory syndrome and variant gene simultaneously, the high corresponding gene that causes a disease pig blue-ear disease variation strain of described China is the latter half of the Chinese highly pathogenic PRRS GP2 of variation strain gene, the GP3 gene, the GP4 gene, the GP5 gene, the M protein gene, all nucleotide sequences of N protein gene and intervening sequence thereof and 3 ' non-coding region, and the purpose nucleotide sequence total length that inserts is 2421bp, the structural protein of the PRRSV that mainly encodes and 3 ' non-coding region.
Described porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain is produced the method for living vaccine, carries out according to the following steps:
(1) seedling going down to posterity and cultivating with cell: Marc145 clone through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, continue is cultivated with cell growth medium, when forming good individual layer, standby; Be used to continue to go down to posterity or virus inoculation;
(2) breeding of cell seed culture of viruses: the cell bottle that PC strain virus liquid has been grown into good cell monolayer by 1/10 volume access, adsorb after 1 hour, add cell maintenance medium in the clone individual layer, continue to cultivate, after appearring in the 70-80% cell, pathology gathers in the crops, 2-3 rearmounted preservation below-15 ℃ of the venom freeze thawing of results takes a morsel and does the inspection of semifinished product;
(3) join seedling, packing and freeze-drying: with the virus-culturing fluid that is up to the standards, mix in a container by 1: 1 volume ratio, fully shake up, quantitatively packing with conventional stablizer; Every part contains the cell venom and is no less than 10
5.0TCID
50, carry out lyophilize after the packing rapidly and get product.
The characteristic of vaccine strain on cell that the present invention produces: with the PB13117 plasmid is carrier, insert the chimeric poison PC strain that regains behind Chinese high-pathogenicity porcine reproductive and respiratory syndrome virus GD strain ORF2 latter half and the ORF3～ORF7 nucleotide sequence, can breed on Marc145 cell inner stablity ground, make cell about 72 hours, produce the hauling type pathology, 20 generations of continuous passage on cell, the multiplication characteristic of virus does not change, and gene structure does not change.
One, vaccine is to the security of this animal:
1, pig blue-ear disease recombiant vaccine (the 4th generation) (TCID
50=10
5.25) infecting 10 healthy piglets of 33 ages in days respectively, infective dose is a 3ml/ pig, collunarium 2ml, musculi colli injection 1ml infects the back and respectively surveys body temperature 1 time morning and afternoon every day, observes clinical symptom, after 30 days, get 5 and slaughter the variation that its lungs of observation and other organs are dissected in the back.
All 10 pigs all do not have fervescence as a result, and all pig is strong lives.5 all internal organs of pig of dissecting do not have the visible pathological change of naked eyes.
2, with pig blue-ear disease recombiant vaccine (the 5th generation) (TCID
50=10
5.0) infecting 5 healthy piglets of 35 ages in days respectively, infective dose is a 3ml/ pig, collunarium 2ml, musculi colli injection 1ml infects the back and respectively surveys body temperature 1 time morning and afternoon every day, observes clinical symptom, after 21 days, all slaughter the variation that its lungs of observation and other organs are dissected in the back.
All 5 pigs all do not have fervescence as a result, and all pig is strong lives.5 all internal organs of pig of dissecting do not have the visible pathological change of naked eyes
3, virulence is returned strong ability.
PRRSV PC strain was returned for 5 generations in the pig body continuously, do not find that now virulence is not returned strong phenomenon.
The result shows: use the pig blue-ear disease recombiant vaccine to pig safety.
Two, pig blue-ear disease PC strain vaccine potency test:
1, uses pig blue-ear disease recombiant vaccine (the 4th generation of PC strain) (TCID
50=10
5.25) 15 healthy piglets of immunity, attack poison after 35 days, attack the poison back and observed 21 days, slaughter the variation that its lungs of observation and other organs are dissected in the back.Test-results is as follows:
Security: in all immune swines 35 days after vaccine inoculation, all do not have body temperature rise.There is not clinical symptom yet.The proof vaccine safety.
Immunoprotection: back 14 days antibody test 100% positives of immunity; After attacking strong poison, all immune swines are all strong lives, and the weight increase of three groups of immune swines is apparently higher than the contrast pig.The pulmonary lesion protection ratio of immune swine is 78%.
2, use two groups of pig blue-ear disease recombiant vaccines (the 5th generation of PC strain) (TCID
50=10
5.25), after 5 times of dilutions, the healthy piglet of 5 36 ages in days of every batch of each immunity, musculi colli injection 1ml/ head after 21 days, is attacked poison together with 5 first of contrasts, every some nose 0.5ml, musculi colli injection 2ml.Every day, thermometric was observed clinical symptom, slaughtered the back and dissected the variation of observing its lungs and other organs in 21 days.The result is as follows.
One group of 2/5 morbidity of immune swine, 1/5 death, another group 1/5 morbidity, 0/5 death, 5/5 morbidity of contrast pig, 4/5 death.
The result shows: can produce the excellent protection effect with pig blue-ear disease recombiant vaccine immune swine.
＜110〉Wuhan Chopper Biology Co., Ltd. of China Veterinery Drug Inspection Office U.S. PTOTATEK Internaional, Inc
＜120〉method of porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain and production living vaccine thereof
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<141>2009-05-18
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<212>DNA
＜213〉the purpose nucleic acid of porcine reproductive and respiratory syndrome virus variant GD pnca gene group 3 ' end insertion
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CTAGTGATTACGCCTGGCTGGCGTTCCTGTCCTTCAGCTACACGGCCCAGTTCCATCCCG 60
AGATATTTGGGATAGGGAATGTGAGTCAAGTTTATGTTGACATCAAGCACCAATTCATCT 120
GCGCTGTTCACGACGGGGATAACGCCACCTTGCCTCGCCATGACAATATTTCAGCCGTAT 180
TTCAGACCTACTACCAACACCAGGTCGACGGCGGCAATTGGTTTCACCTGGAATGGCTGC 240
GTCCTTTCTTTTCCTCTTGGTTGGTTTTAAATGTTTCGTGGTTTCTCAGGCGTTCGCCTG 300
CAAGCCATGTTTCAGTTCGAGTCTTTCGGACATCAAAACCAACACCACCGCAGCATCAGA 360
CTTCGTTGTCCTCCAGGACATCAGCTGCCTTAGGCATGGCGACTCGTCCTCTCCGACGAT 420
TCGCAAAATTTCTCAGTGCCGCACGGCGATAGGGACGCCCGTGTACATCACCATCACTGC 480
CAATGTCACAGATGAAAATTATCTACATTCTTCTGATCTCCTCATGCTTTCTTCTTGCCT 540
TTTCTATGCTTCCGAGATGAGTGAAAAGGGATTCAAAGTGGTGTTTGGCAATGTGTCAGG 600
CACTGTGGCTGTGTGCGTCAACTTTACCAGCTACGTCCAACATGTCAAGGAGTTTACCCA 660
ACGCTCCTTAGTGGTCGATCATGTGCGACTGCTTCATTTCATGACACCTGAGACCATGAG 720
GTGGGCAACCGTTTTAGCCTGTCTTTTTGCCATCCTACTGGCAATTTGAATGTTCAAGTA 780
TGTTGGGGAAGTGCTTGACCGCGTGCTGTTGCTCGCGATTGCTTTTTTTGTGGTGTATCG 840
TGCCGTTCTATCTTGCTGTGCTCGTCAACGCCAGCAACAACAACAGCTCTCATATTCAGT 900
TGATTTATAACTTAACGTTATGTGAGCTGAATGGCACAGATTGGCTGGCACAAAAATTTG 960
ACTGGGCAGTGGAGACTTTTGTCATCTTCCCCGTGTTGACTCACATTGTTTCCTATGGGG 1020
CACTCACCACCAGCCATTTCCTTGACACAGTTGGTTTGGCCACTGTGTCTACCGCCGGAT 1080
ATTATCACGGGCGGTATGTCTTGAGTAGCATTTACGCAGTCTGTGCTCTGGCTGCGCTGA 1140
TTTGCTTTGTCATTAGGCTTGCGAAGAACTGCATGTCCTGGCGCTACTCTTGTACCAGAT 1200
ATACCAACTTCCTTCTGGACACTAAGGGCAGACTCTATCGTTGGCGGTCGCCCGTCATTG 1260
TGGAGAAAGGGGGTAAGGTTGAGGTCGAAGGTCACCTGATCGACCTCAAGAGAGTTGTGC 1320
TTGATGGTTCCGCGGCAACCCCTTTAACCAGAGTTTCAGCGGAACGATGGGGTCGTCTCT 1380
AGACGACTTCTGCAATGATAGCACAGCTCCACAGAAGGTGCTTTTGGCGTTTTCCATTAC 1440
CTACACGCCAGTGATGATATATGCTCTAAAGGTAAGTCGCGGCCGACTGCTAGGGCTTCT 1500
GCACCTTTTGATCTTTCTGAATTGTGCTTTTACCTTCGGGTACATGACATTCGTGCACTT 1560
TGAGAGCACAAATAGGGTCGCGCTCACTATGGGAGCAGTAGTTGCACTTCTTTGGGGAGT 1620
GTACTCAGCCATAGAAACCTGGAAATTCATCACCTCCAGATGCCGTTTGTGCTTGCTAGG 1680
CCGCAAGTACATTCTGGCCCCTGCCCACCACGTCGAAAGTGCCGCGGGCTTTCATCCGAT 1740
TGCGGCAAATGATAACCACGCATTTGTCGTCCGGCGTCCCGGCTCCACTACGGTCAACGG 1800
CACATTGGTGCCCGGGTTGAAAAGCCTCGTGTTGGGTGGCAGAAAAGCTGTTAAGCAGGG 1860
AGTGGTAAACCTTGTTAAATATGCCAAATAACAACGGCAAGCAGCAAAAGAAAAAGAAGG 1920
GGAATGGCCAGCCAGTCAATCAGCTGTGCCAAATGCTGGGTAAGATCATCGCCCAACAAA 1980
ACCAGTCCAGAGGCAAGGGACCGGGGAAGAAAAATAGGAAGAAAAACCCGGAGAAGCCCC 2040
ATTTCCCTCTAGCGACTGAAGATGACGTCAGGCATCACTTTACCCCTAGTGAGCGGCAAT 2100
TGTGTCTGTCGTCGATCCAGACTGCCTTCAATCAGGGCGCTGGAACTTGTGCCCTGTCAG 2160
ATTCAGGGAGGATAAGTTACACTGTGGAGTTTAGTTTGCCGACGCAACATACTGTGCGTC 2220
TGATCCGCGCCACAGCATCACCCTCAGCATGATGGGCTGGCATTCTTTGGCACCTCAGTG 2280
TTAGAATTGGGAGAATGTGTGGTGAATGGCACTGATTGACACTGTGCCTCTAAGTCACCT 2340
ATTCAATTAGGGCGACCGTGTGGGGGTAAAGTTTAATTGGCGAGAACCATGCGGCCGTAA 2400
TTAAAAAAAAAAAAAAAAAAA 2421
<210>2
<211>15562
<212>DNA
＜213〉porcine reproductive and respiratory syndrome virus PC pnca gene group complete sequence
<400>2
TATGACGTAT?AGGTGTTGGC?TCTATGCCTT?GACATTTGTA?TTGTCAGGAG?CTGTGATCAT 60
TGACACAGCC?CAAAGCTTGC?TGCACAGAAA?CACCCTTCTG?TGACAGCCTC?CTTCAGGGGA 120
GTTTAGGGGT?CTGTCCCTAG?CACCTTGCTT?CCGGAGTTGC?ACTGCTTTAC?GGTCTCTCCA 180
CCCCTTTAAC?CATGTCTGGG?ATACTTGATC?GGTGCACGTG?TACCCCCAAT?GCCAGGGTGT 240
TCATGGCGGA GGGCCAAGTC TACTGCACAC GATGCCTCAG TGCACGGTCT CTCCTTCCTC 300
TGAATCTCCA AGTTCCTGAA CTCGGGGTGC TGGGCCTATT CTATAGGCCC GAAGAGCCAC 360
TCCGGTGGAC GTTGCCACGT GCATTCCCCA CTGTTGAGTG CTCCCCCGCC GGAGCCTGCT 420
GGCTTTCTGC CATCTTTCCA ATTGCACGAA TGACCAGTGG AAATCTGAAC TTTCAACAAA 480
GAATGGTGCG GGTCGCAGCT GAGCTTTACA GAGCCGGCCA GCTCACCCCT GCAGTCTTGA 540
AGACTCTACA AGTTTATGAA CGGGGTTGCC ACTGGTACCC CATTGTTGGA CCTGTTCCTG 600
GAGTGGCCGT TTATGCCAAC TCCCTACATG TGAGTGATAA ACCTTTCCCG GGAGCAACTC 660
ACGTGTTAAC CAACCTGCCG CTCCCGCAGA GACCCAAGCC TGATGATTTT TGCCCCTTTG 720
AGTGTGCTAT GGCTACTGTC TATGACATTG GTCATGACGC CGTCATGTAT GTGGCCGAAG 780
AGAAAGTCTC CTGGGCCCCT CGTGGCGGGG ATGAAGTGAA ATTCGAACCT GTCCCCGGGG 840
AGTTGAAGTT GATTGCGAAC CGACTCCGCA CCTCCTTCCC GCCCCACCAC GCAGTGGACA 900
TGTCTAAGTT CACCTTCACA GCCCCTGGGC GTGGTGTTTC TATGCGGGTC GAACGCCAAC 960
ACGGCTGCCT CCCCGCTGAC ACAGTTCCTG AAGGCAACTG CTGGTGGAGC TTGTTCAACT 1020
TGCTCCCACT GGAAGTTCAG AACAAAGAAA TTCGTCATGC CGGCCAATTT GGCTACCAGA 1080
CTAAGCATGG TGTCTCTGGC AAGTACCTAC AGCGGAGGCT GCAAGTTAAT GGTCTTCGAG 1140
CAGTAACTGA CCTAAATGGA CCTATCGTCG TACAGTGCTT CTCCGTTAAG GAGAGTTGGA 1200
TCCGCCACTT GAAACTGGCG GAAGAACCCA GCTACCCTGG GTTTGAGGAC CTCCTCAGAA 1260
TAAGGGTTGA GCCCAACACG TCGCCATTGG CTGACAAGGA TGAAAAAATT TTCCGGTTTG 1320
GCAATCACAA GTGGTATGGC GCTGGAAAGA GAGCAAGGAA AGCACGCTCT AGTGCGACTG 1380
CTACAGTCGC TGGCCGCGCT TTGCCCGTTC GTGAAACCCG GCAGGTCGAG GAGCACGAGG 1440
TTGCCGGCGC CAACAAGGCT GAGCACCTCA AACACTACTC CCCGCCTGCC GAAGGGAATT 1500
GTGGTTGGCA CTGCATTTCC GCCATCGGCA ACCGGATGTT GAATTCCAAA TTTGAAACCA 1560
CCCTTCCCGA AAGAGTGAGA CCTCCAGATG ACTGGGCTAC TGATGAGGAT CTTGTGAATG 1620
CCATCCAAAT CCTCAGACTC CCTGCGGCCT TGGACAGGAA CGGTGCTTGT GCTAGCGCCA 1680
AGTACGTACT TAAGCTGGAA GGTGAGCATT GGACTGTCAC TGTGACCCCT GGGATGTCCC 1740
CTTCTTTGCT CCCTCTTGAA TGTGTTCAGG GCTGTTGCGA GCATAAGGGC GGTCTTGGTT 1800
CCCCAGATGC AGTCGAGGTT TTCGGATTTG ACCCTGCCTG CCTTGACTGG CTGGCTGAGG 1860
TGATGCACTT GCCTAGCAAT GCCATCCCAG CCGCTCTGGC CGAAATGTCC GGCGATTCCA 1920
ATCGTCCGGC TTCCCCGGTC ACCACCGTGT GGACTGTTTC GCAGTTCTTA GCCCGCCACA 1980
ACGGAGGGAA TCACCCTGAC CAAATACGCT TAGGGAAAAT TATCAGCCTT TGTCAGGTGA 2040
TTGAGGACTG CTGCTGTTCC CAGAACAAAA CCAACCGGGT CACCCCGGAG GAGGTCGCAG 2100
CAAAGATTGA CCTGTACCTC CGTGGTGCAA CAAATCTTGA AGAATGCTTG GCCAGGCTTG 2160
AGAAAGCGCG CCCGCCACGC GTAATGGACA CCTCCTTTGA TTGGGATGTT GTGCTCCCTG 2220
GGGTTGAGGC GGCAACTCAA ACGACCGAAC TGCCCCAAGT CAACCAGTGT CGCGCTCTGG 2280
TCCCTGTTGT GACTCAAAAG TCCTTGGACA ACAACTCGGT CCCTCTGACC GCCTTTTCAC 2340
TGGCTAACTA CTACTACCGC GCGCAAGGTG ACGAAGTTCG TCACCGTGAA AGACTAACCA 2400
CCGTGCTCTC TAAGTTGGAA GGGGTTGTTC GAGAAGAATA CGGGCTCATG CCAACCGGGC 2460
CTGGTCCACG GCCCACACTG CCACGCGGGC TCGACGAACT CAAGGACCAG ATGGAGGTGG 2520
ACTTGCTGAA ACTGGCTAAC GCCCAGATGA CTTCGGACAT GATGGCCTGG GCAGTCGAGC 2580
AGGTTGACCT AAAGACTTGG GTCAAGAACT ATCCGCGGTG GACACCACCA CCTCCTCCGC 2640
CAATAGTTCA GCCTCGAAAA ACGAAGCTTG TCAAGAGCTT ACCAGAGAGC AAGCCTGTTC 2700
CTGCACCGCG TAGGAAGGTC AGGTCCGATT GTGACTGCCC CACCCTATCG GGCAACAATC 2760
TTCCTGACAG TTGGGAAGAT TTGGCTGTTG GTTGCCCCTC TGATCTCCCT ACCTCACCTG 2820
AGCCGGTAAC ACCTTTGAGT GAGCCGGCAT CTGTGTCCGC ACCGCGACGC TCTTTTAGGC 2880
CGGTGAAGCC TTTGAGTGAA CCAGTTCCAG TCCCTGCACC GCGCAAGACT GTGTCCCGAC 2940
CGGCAACACC TCTGAGTGAG CCGATCCCTG TGCCCGCACC GCGACGCAAG TTTCAGCAGG 3000
TAGAAAAAGT GAATCCGGCG GCGGCAACCC TGGCGTGCCA AGACGAGTTT CCAGATTTGT 3060
CTGCATCCTC GCATACTGAA TATGAGGCGT CTCCCCTTGT ACTACCGCAG AACGGGGACG 3120
TTCTTGAAGT GGAGGAGCGG GAAGCTGAGG AAATCCTGAG TGGAATCTCA GACATACTGG 3180
ATGCCATCAA ACCGGCATCT GCATCATCAA GCAGCTCCCT GTCAAGTGTG GCGATCACAC 3240
GCCCGAAATA CTCAGCTCAA GCCATCATTG ACTCGGGTGG GCCCTACAGC GGGCATCTCC 3300
AAGAGGTGAA GGAAACATGC CTAAGCATCA TGAGTGAGGC ATGTGATGTG ACCAAGCTTG 3360
ATGACCCTGC CACGCAGGAA TGGCTTTCTC GCATGTGGGA TAGGGTGGAC ATGCTGACTT 3420
GGCGCAATAC GTCTGTTCAC CAGGCGTCTC GCACCTTGGA CGACAGATTT AAGTTTCTCC 3480
CGAAGATGAT ACTTGAAACA CCGCCGCCCT ACCCGTGTGG GTTCGTGATG ATGCCTCGCA 3540
CACCTGCACC CTCCGTGGGT GCGGAGAGCG ACCTCACTAT TGGCTCAGTC GCTACTGAGG 3600
ACGTTCCACG CATCTTCGGG AAAGTAAATG?ATGTCTGCAA GATGATCGAC CAGAGACCCT 3660
TGGTACTCTT TGAAAATGAG CTGGCAGATG ACCAACCTGC CAGAGATCCT CGGACATCAT 3720
CGCAGAGGTT TGACGGGAGC ACACCAGCTC CGCCCGCAGG CACGGATGGC ACCGGTTTGG 3780
CTTCGGGCCC TGGAGTGAGA GAAGTGGATT CATGTGAGGC GAGCTCAACC GAGAAAAATG 3840
AACAGCCCTT CGTGTTGAAC GGCGGCGCCA GCACACAGGC GTCAACGTTT ACCAATTTGC 3900
CGCCTCCAGG CGGTATAGAT GCGGGCGGGA GTGGGCCGTT ACAAACGGTG CGAAAGAAGG 3960
CTGAACGGTT CTTTGACCTA CTAAGCCGTC AGGTTTTTAA TCTCGTCTCC CATCTCCCTG 4020
TTTTCTTCTC ACGCCTTTTC AAACCTGGCG GTGACTATTC TCCGGGTGAT TGGGGTTTTG 4080
CAGCTTTTAC TTTATTGTGC CTCTTTTTGT GTTACAGTTA CCCGGCCTTT GGTGCTGTTC 4140
CCCTCTTGGG TGTATTTTCT GGGTCTTCTC GGCGTGTTCG AATGGGGGTT TTTGGCTGCT 4200
GGTTGGCTTT TGCTGTTAGT CTGTTCAAAC CTGTGTCCGA CCCAGTCGGC GCTGCTTGTG 4260
AATTTGATTC GCCAGAGTGT AGAAACATCC TTCATTCTTT TGAGCTTCTC AAACCTTGGG 4320
ACCCTGTTCG CGGCCTTGTT GTGGGCCCCG TCGGTCTCAG TCTTGCCATT TTTGGCAGGT 4380
TATTGGGCGG GGCACGCCAC ATCTGGCACT TTTTGCTTAG GTTTGGCATT GTTGCAGATT 4440
GTATCTTGGC TGGAGCTTAT GTGCTTTCTC AAGGCAGGTG TAAAAAGTGC TGGGGATCTT 4500
GTATAAGAAC TGCTCCTAAT GAGGTCGCCT TTAACGTGTT TCCTTTTACA CGTGCGACCA 4560
GGTCGTCACT TATCGACCTG TGCAATCGGT TTTGCGCGCC AAAAGGTATG GACCCCATTT 4620
TCCTCGCCAC TGGGTGGCGC GGGTGCTGGA CCGGCCGAAG CCCCATTGAG CAACCCTCTG 4680
AAAAACCCAT CGCGTTTGCC CAGTTGGATG AAAAAAAGAT TACGGCTAGG ACTGTGGTCG 4740
CCCAGCCCTA TGACCCCAAC CAAGCTGTAA AGTGCTTGCG GGTATTGCAG GCGGGCGGGG 4800
TGATGGTGGC TGAGGCAGTC CCAAAAGTGG TCAAAGTTTC TGCTGTTCCA TTCCGAGCCC 4860
CCTTCTTTCC CACCGGAGTG AAAGTTGATC CTGAATGCAG GATTGTGGTT GACCCCGACA 4920
CTTTCACTGC AGCCCTCCGA TCTGGCTACT CCACCACAAA CCTCGTCCTT GGTGTGGGGG 4980
ACTTTGCCCA GCTGAATGGA TTAAAAATCA GGCAAATTTC CAAGCCTTCA GGGGGAGGTC 5040
CACACCTCAT GGCTGCCCTG CATGTTGCTT GCTCTATGGC TCTGCACATG CTTGTTGGGA 5100
TTTATGTGAC TGCTGTGGGT TCTTGCGGCA CCGGCACTAA CGATCCGTGG TGCGCCAACC 5160
CGTTTGCCGT CCCTGGCTAC GGACCTGGCT CTCTCTGCAC GTCCAGATTG TGCATCTCCC 5220
AACATGGCCT TACCCTACCC TTGACAGCAC TTGTGGCGGG ATTCGGCTTT CAGGAAATTG 5280
CCTTGGTTAT TTTGATTTTT GTTTCTATCG GAGGCATGGC TCATAGATTG AGTTGCAAGG 5340
CTGATATGCT GTGTATTTTG TTTGCAATCG CCAGCTATGT TTGGGTACCT CTTACCTGGT 5400
TGCTTTGTGT GTTTCCTTGC TGGTTGCGCT GTTTTTCGTT GCACCCCCTC ACCATCCTAT 5460
GGTTGGTGTT TTTCCTGATT TCTGTAAATA TGCCTTCAGG AATCTTGGCC TTGGTGTTGT 5520
TGATTTCTCT CTGGCTTCTT GGTCGTTATA CCAACGTTGC CGGTCTTGTC ACTCCCTATG 5580
ACATTCATCA TTACACCAGT GGCCCCCGCG GTGTTGCCGC CTTGGCTACC GCACCAGATG 5640
GGACCTACTT GGCCGCTGTC CGCCGCGCCG CGTTGACTGG TCGCACCATG CTGTTTACCC 5700
CGTCTCAGCT CGGGTCCCTT CTTGAGGGCG CTTTCAGAAC TCGAAAGCCC TCACTGAACA 5760
CCGTTAATGT GGTCGGGTCC TCCATGGGCT CTGGCGGGGT GTTCACCATC GACGGGAAAA 5820
TTAAGTGCGT AACTGCTGCA CATGTCCTTA CGGGTAATTC AGCTAGGGTT TCCGGGGTTG 5880
GCTTCAATCA AATGCTTGAC TTCGATGTGA AAGGAGACTT CGCCATAGCC GATTGCCCAG 5940
ACTGGCAAGG GGCTGCTCCC AAGACCCAAT TCTGCGAGGA AGGATGGACT GGCCGGGCCT 6000
ATTGGCTAAC GTCTTCTGGT GTCGAACCCG GCGTCATTGG AAAAGGATTC GCCTTCTGCT 6060
TCACCGCGTG CGGCGATTCC GGATCCCCAG TAATCACCGA GGCCGGCGAG CTTATCGGCG 6120
TTCACACGGG GTCAAATAAA CAAGGAGGAG GCATCGTCAC GCGCCCCTCA GGCCAGTTTT 6180
GTAGTGTGGC ACCCGTCAAA TTAAGCGAAC TAAGTGAATT CTTTGCAGGG CCTAAGGTCC 6240
CGCTCGGTGA TGTGAAAGTT GGCAGCCACA TAATTGAAGA CGTAGGCGAG GTGCCTTCAG 6300
ATCTTTGCGC CTTGCTTGCT GCCAAACCTG AACTGGAAGG AGGCCTCTCC ACCGTTCAAC 6360
TTCTGTGTGT GTTTTTCCTC CTGTGGAGAA TGATGGGACA TGCCTGGACG CCCTTGGTTG 6420
CCGTAGGGTT TTTTATCTTG AATGAGGTCC TCCCAGCTGT CCTGGTCCGG AGTGTTTTCT 6480
CCTTTGGAAT GTTTGTGCTA TCCTGGCTCA CACCATGGTC TGCGCAAGTT CTGATGATCA 6540
GGCTTCTAAC AGCAGCTCTT AACAGGAATA GATGGTCACT TGCCTTTTAC AGCCTCGGTG 6600
CAATGACTGG TTTTGTCGCA GATCTCGCGG CTACTCAGGG GTATCCGTTG CAGGCAGTGA 6660
TGAGTTTGAG CACTTATGCA TTCCTGCCTC GGATAATGGT TGTGACTTCA CCAGTCCCAG 6720
TGGTTGCGTG TGGTGTTGTG CACCTACTTG CCATCATTTT GTACTTGTTT AAGTACCGCT 6780
GCCTGCACAA CATCCTTGTT GGCGATGGAG TGTTCTCTGC GGCTTTCTTC CTGCGATATT 6840
TTGCCGAGGG AAAGTTGAGG GAGGGGGTGT CGCAATCCTG CGGGATGAAT CATGAGTCAC 6900
TTACCGGTGC CCTCGCTATG AGACTCAATG ACGAGGACTT GGATTTCCTC ACGAAATGGA 6960
CTGATTTCAA GTGCTTTGTT TCTGCGTCCA ACATGAGAAA TGCTGCGGGC CAATTCATCG 7020
AGGCTGCCTA TGCTAAAGCA CTTAGAGTAG AACTTGCCCA GTTGGTGCAG GTTGATAAGG 7080
TTCGGGGTAC TTTGGCCAAA CTTGAAGCTT TTGCCGACAC CGTGGCACCC CAACTCTCGC 7140
CCGGTGACAT TGTTGTCGCT CTTGGCCATA CGCCTGTTGG CAGTATCTTC GACCTGAAGG 7200
TTGGTAACAC CAAGCACACT CTCCAAGCCA TCGAGACCAG GGTCCTTGCT GGGTCCAAAA 7260
TGACCGTGGC GCGCGTCGTC GATCCGACCC CCACGCCCCC ACCCGCACCC GTGCCCATCC 7320
CCCTCCCACC GAAGGTTTTG GAGAACGGTC CAAACGCTTG GGGGGATGAA GACCGTTTGA 7380
ATAAAAAGAG GAGGCGCAGG ATGGAAGCCC TCGGCATCTA TGTTATGGGC GGGAAAAAGT 7440
ACCAGAAATT TTGGGACAAG AATTCCGGTG ACGTGTTTTA TGAGGAGGTC CATAACAACA 7500
CAGATGAGTG GGAGTGCCTC AGAGTTGGCG ACCCTGCCGA CTTTGACCCT GAGAAGGGAA 7560
CTTTGTGTGG GCATGTCACC ATTGAAGATA GGGCTTACCA TGTTTACACC TCCCCATCTG 7620
GTAAGAAATT CCTAGTCCCC GTCAACCCAG AGAACGGAAG AGTTCAATGG GAGGCTGCAA 7680
AGCTTTCCGT TGAGCAGGCC CTTGGTATGA TGAACGTCGA CGGCGAGCTG ACTGCCAAGG 7740
AACTGGAGAA ACTGAAAAGA ATAATTGACA AACTCCAGGG CCTGACTAAG GAGCAGTGTT 7800
TAAACTGCTA GCCGCTAGCG GCTTGACCCG CTGTGGTCGC GGCGGCTTGG TTGTTACTGA 7860
GACAGCGGTA AAAATAGTCA AATTTCACAA CCGGACCTTC ACCTTGGGAC CTGTAAATTT 7920
AAAAGTGGCT AGTGAGGTTG AGCTAAAAGA CGCGGTCGAG CACAACCAAC ACCCGGTTGC 7980
TAGACCAGTC GATGGTGGTG TTGTGCTCCT GCGTTCCGCG GTTCCCTCGC TTATAGATGT 8040
CCTGATCTCC GGTGCTGATG CATCTCCCAA ATTACTTGCC CATCACGGAC CGGGCAACAC 8100
CGGGATCGAT GGCACGCTCT GGGATTTTGA GTCCGAAGCC ACTAAAGAGG AAGTCGCACT 8160
CAGCGCGCAA ATAATACAGG CTTGTGACAT TAGGCGCGGC GACGCACCTG AAATTGGTCT 8220
CCCTTACAAG CTGTACCCTA TTAGGGGCAA CCCTGAGCGG GTAAAGGGAG TTTTACGGAA 8280
TACAAGGTTT GGAGACATAC CTTATAAAAC CCCCAGTGAC ACTGGAAGCC CAGTGCACGC 8340
GGCTGCCTGC CTCACGCCTA ACACCACTCC GGTGACTGAC GGGCGCTCCG TCTTGGCCAC 8400
GACCATGCCC TCCGGGTTTG AGTTGTATGT ACCGACCATA CCAGCGTCTG TCCTTGACTA 8460
TCTTGATTCT AGGCCCGACT GCCCTAAACA GTTGACGGAG CACGGCTGTG AAGATGCCGC 8520
ACTGAAAGAC CTCTCCAAGT ATGACTTGTC CACCCAAGGC TTTGTTTTGC CTGGAGTTCT 8580
TCGCCTTGTG CGGAAGTACC TGTTTGCCCA TGTGGGCAAA TGCCCGCCCG TTCATCGGCC 8640
TTCCACTTAC CCTGCCAAGA ATTCTATGGC TGGAATAAAT GGGAACAGGT TTCCAACCAA 8700
GGACATCCAG AGCGTCCCTG AAATCGACGT TCTGTGCGCA CAGGCTGTGC GAGAAAATTG 8760
GCAAACTGTT ACCCCTTGTA CCCTCAAGAA ACAGTATTGT GGGAAGAAGA AGACTAGGAC 8820
CATACTCGGC ACCAATAACT TTATAGCGCT GGCTCACCGT GCAGCGTTGA GTGGTGTCAC 8880
CCAGGGCTTC ATGAAAAAGG CGTTTAACTC GCCTATCGCC CTCGGGAAAA ACAAATTTAA 8940
GGAGCTACAG ACTCCGGTCC TAGGCAGGTG CCTTGAAGCT GATCTTGCAT CCTGCGATCG 9000
ATCTACACCT GCAATTGTCC GCTGGTTTGC CGCCAATCTT CTTTATGAGC TTGCCTGTGC 9060
TGAAGAGCAT CTACCGTCGT ATGTGCTGAA CTGCTGTCAC GACTTACTGG TCACGCAGTC 9120
CGGTGCAGTG ACTAAGAGAG GCGGCCTGTC GTCTGGCGAC CCGATAACCT CTGTGTCTAA 9180
TACCATTTAC AGCTTGGTGA TCTATGCACA GCACATGGTG CTTAGTTGCT TCAAAAGTGG 9240
TCACCCCCAT GGCCTTCTGT TCTTACAAGA CCAGCTAAAG TTTGAGGACA TGCTCAAGGT 9300
TCAACCCCTA ATCGTCTATT CGGACGACCT CGTGCTGTAT GCCGAGTCTC CCACCATGCC 9360
AAACTACCAT TGGTGGGTTG AACATCTGAA TTTGATGCTG GGGTTTCAGA CGGACCCAAA 9420
GAAAACAGCC ATAACAGACT CGCCATCATT TCTAGGCTGT AGAATAATAA ATGGGCGCCA 9480
GCTAGTCCCC AACCGTGACA GGATTCTCGC GGCCCTCGCC TACCACATGA AGGCGAGTAA 9540
TGTTTCTGAA TACTACGCCT CAGCGGCTGC AATACTCATG GACAGCTGTG CTTGTTTGGA 9600
GTATGATCCT GAATGGTTTG AAGAACTTGT AGTTGGGATA GCGCAGTGCG CCCGCAAAGA 9660
CGGCTACAGC TTTCCCGGCA CGCCGTTCTT CATGTCCATG TGGGAAAAAC TCAGGTCCAA 9720
TTATGAGGGG AAGAAGTCGA GAGTGTGCGG GTACTGCGGG GCCCCGGCCC CGTACGCTAC 9780
TGCCTGTGGC CTCGACGTCT GCATTTACCA CACCCACTTC CACCAGCATT GTCCAGTCAC 9840
AATCTGGTGT GGCCATCCAG CGGGTTCTGG TTCTTGTAGT GAGTGCAAAT CCCCTGTAGG 9900
GAAAGGCACA AGCCCTTTAG ACGAGGTGCT GGAACAAGTC CCGTATAAGC CCCCACGGAC 9960
CGTTATCATG CATGTGGAGC AGGGACTTAC CCCCCTTGAC CCAGGTAGAT ACCAGACTCG 10020
CCGCGGATTA GTCTCTGTCA GGCGTGGAAT CAGGGGAAAT GAAGTTGAAC TACCAGACGG 10080
TGATTATGCT AGTACCGCCT TGCTCCCTAC CTGTAAAGAG ATCAACATGG TCGCTGTCGC 10140
TTCTAATGTA TTGCGCAGCA GGTTCATCAT CGGTCCACCC GGTGCTGGGA AAACATACTG 10200
GCTCCTTCAA CAGGTTCAGG ATGGTGATGT TATTTACACA CCAACTCACC AGACCATGCT 10260
TGACATGATT AGAGCTTTGG GGACGTGCCG GTTCAACGTT CCGGCAGGCA CAACGCTGCA 10320
ATTCCCCGTC CCCTCCCGTA CCGGTCCGTG GGTTCGCATT CTAGCCGGCG GTTGGTGTCC 10380
TGGCAAGAAT TCCTTCCTGG ATGAAGCAGC ATATTGCAAT CACCTTGATG TTTTGAGGCT 10440
TCTTAGCAAA ACTACCCTCA CCTGTCTAGG AGACTTCAAA CAACTCCACC CAGTGGGTTT 10500
TGATTCTCAT TGCTATGTTT TTGACATCAT GCCTCAAACT CAACTGAAGA CCATCTGGAG 10560
GTTTGGACAG AATATCTGTG ATGCCATCCA GCCAGATTAC AGGGACAAAC TCATGTCCAT 10620
GGTCAACACA ACCCGTGTGA CCTACGTGGA AAAACCTATC AGGTATGGGC AAGTCCTCAC 10680
CCCCTACCAC AGAGATCGAG AGGACGACGC CATCACTATT GACTCCAGTC AAGGCGCCAC 10740
ATTCGATGTG GTTACACTGC ATTTGCCCAC TAAAGATTCA CTCAACAGGC AAAGAGCCCT 10800
TGTTGCTATC ACCAGGGCCA GACATGCTAT CTTTGTGTAT GACCCACACA GGCAGCTGCA 10860
GAGCTTGTTT GATCTTCCTG AAAAAGGCAC ACCCGTTAAC CTCGCAGTGC ACCGCGACGG 10920
GCAGCTGATC GTGCTGGATA GAAATAACAA AGAATGCACG GTTGCTCAAG CTCTAGGCAA 10980
CGGGGATAAA TTTAGGGCCA CAGACAAGCG TGTTGTAGAT TCTCTCCGCG CTATTTGTGC 11040
TGACCTAGAA GGGTCGAGCT CTCCGCTCCC CAAGGTCGCA CACAACTTGG GATTTTATTT 11100
CTCACCTGAT TTAACACAGT TTGCCAAACT CCCAGCAGAA CTTGCACCTC ACTGGCCTGT 11160
GGTGACAACC CAGAACAATG AAAATTGGCC AGATCGGCTG GTTGCCAGCC TTCGTCCTAT 11220
CCATGAGTAT AGCCGCGCGT GCATCGGTGC CGGCTATATG GTTGGCCCCT CGGTGTTTCT 11280
AGGCACCCCC GGGGTCGTGT CATACTATCT CACAAAATTT GTTAAGGGCG AGGCTCAAGT 11340
GCTTCCGAAG ACGGTCTTCA GCACCGGCCG AATTGAGGTA GATTGCCGGG AATATCTTGA 11400
CGATCGGGAG CGAGAAGTTG CTGCGTCCCT CCCACATGCC TTCATTGGTG ACGTCAAAGG 11460
CACTACCGTT GGAGGATGTC ATCATGTCAC CTCCAGATAC CTCCCGCGCT TCCTCCCCAA 11520
GGAATCAGTT GCTGTGGTCG GGGTTTCAAG TCCCGGAAAA GCCGCGAAAG CATTGTGCAC 11580
ACTGACAGAT GTGTACCTCC CAGACCTTGA AGCCTATCTC CACCCGGAGA CCCAGTCCAA 11640
GTGCTGGAAA ATGATGTTGG ACTTCAAGGA AGTTCGACTA ATGGTCTGGA AAGACAAAAC 11700
AGCCTATTTC CAACTTGAAG GACGCTATTT CACCTGGTAT CAGCTTGCTA GCTATGCCTC 11760
GTACATCCGT GTTCCTGTCA ACTCTACTGT GTACTTGGAC CCCTGCATGG GCCCCGCCCT 11820
TTGCAACAGG AGAGTCGTTG GGTCCACTCA CTGGGGGGCT GACCTCGCAG TCACCCCTTA 11880
TGATTACGGC GCTAAAATTA TCCTGTCTAG CGCGTACCAT GGTGAAATGC CCCCCGGATA 11940
CAAAATTCTG GCGTGCGCGG AATTCTCGTT GGATGACCCA GTCAAGTACA AACATACTTG 12000
GGGGTTTGAA TCGGATACAG CGTATCTGTA TGAGTTCACC GGAAACGGTG AGGATTGGGA 12060
GGATTACAAT GATGCGTTTC GTGCGCGCCA GGAAGGGAAA ATTTATAAGG CCACTGCCAC 12120
CAGCATGAAG TTTTATTTTC CCCCGGGCCC TGTCATTGAA CCAACTTTAG GCCTGAATTG 12180
ATTAATTAAT TTAAATGGCG CGCCAATGAA ATGGGGTCCA TGCAAAGCCT TTTTGACAAA 12240
ATTGGTCAAC TTTTTGTGGA TGCTTTCACG GAATTCTTGG TGTCCATTGT TGATATCATC 12300
ATATTTTTGG CCATTTTGTT TGGCTTCACC GTCGCCGGTT GGCTGGTGGT CTTTTGCATC 12360
AGATTGGTTT GCTCCGCGAT ACTCCGTGCA CGCCCTGCCT TTCACTCTGA GCAGTTACAG 12420
AAGATCCTAT GAGGCCTTTC TTTCCCTGTG TCAGGTGGAC ATTCCCACCT GGGGAATCAA 12480
ACATCCTCTG GGGGTGCTTT GGCACCATAA GGTGTCAACC CTGATTGATG AAATGGTGTC 12540
GCGTCGAATG TACCGCATCA TGGAAAAAGC AGGACAGGCT GCCTGGAAAC AGGTGGTGAG 12600
CGAGGCCACG CTGTCTCGCA TTAGTAGTTT GGATGTGGTG GCTCATTTTC AACATCTTGC 12660
CGCCGTTGAA GCCGAGACCT GTAAATATTT GGCCTCTCGG CTACCCATGC TACACAACCT 12720
GCGCATGACG GGGTCAAATG TAACCATAGT ATATAATGGT ACTTTGAATC AGGTGTTTGC 12780
CATTTTCCCG ACCCCTGGTT CCCGGCCAAA GCTTCATGAT TTTCAGCAAT GGCTGATAGC 12840
TGTGCATTCG TCCATATTTT CCTCTGTTGC AGCTTCTTGT ACTCTGTTTG TTGTACTGTG 12900
GTTGCGGGTC CCAATGCTAC GTACTGTTTT TGGTTTCCGC TGGTTAGGGG CAATTTTTCC 12960
TTCGAGCTCT TGGTGAATTA CACGGTGTGC CCACCTTGCC TCACCCGGCA AGCAGCCGCA 13020
CAGCGCTACG AACCTGGCAA GGCTCTTTGG TGCAGAATTG GGTACGATCG ATGTGAGGAG 13080
GACGATCACG ACGAGCTAGG GTTCGTGATA CCGTCTGGCC TCTCCAGCGA AGGCCACTTG 13140
ACTAGTGATT ACGCCTGGCT GGCGTTCCTG TCCTTCAGCT ACACGGCCCA GTTCCATCCC 13200
GAGATATTTG GGATAGGGAA TGTGAGTCAA GTTTATGTTG ACATCAAGCA CCAATTCATC 13260
TGCGCTGTTC ACGACGGGGA TAACGCCACC TTGCCTCGCC ATGACAATAT TTCAGCCGTA 13320
TTTCAGACCT ACTACCAACA CCAGGTCGAC GGCGGCAATT GGTTTCACCT GGAATGGCTG 13380
CGTCCTTTCT TTTCCTCTTG GTTGGTTTTA AATGTTTCGT GGTTTCTCAG GCGTTCGCCT 13440
GCAAGCCATG TTTCAGTTCG AGTCTTTCGG ACATCAAAAC CAACACCACC GCAGCATCAG 13500
ACTTCGTTGT CCTCCAGGAC ATCAGCTGCC TTAGGCATGG CGACTCGTCC TCTCCGACGA 13560
TTCGCAAAAT TTCTCAGTGC CGCACGGCGA TAGGGACGCC CGTGTACATC ACCATCACTG 13620
CCAATGTCAC AGATGAAAAT TATCTACATT CTTCTGATCT CCTCATGCTT TCTTCTTGCC 13680
TTTTCTATGC TTCCGAGATG AGTGAAAAGG GATTCAAAGT GGTGTTTGGC AATGTGTCAG 13740
GCACTGTGGC TGTGTGCGTC AACTTTACCA GCTACGTCCA ACATGTCAAG GAGTTTACCC 13800
AACGCTCCTT AGTGGTCGAT CATGTGCGAC TGCTTCATTT CATGACACCT GAGACCATGA 13860
GGTGGGCAAC CGTTTTAGCC TGTCTTTTTG CCATCCTACT GGCAATTTGA ATGTTCAAGT 13920
ATGTTGGGGA AGTGCTTGAC CGCGTGCTGT TGCTCGCGAT TGCTTTTTTT GTGGTGTATC 13980
GTGCCGTTCT ATCTTGCTGT GCTCGTCAAC GCCAGCAACA ACAACAGCTC TCATATTCAG 14040
TTGATTTATA ACTTAACGTT ATGTGAGCTG AATGGCACAG ATTGGCTGGC ACAAAAATTT 14100
GACTGGGCAG TGGAGACTTT TGTCATCTTC CCCGTGTTGA CTCACATTGT TTCCTATGGG 14160
GCACTCACCA CCAGCCATTT CCTTGACACA GTTGGTTTGG CCACTGTGTC TACCGCCGGA 14220
TATTATCACG GGCGGTATGT CTTGAGTAGC ATTTACGCAG TCTGTGCTCT GGCTGCGCTG 14280
ATTTGCTTTG TCATTAGGCT TGCGAAGAAC TGCATGTCCT GGCGCTACTC TTGTACCAGA 14340
TATACCAACT TCCTTCTGGA CACTAAGGGC AGACTCTATC GTTGGCGGTC GCCCGTCATT 14400
GTGGAGAAAG GGGGTAAGGT TGAGGTCGAA GGTCACCTGA TCGACCTCAA GAGAGTTGTG 14460
CTTGATGGTT CCGCGGCAAC CCCTTTAACC AGAGTTTCAG CGGAACGATG GGGTCGTCTC 14520
TAGACGACTT CTGCAATGAT AGCACAGCTC CACAGAAGGT GCTTTTGGCG TTTTCCATTA 14580
CCTACACGCC AGTGATGATA TATGCTCTAA AGGTAAGTCG CGGCCGACTG CTAGGGCTTC 14640
TGCACCTTTT GATCTTTCTG AATTGTGCTT TTACCTTCGG GTACATGACA TTCGTGCACT 14700
TTGAGAGCAC AAATAGGGTC GCGCTCACTA TGGGAGCAGT AGTTGCACTT CTTTGGGGAG 14760
TGTACTCAGC CATAGAAACC TGGAAATTCA TCACCTCCAG ATGCCGTTTG TGCTTGCTAG 14820
GCCGCAAGTA CATTCTGGCC CCTGCCCACC ACGTCGAAAG TGCCGCGGGC TTTCATCCGA 14880
TTGCGGCAAA TGATAACCAC GCATTTGTCG TCCGGCGTCC CGGCTCCACT ACGGTCAACG 14940
GCACATTGGT GCCCGGGTTG AAAAGCCTCG TGTTGGGTGG CAGAAAAGCT GTTAAGCAGG 15000
GAGTGGTAAA CCTTGTTAAA TATGCCAAAT AACAACGGCA AGCAGCAAAA GAAAAAGAAG 15060
GGGAATGGCC AGCCAGTCAA TCAGCTGTGC CAAATGCTGG GTAAGATCAT CGCCCAACAA 15120
AACCAGTCCA GAGGCAAGGG ACCGGGGAAG AAAAATAGGA AGAAAAACCC GGAGAAGCCC 15180
CATTTCCCTC TAGCGACTGA AGATGACGTC AGGCATCACT TTACCCCTAG TGAGCGGCAA 15240
TTGTGTCTGT CGTCGATCCA GACTGCCTTC AATCAGGGCG CTGGAACTTG TGCCCTGTCA 15300
GATTCAGGGA GGATAAGTTA CACTGTGGAG TTTAGTTTGC CGACGCAACA TACTGTGCGT 15360
CTGATCCGCG CCACAGCATC ACCCTCAGCA TGATGGGCTG GCATTCTTTG GCACCTCAGT 15420
GTTAGAATTG GGAGAATGTG TGGTGAATGG CACTGATTGA CACTGTGCCT CTAAGTCACC 15480
TATTCAATTA GGGCGACCGT GTGGGGGTAA AGTTTAATTG GCGAGAACCA TGCGGCCGTA 15540
ATTAAAAAAA?AAAAAAAAAA?AA 15562
Claims (1)
1. a porcine reproductive and respiratory syndrome virus (PRRSV) chimeric recombinant vaccine strain, it is to utilize reverse genetic manipulation and gene recombination technology latter half and the GP3～GP5 gene with the GP2 gene of the classical malicious PTK strain of porcine reproductive and respiratory syndrome virus, the M protein gene, behind all nucleotide sequence Delete Alls of N protein gene and intervening sequence thereof and 3 ' non-coding region, replace to the corresponding gene of the high pig blue-ear disease variation strain of causing a disease of China, reassemble into a chimeric recombinant vaccine strain that contains classical strain of porcine reproductive and respiratory syndrome and variant gene simultaneously, the complete genome sequence of described chimeric recombinant vaccine strain is shown in SEQ ID NO:2.
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| CN102304496B (en) * | 2010-02-05 | 2012-12-26 | 中国兽医药品监察所 | Attenuated culture of high-pathogenic porcine reproductive and respiratory syndrome virus attenuated vaccine GDr strain |
| CN101899421B (en) * | 2010-03-01 | 2012-09-19 | 中国农业科学院上海兽医研究所 | Recombinant baculovirus and construction method and application thereof |
| CA2800824A1 (en) * | 2010-06-02 | 2011-12-08 | Virginia Tech Intellectual Properties, Inc. | Novel modified live-attenuated vaccines (mlv) and subunit vaccines created by dna shuffling against porcine reproductive and respiratory syndrome virus (prrsv) |
| CN102115731B (en) * | 2010-12-01 | 2012-10-17 | 北京济福霖生物技术有限公司 | Method for preparing porcine reproductive and respiratory syndrome resistant transgenic pig |
| RU2628313C2 (en) | 2011-05-27 | 2017-08-15 | Синовет (Бейджинг) Байотекнолоджи Ко.,Лтд | Combined vaccines for prevention of pigs viral infections |
| CN102908617A (en) * | 2011-08-04 | 2013-02-06 | 广州格拉姆生物科技有限公司 | Porcine reproductive and respiratory syndrome virus M protein and CD40L fusion vaccine and preparation method |
| CN103555680B (en) * | 2013-11-04 | 2016-01-20 | 武汉中博生物股份有限公司 | One has immunogenic PRRSV virus-like particle and preparation and application thereof |
| CN104250638B (en) * | 2014-04-03 | 2017-01-25 | 普莱柯生物工程股份有限公司 | Porcine reproductive and respiratory syndrome virus attenuated strain, vaccine composition and preparation method and application thereof |
| CN104561092B (en) * | 2014-12-19 | 2018-12-11 | 中国农业科学院上海兽医研究所 | Express construction method and the application of CSFV E 2 protein recombination PRRS viral genetic engineering vaccine |
| CN104593336B (en) * | 2014-12-25 | 2019-05-17 | 中国农业大学 | Porcine reproductive and respiratory syndrome embedded virus RvHBJX and its application |
| CN106924726B (en) * | 2015-12-29 | 2021-02-09 | 普莱柯生物工程股份有限公司 | Vaccine composition for preventing porcine reproductive and respiratory syndrome and preparation method and application thereof |
| CN113151195B (en) * | 2020-12-10 | 2022-07-12 | 武汉科前生物股份有限公司 | Porcine reproductive and respiratory syndrome chimeric recombinant vaccine strain and application thereof |
| CN116036253B (en) * | 2022-11-24 | 2023-12-26 | 畜科生物工程有限公司 | Live vaccine for porcine reproductive and respiratory syndrome and preparation method thereof |
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