CN101603035A - Porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain and the method for producing living vaccine thereof - Google Patents
Porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain and the method for producing living vaccine thereof Download PDFInfo
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Abstract
The method that the present invention relates to the porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain and produce living vaccine, 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, and it is low to utilize chimeric recombinant vaccine PC strain production living vaccine to have virulence, passback pig 5 generation virulence is not returned by force, and heritability is stable; Have good immunogenicity, can protect the infection of pig blue-ear disease classical strains and highly pathogenic mutant strain 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.
Annex 1:
CTAGTGA
TTACGCCTGGCTGGCGTTCCTGTCCTTCAGCTACACGGCCCAGTTCCATCCCGAGATATTTGGGATAGGGAATGTGA
GTCAAGTTTATGTTGACATCAAGCACCAATTCATCTGCGCTGTTCACGACGGGGATAACGCCACCTTGCCTCGCCATGACAATA
TTTCAGCCGTA
TTTCAGACCTACTACCAACACCAGGTCGACGGCGGCA
ATTGGTTTCACCTGG
AATGGCTGCGTCCTTTCTTTT
CCTCTTGGTTGGTTTTAAATGTTTCGTGGTTTCTCAGGCGTTCGCCTGCAAGCCATGTTTCAGTTCGAGTCTTTCGGACATCAA
AACCAACACCACCGCAGCATCAGACTTCGTTGTCCTCCAGGACATCAGCTGCCTTAGGCATGGCGACTCGTCCTCTCCGACGAT
TCGCAAAATTTCTCAGTGCCGCACGGCGATAGGGACGCCCGTGTACATCACCATCACTGCCAATGTCACAGATGAAAATTATCT
ACATTCTTCTGATCTCCTCATGCTTTCTTCTTGCCTTTTCTATGCTTCCGAGATGAGTGAAAAGGGATTCAAAGTGGTGTTTGG
CAATGTGTCAGGCACTGTGGCTGTGTGCGTCAACTTTACCAGCTACGTCCAACATGTCAAGGAGTTTACCCAACGCTCCTTAGT
GGTCGATCATGTGCGACTGCTTCATTTCATGACACCTGAGACCATGAGGTGGGCAACCGTTTTAGCCTGTCTTTTTGCCATCCT
ACTGGCAATTTGAATGTTCAAGTATGTTGGGGAAGTGCTTGACCGCGTGCTGTTGCTCGCGATTGCTTTTTTTGTGGTGTATCG
TGCCGTTCTATCTTGCTGTGCTCGTCAACGCCAGCAACAACAACAGCTCTCATATTCAGTTGATTTATAACTTAACGTTATGTG
AGCTGAATGGCACAGATTGGCTGGCACAAAAATTTGACTGGGCAGTGGAGACTTTTGTCATCTTCCCCGTGTTGACTCACATTG
TTTCCTATGGGGCACTCACCACCAGCCATTTCCTTGACACAGTTGGTTTGGCCACTGTGTCTACCGCCGGATATTATCACGGGC
GGTATGTCTTGAGTAGCATTTACGCAGTCTGTGCTCTGGCTGCGCTGATTTGCTTTGTCATTAGGCTTGCGAAGAACTGCATGT
CCTGGCGCTACTCTTGTACCAGATATACCAACTTCCTTCTGGACACTAAGGGCAGACTCTATCGTTGGCGGTCGCCCGTCATTG
TGGAGAAAGGGGGTAAGGTTGAGGTCGAAGGTCACCTGATCGACCTCAAGAGAGTTGTGCTTGATGGTTCCGCGGCAACCCCTT
TAACCAGAGTTTCAGCGGAACGATGGGGTCGTCTCTAGACGACTTCTGCAATGATAGCACAGCTCCACAGAAGGTGCTTTTGGC
GTTTTCCATTACCTACACGCCAGTGATGATATATGCTCTAAAGGTAAGTCGCGGCCGACTGCTAGGGCTTCTGCACCTTTTGAT
CTTTCTGAATTGTGCTTTTACCTTCGGGTACATGACATTCGTGCACTTTGAGAGCACAAATAGGGTCGCGCTCACTATGGGAGC
AGTAGTTGCACTTCTTTGGGGAGTGTACTCAGCCATAGAAACCTGGAAATTCATCACCTCCAGATGCCGTTTGTGCTTGCTAGG
CCGCAAGTACATTCTGGCCCCTGCCCACCACGTCGAAAGTGCCGCGGGCTTTCATCCGATTGCGGCAAATGATAACCACGCATT
TGTCGTCCGGCGTCCCGGCTCCACTACGGTCAACGGCACATTGGTGCCCGGGTTGAAAAGCCTCGTGTTGGGTGGCAGAAAAGC
TGTTAAGCAGGGAGTGGTAAACCTTGTTAAATATGCCAAATAACAACGGCAAGCAGCAAAAGAAAAAGAAGGGGAATGGCCAGC
CAGTCAATCAGCTGTGCCAAATGCTGGGTAAGATCATCGCCCAACAAAACCAGTCCAGAGGCAAGGGACCGGGGAAGAAAAATA
GGAAGAAAAACCCGGAGAAGCCCCATTTCCCTCTAGCGACTGAAGATGACGTCAGGCATCACTTTACCCCTAGTGAGCGGCAAT
TGTGTCTGTCGTCGATCCAGACTGCCTTCAATCAGGGCGCTGGAACTTGTGCCCTGTCAGATTCAGGGAGGATAAGTTACACTG
TGGAGTTTAGTTTGCCGACGCAACATACTGTGCGTCTGATCCGCGCCACAGCATCACCCTCAGCATGATGGGCTGGCATTCTTT
GGCACCTCAGTGTTAGAATTGGGAGAATGTGTGGTGAATGGCACTGATTGACACTGTGCCTCTAAGTCACCTATTCAATTAGGG
CGACCGTGTGGGGGTAAAGTTTAATTGGCGAGAACCATGCGGCCGTAATTAAAAAAAAAAAAAAAAAAA
Annex 2:
TATGACGTATAGGTGTTGGCTCTATGCCTTGACATTTGTATTGTCAGGAGCTGTGATCATTGACACAGCCCAAAGCTTGCTGCACAGAAACACCCTTC
TGTGACAGCCTCCTTCAGGGGAGTTTAGGGGTCGTCCCTAGCACCTTGCTTCCGGAGTTGCACTGCTTTACGGTCTCTCCACCCCTTTAACCATGTCT
G
GGATACTTGATCGGTGC
ACGTGTACCCCCAATGCCAGGGTGTTCATGGCGGAGGGCCAAG
TCTACTGCACACGATG
CCTCAGTGCACGGTCTCTCCT
TCCTCTGAATCTCCAAGTTCCTGAACTCGGGGTGCTGGGCCTATTCTATAGGCCCGAAGAGCCACTCCGGTGGACGTTGCCACGTGCATTCCCCACTG
TTGAGTGCTCCCCCGCCGGAGCCTGCTGGCTTTCTGCCATCTTTCCAATTGCACGAATGACCAGTGGAAATCTGAACTTTCAACAAAGAATGGTGCGG
GTCGCAGCTGAGCTTTACAGAGCCGGCCAGCTCACCCCTGCAGTCTTGAAGACTCTACAAGTTTATGAACGGGGTTGCCACTGGTACCCCATTGTTGG
ACCTGTTCCTGGAGTGGCCGTTTATGCCAACTCCCTACATGTGAGTGATAAACCTTTCCCGGGAGCAACTCACGTGTTAACCAACCTGCCGCTCCCGC
AGAGACCCAAGCCTGATGATTTTTGCCCCTTTGAGTGTGCTATGGCTACTGTCTATGACATTGGTCATGACGCCGTCATGTATGTGGCCGCAGAGAAA
GTCTCCTGGGCCCCTCGTGGCGGGGATGAAGTGAAATTCGAACCTGTCCCCGGGGAGTTGAAGTTGATTGCGAACCGACTCCGCACCTCCTTCCCGCC
CCACCACGCAGTGGACATGTCTAAGTTCACCTTCACAGCCCCTGGGCGTGGTGTTTCTATGCGGGTCGAACGCCAACACGGCTGCCTCCCCGCTGACA
CAGTTCCTGAAGGCAACTGCTGGTGGAGCTTGTTCAACTTGCTCCCACTGGAAGTTCAGAACAAAGAAATTCGTCATGCCGGCCAATTTGGCTACCAG
ACTAAGCATGGTGTCTCTGGCAAGTACCTACAGCGGAGGCTGCAAGTTAATGGTCTTCGAGCAGTAACTGACCTAAATGGACCTATCGTCGTACAGTG
CTTCTCCGTTAACGAGAGTTGGATCCGCCACTTGAAACTGGCGGAAGAACCCAGCTACCCTGGGTTTGAGGACCTCCTCAGAATAAGGGTTGAGCCCA
ACACGTCGCCATTGGCTGACAAGGATGAAAAAATTTTCCGGTTTGGCAATCACAAGTGGTATGGCGCTGGAAAGAGAGCAAGGAAAGCACGCTCTAGT
GCGACTGCTACAGTCGCTGGCCGCGCTTTGCCCGTTCGTGAAACCCGGCAGGTCGAGGAGCACGAGGTTGCCGGCGCCAACAAGGCTGAGCACCTCAA
ACACTACTCCCCGCCTGCCGAAGGGAATTGTGGTTGGCACTGCATTTCCGCCATCGGCAACCGGATGTTGAATTCCAAATTTGAAACCACCCTTCCCG
AAAGAGTGAGACCTCCAGATGACTGGGCTACTGATGAGGATCTTGTGAATGCCATCCAAATCCTCAGACTCCCTGCGGCCTTGGACAGGAACGGTGCT
TGTGCTAGCGCCAAGTACGTACTTAAGCTGGAAGGTGAGCATTGGACTGTCACTGTGACCCCTGGGATGTCCCCTTCTTTGCTCCCTCTTGAATGTGT
TCAGGGCTGTTGCGAGCATAAGGGCGGTCTTGGTTCCCCAGATGCAGTCGAGGTTTTCGGATTTGACCCTGCCTGCCTTGACTGGCTGGCTGAGGTGA
TGCACTTGCCTAGCAATGCCATCCCAGCCGCTCTGGCCGAAATGTCCGGCGATTCCAATCGTCCGGCTTCCCCGGTCACCACCGTGTGGACTGTTTCG
CAGTTCTTAGCCCGCCACAACGGAGGGAATCACCCTGACCAAATACGCTTAGGGAAAATTATCAGCCTTTGTCAGGTGATTGAGGACTGCTGCTGTTC
CCAGAACAAAACCAACCGGGTCACCCCGGAGGAGGTCGCAGCAAAGATTGACCTGTACCTCCGTGGTGCAACAAATCTTGAAGAATGCTTGGCCAGGC
TTGAGAAAGCGCGCCCGCCACGCGTAATGGACACCTCCTTTGATTGGGATGTTGTGCTCCCTGGGGTTGAGGCGGCAACTCAAACGACCGAACTGCCC
CAAGTCAACCAGTGTCGCGCTCTGGTCCCTGTTGTGACTCAAAAGTCCTTGGACAACAACTCGGTCCCTCTGACCGCCTTTTCACTGGCTAACTACTA
CTACCGCGCGCAAGGTGACGAAGTTCGTCACCGTGAAAGACTAACCACCGTGCTCTCTAAGTTGGAAGGGGTTGTTCGAGAAGAATACGGGCTCATGC
CAACCGGGCCTGGTCCACGGCCCACACTGCCACGCGGGCTCGACGAACTCAAGGACCAGATGGAGGTGGACTTGCTGAAACTGGCTAACGCCCAGATG
ACTTCGGACATGATGGCCTGGGCAGTCGAGCAGGTTGACCTAAAGACTTGGGTCAAGAACTATCCGCGGTGGACACCACCACCTCCTCCGCCAATAGT
TCAGCCTCGAAAAACGAAGCTTGTCAAGAGCTTACCAGAGAGCAAGCCTGTTCCTGCACCGCGTAGGAAGGTCAGGTCCGATTGTGACTGCCCCACCC
TATCGGGCAACAATCTTCCTGACAGTTGGGAAGATTTGGCTGTTGGTTGCCCCTCTGATCTCCCTACCTCACCTGAGCCGGTAACACCTTTGAGTGAG
CCGGCATCTGTGTCCGCACCGCGACGCTCTTTTAGGCCGGTGAAGCCTTTGAGTGAACCAGTTCCAGTCCCTGCACCGCGCAAGACTGTGTCCCGACC
GGCAACACCTCTGAGTGAGCCGATCCCTGTGCCCGCACCGCGACGCAAGTTTCAGCAGGTAGAAAAAGTGAATCCGGCGGCGGCAACCCTGGCGTGCC
AAGACGAGTTTCCAGATTTGTCTGCATCCTCGCATACTGAATATGAGGCGTCTCCCCTTGTACTACCGCAGAACGGGGACGTTCTTGAAGTGGAGGAG
CGGGAAGCTGAGGAAATCCTGAGTGGAATCTCAGACATACTGGATGCCATCAAACCGGCATCTGCATCATCAAGCAGCTCCCTGTCAAGTGTGGCGAT
CACACGCCCGAAATACTCAGCTCAAGCCATCATTGACTCGGGTGGGCCCTACAGCGGGCATCTCCAAGAGGTGAAGGAAACATGCCTAAGCATCATGA
GTGAGGCATGTGATGTGACCAAGCTTGATGACCCTGCCACGCAGGAATGGCTTTCTCGCATGTGGGATAGGGTGGACATGCTGACTTGGCGCAATACG
TCTGTTCACCAGGCGTCTCGCACCTTGGACGACAGATTTAAGTTTCTCCCGAAGATGATACTTGAAACACCGCCGCCCTACCCGTGTGGGTTCGTGAT
GATGCCTCGCACACCTGCACCCTCCGTGGGTGCGGAGAGCGACCTCACTATTGGCTCAGTCGCTACTGAGGGCGTTCCACGCATCTTCGGGAAAGTAA
ATGATGTCTGCAAGATGATCGACCAGAGACCCTTGGTACTCTTTGAAAATGAGCTGGCAGATGACCAACCTGCCAGAGATCCTCGGACATCATCGCAG
AGGTTTGACGGGAGCACACCAGCTCCGCCCGCAGGCACGGATGGCACCGGTTTGGCTTCGGGCCCTGGAGTGAGAGAAGTGGATTCATGTGAGGCGAG
CTCAACCGAGAAAAATGAACAGCCCTTCGTGTTGAACGGCGGCGCCAGCACACAGGCGTCAACGTTTACCAATTTGCCGCCTCCAGGCGGTATAGATG
CGGGCGGGAGTGGGCCGTTACAAACGGTGCGAAAGAAGGCTGAACGGTTCTTTGACCTACTAAGCCGTCAGGTTTTTAATCTCGTCTCCCATCTCCCT
GTTTTCTTCTCACGCCTTTTCAAACCTGGCGGTGACTATTCTCCGGGTGATTGGGGTTTTGCAGCTTTTACTTTATTGTGCCTCTTTTTGTGTTACAG
TTACCCGGCCTTTGGTGCTGTTCCCCTCTTGGGTGTATTTTCTGGGTCTTCTCGGCGTGTTCGAATGGGGGTTTTTGGCTGCTGGTTGGCTTTTGCTG
TTAGTCTGTTCAAACCTGTGTCCGACCCAGTCGGCGCTGCTTGTGAATTTGATTCGCCAGAGTGTAGAAACATCCTTCATTCTTTTGAGCTTCTCAAA
CCTTGGGACCCTGTTCGCGGCCTTGTTGTGGGCCCCGTCGGTCTCAGTCTTGCCATTTTTGGCAGGTTATTGGGCGGGGCACGCCACATCTGGCACTT
TTTGCTTAGGTTTGGCATTGTTGCAGATTGTATCTTGGCTGGAGCTTATGTGCTTTCTCAAGGCAGGTGTAAAAAGTGCTGGGGATCTTGTATAAGAA
CTGCTC
CTAATGAGGTCGCCTT
TAACGTGTTTCCTTTTACACGTGCGACCAGGTCGTCACTTATC
GACCTGTGCAATCGGTT
TTGCGCGCCAAAAGGT
ATGGACCCCATTTTCCTCGCCACTGGGTGGCGCGGGTGCTGGACCGGCCGAAGCCCCATTGAGCAACCCTCTGAAAAACCCATCGCGTTTGCCCAGTT
GGATGAAAAAAAGATTACGGCTAGGACTGTGGTCGCCCAGCCCTATGACCCCAACCAAGCTGTAAAGTGCTTGCGGGTATTGCAGGCGGGCGGGGTGA
TGGTGGCTGAGGCAGTCCCAAAAGTGGTCAAAGTTTCTGCTGTTCCATTCCGAGCCCCCTTCTTTCCCACCGGAGTGAAAGTTGATCCTGAATGCAGG
ATTGTGGTTGACCCCGACACTTTCACTGCAGCCCTCCGATCTGGCTACTCCACCACAAACCTCGTCCTTGGTGTGGGGGACTTTGCCCAGCTGAATGG
ATTAAAAATCAGGCAAATTTCCAAGCCTTCAGGGGGAGGTCCACACCTCATGGCTGCCCTGCATGTTGCTTGCTCTATGGCTCTGCACATGCTTGTTG
GGATTTATGTGACTGCTGTGGGTTCTTGCGGCACCGGCACTAACGATCCGTGGTGCGCCAACCCGTTTGCCGTCCCTGGCTACGGACCTGGCTCTCTC
TGCACGTCCAGATTGTGCATCTCCCAACATGGCCTTACCCTACCCTTGACAGCACTTGTGGCGGGATTCGGCTTTCAGGAAATTGCCTTGGTTATTTT
GATTTTTGTTTCTATCGGAGGCATGGCTCATAGATTGAGTTGCAAGGCTGATATGCTGTGTATTTTGTTTGCAATCGCCAGCTATGTTTGGGTACCTC
TTACCTGGTTGCTTTGTGTGTTTCCTTGCTGGTTGCGCTGTTTTTCGTTGCACCCCCTCACCATCCTATGGTTGGTGTTTTTCCTGATTTCTGTAAAT
ATGCCTTCAGGAATCTTGGCCTTGGTGTTGTTGATTTCTCTCTGGCTTCTTGGTCGTTATACCAACGTTGCCGGTCTTGTCACTCCCTATGACATTCA
TCATTACACCAGTGGCCCCCGCGGTGTTGCCGCCTTGGCTACCGCACCAGATGGGACCTACTTGGCCGCTGTCCGCCGCGCCGCGTTGACTGGTCGCA
CCATGCTGTTTACCCCGTCTCAGCTCGGGTCCCTTCTTGAGGGCGCTTTCAGAACTCGAAAGCCCTCACTGAACACCGTTAATGTGGTCGGGTCCTCC
ATGGGCTCTGGCGGGGTGTTCACCATCGACGGGAAAATTAAGTGCGTAACTGCTGCACATGTCCTTACGGGTAATTCAGCTAGGGTTTCCGGGGTTGG
CTTCAATCAAATGCTTGACTTCGATGTGAAAGGAGACTTCGCCATAGCCGATTGCCCAGACTGGCAAGGGGCTGCTCCCAAGACCCAATTCTGCGAGG
AAGGATGGACTGGCCGGGCCTATTGGCTAACGTCTTCTGGTGTCGAACCCGGCGTCATTGGAAAAGGATTCGCCTTCTGCTTCACCGCGTGCGGCGAT
TCCGGATCCCCAGTAATCACCGAGGCCGGCGAGCTTATCGGCGTTCACACGGGGTCAAATAAACAAGGAGGAGGCATCGTCACGCGCCCCTCAGGCCA
GTTTTGTAGTGTGGCACCCGTCAAATTAAGCGAACTAAGTGAATTCTTTGCAGGGCCTAAGGTCCCGCTCGGTGATGTGAAAGTTGGCAGCCACATAA
TTGAAGACGTAGGCGAGGTGCCTTCAGATCTTTGCGCCTTGCTTGCTGCCAAACCTGAACTGGAAGGAGGCCTCTCCACCGTTCAACTTCTGTGTGTG
TTTTTCCTCCTGTGGAGAATGATGGGACATGCCTGGACGCCCTTGGTTGCCGTAGGGTTTTTTATCTTGAATGAGGTCCTCCCAGCTGTCCTGGTCCG
GAGTGTTTTCTCCTTTGGAATGTTTGTGCTATCCTGGCTCACACCATGGTCTGCGCAAGTTCTGATGATCAGGCTTCTAACAGCAGCTCTTAACAGGA
ATAGATGGTCACTTGCCTTTTACAGCCTCGGTGCAATGACTGGTTTTGTCGCAGATCTCGCGGCTACTCAGGGGTATCCGTTGCAGGCAGTGATGAGT
TTGAGCACTTATGCATTCCTGCCTCGGATAATGGTTGTGACTTCACCAGTCCCAGTGGTTGCGTGTGGTGTTGTGCACCTACTTGCCATCATTTTGTA
CTTGTTTAAGTACCGCTGCCTGCACAACATCCTTGTTGGCGATGGAGTGTTCTCTGCGGCTTTCTTCCTGCGATATTTTGCCGAGGGAAAGTTGAGGG
AGGGGGTGTCGCAATCCTGCGGGATGAATCATGAGTCACTTACCGGTGCCCTCGCTATGAGACTCAATGACGAGGACTTGGATTTCCTCACGAAATGG
ACTGATTTCAAGTGCTTTGTTTCTGCGTCCAACATGAGAAATGCTGCGGGCCAATTCATCGAGGCTGCCTATGCTAAAGCACTTAGAGTAGAACTTGC
CCAGTTGGTGCAGGTTGATAAGGTTCGGGGTACTTTGGCCAAACTTGAAGCTTTTGCCGACACCGTGGCACCCCAACTCTCGCCCGGTGACATTGTTG
TCGCTCTTGGCCATACGCCTGTTGGCAGTATCTTCGACCTGAAGGTTGGTAACACCAAGCACACTCTCCAAGCCATCGAGACCAGGGTCCTTGCTGGG
TCCAAAATGACCGTGGCGCGCGTCGTCGATCCGACCCCCACGCCCCCACCCGCACCCGTGCCCATCCCCCTCCCACCGAAGGTTTTGGAGAACGGTCC
AAACGCTTGGGGGGATGAAGACCGTTTGAATAAAAAGAGGAGGCGCAGGATGGAAGCCCTCGGCATCTATGTTATGGGCGGGAAAAAGTACCAGAAAT
TTTGGGACAAGAATTCCGGTGACGTGTTTTATGAGGAGGTCCATAACAACACAGATGAGTGGGAGTGCCTCAGAGTTGGCGACCCTGCCGACTTTGAC
CCTGAGAAGGGAACTTTGTGTGGGCATGTCACCATTGAAGATAGGGCTTACCATGTTTACACCTCCCCATCTGGTAAGAAATTCCTAGTCCCCGTCAA
CCCAGAGAACGGAAGAGTTCAATGGGAGGCTGCAAAGCTTTCCGTTGAGCAGGCCCTTGGTATGATGAACGTCGACGGCGAGCTGACTGCCAAGGAAC
TGGAGAAACTGAAAAGAATAATTGACAAACTCCAGGGCCTGACTAAGGAGCAGTGTTTAAACTGCTAGCCGCTAGCGGCTTGACCCGCTGTGGTCGCG
GCGGCTTGGTTGTTACTGAGACAGCGGTAAAAATAGTCAAATTTCACAAGCGGACCTTCACCTTGGGACCTGTAAATTTAAAAGTGGCTAGTGAGGTT
GAGCTAAAAGACGCGGTCGAGCACAACCAACACCCGGTTGCTAGACCAGTCGATGGTGGTGTTGTGCTCCTGCGTTCCGCGGTTCCCTCGCTTATAGA
TGTCCTGATCTCCGGTGCTGATGCATCTCCCAAATTACTTGCCCATCACGGACCGGGCAACACCGGGATCGATGGCACGCTCTGGGATTTTGAGTCCG
AAGCCACTAAAGAGGAAGTCGCACTCAGCGCGCAAATAATACAGGCTTGTGACATTAGGCGCGGCGACGCACCTGAAATTGGTCTCCCTTACAAGCTG
TACCCTATTAGGGGCAACCCTGAGCGGGTAAAGGGAGTTTTACGGAATACAAGGTTTGGAGACATACCTTATAAAACCCCCAGTGACACTGGAAGCCC
AGTGCACGCGGCTGCCTGCCTCACGCCTAACACCACTCCGGTGACTGACGGGCGCTCCGTCTTGGCCACGACCATGCCCTCCGGGTTTGAGTTGTATG
TACCGACCATACCAGCGTCTGTCCTTGACTATCTTGATTCTAGGCCCGACTGCCCTAAACAGTTGACGGAGCACGGCTGTGAAGATGCCGCACTGAAA
GACCTCTCCAAGTATGACTTGTCCACCCAAGGCTTTGTTTTGCCTGGAGTTCTTCGCCTTGTGCGGAAGTACCTGTTTGCCCATGTGGGCAAATGCCC
GCCCGTTCATCGGCCTTCCACTTACCCTGCCAAGAATTCTATGGCTGGAATAAATGGGAACAGGTTTCCAACCAAGGACATCCAGAGCGTCCCTGAAA
TCGACGTTCTGTGCGCACAGGCTGTGCGAGAAAATTGGCAAACTGTTACCCCTTGTACCCTCAAGAAACAGTATTGTGGGAAGAAGAAGACTAGGACC
ATACTCGGCA
CCAATAACTTTATAGCG
CTGGCTCACCGTGCAGCGTTGAGTGGTGTCACCCAGGGCTTCA
TGAAAAAGGCGTTTAAC
TCGCCTATCGC
CCTCGGGAAAAACAAATTTAAGGAGCTACAGACTCCGGTCCTAGGCAGGTGCCTTGAAGCTGATCTTGCATCCTGCGATCGATCTACACCTGCAATTG
TCCGCTGGTTTGCCGCCAATCTTCTTTATGAGCTTGCCTGTGCTGAAGAGCATCTACCGTCGTATGTGCTGAACTGCTGTCACGACTTACTGGTCACG
CAGTCCGGTGCAGTGACTAAGAGAGGCGGCCTGTCGTCTGGCGACCCGATAACCTCTGTGTCTAATACCATTTACAGCTTGGTGATCTATGCACAGCA
CATGGTGCTTAGTTGCTTCAAAAGTGGTCACCCCCATGGCCTTCTGTTCTTACAAGACCAGCTAAAGTTTGAGGACATGCTCAAGGTTCAACCCCTAA
TCGTCTATTCGGACGACCTCGTGCTGTATGCCGAGTCTCCCACCATGCCAAACTACCATTGGTGGGTTGAACATCTGAATTTGATGCTGGGGTTTCAG
ACGGACCCAAAGAAAACAGCCATAACAGACTCGCCATCATTTCTAGGCTGTAGAATAATAAATGGGCGCCAGCTAGTCCCCAACCGTGACAGGATTCT
CGCGGCCCTCGCCTACCACATGAAGGCGAGTAATGTTTCTGAATACTACGCCTCAGCGGCTGCAATACTCATGGACAGCTGTGCTTGTTTGGAGTATG
ATCCTGAATGGTTTGCAGAACTTGTAGTTGGGATAGCGCAGTGCGCCCGCAAAGACGGCTACAGCTTTCCCGGCACGCCGTTCTTCATGTCCATGTGG
GAAAAACTCAGGTCCAATTATGAGGGGAAGAAGTCGAGAGTGTGCGGGTACTGCGGGGCCCCGGCCCCGTACGCTACTGCCTGTGGCCTCGACGTCTG
CATTTACCACACCCACTTCCACCAGCATTGTCCAGTCACAATCTGGTGTGGCCATCCAGCGGGTTCTGGTTCTTGTAGTGAGTGCAAATCCCCTGTAG
GGAAAGGCACAAGCCCTTTAGACGAGGTGCTGGAACAAGTCCCGTATAAGCCCCCACGGACCGTTATCATGCATGTGGAGCAGGGACTTACCCCCCTT
GACCCAGGTAGATACCAGACTCGCCGCGGATTAGTCTCTGTCAGGCGTGGAATCAGGGGAAATGAAGTTGAACTACCAGACGGTGATTATGCTAGTAC
CGCCTTGCTCCCTACCTGTAAAGAGATCAACATGGTCGCTGTCGCTTCTAATGTATTGCGCAGCAGGTTCATCATCGGTCCACCCGGTGCTGGGAAAA
CATACTGGCTCCTTCAACAGGTTCAGGATGGTGATGTTATTTACACACCAACTCACCAGACCATGCTTGACATGATTAGAGCTTTGGGGACGTGCCGG
TTCAACGTTCCGGCAGGCACAACGCTGCAATTCCCCGTCCCCTCCCGTACCGGTCCGTGGGTTCGCATTCTAGCCGGCGGTTGGTGTCCTGGCAAGAA
TTCCTTCCTGGATGAAGCAGCATATTGCAATCACCTTGATGTTTTGAGGCTTCTTAGCAAAACTACCCTCACCTGTCTAGGAGACTTCAAACAACTCC
ACCCAGTGGGTTTTGATTCTCATTGCTATGTTTTTGACATCATGCCTCAAACTCAACTGAAGACCATCTGGAGGTTTGGACAGAATATCTGTGATGCC
ATCCAGCCAGATTACAGGGACAAACTCATGTCCATGGTCAACACAACCCGTGTGACCTACGTGGAAAAACCTATCAGGTATGGGCAAGTCCTCACCCC
CTACCACAGAGATCGAGAGGACGACGCCATCACTATTGACTCCAGTCAAGGCGCCACATTCGATGTGGTTACACTGCATTTGCCCACTAAAGATTCAC
TCAACAGGCAAAGAGCCCTTGTTGCTATCACCAGGGCCAGACATGCTATCTTTGTGTATGACCCACACAGGCAGCTGCAGAGCTTGTTTGATCTTCCT
GAAAAAGGCACACCCGTTAACCTCGCAGTGCACCGCGACGGGCAGCTGATCGTGCTGGATAGAAATAACAAAGAATGCACGGTTGCTCAAGCTCTAGG
CAACGGGGATAAATTTAGGGCCACAGACAAGCGTGTTGTAGATTCTCTCCGCGCTATTTGTGCTGACCTAGAAGGGTCGAGCTCTCCGCTCCCCAAGG
TCGCACACAACTTGGGATTTTATTTCTCACCTGATTTAACACAGTTTGCCAAACTCCCAGCAGAACTTGCACCTCACTGGCCTGTGGTGACAACCCAG
AACAATGAAAATTGGCCAGATCGGCTGGTTGCCAGCCTTCGTCCTATCCATGAGTATAGCCGCGCGTGCATCGGTGCCGGCTATATGGTTGGCCCCTC
GGTGTTTCTAGGCACCCCCGGGGTCGTGTCATACTATCTCACAAAATTTGTTAAGGGCGAGGCTCAAGTGCTTCCGAAGACGGTCTTCAGCACCGGCC
GAATTGAGGTAGATTGCCGGGAATATCTTGACGATCGGGAGCGAGAAGTTGCTGCGTCCCTCCCACATGCCTTCATTGGTGACGTCAAAGGCACTACC
GTTGGAGGATGTCATCATGTCACCTCCAGATACCTCCCGCGCTTCCTCCCCAAGGAATCAGTTGCTGTGGTCGGGGTTTCAAGTCCCGGAAAAGCCGC
GAAAGCATTGTGCACACTGACAGATGTGTACCTCCCAGACCTTGAAGCCTATCTCCACCCGGAGACCCAGTCCAAGTGCTGGAAAATGATGTTGGACT
TCAAGGAAGTTCGACTAATGGTCTGGAAAGACAAAACAGCCTATTTCCAACTTGAAGGACGCTATTTCACCTGGTATCAGCTTGCTAGCTATGCCTCG
TACATCCGTGTTCCTGTCAACTCTACTGTGTACTTGGACCCCTGCATGGGCCCCGCCCTTTGCAACAGGAGAGTCGTTGGGTCCACTCACTGGGGGGC
TGACCTCGCAGTCACCCCTTATGATTACGGCGCTAAAATTATCCTGTCTAGCGCGTACCATGGTGAAATGCCCCCCGGATACAAAATTCTGGCGTGCG
CGGAATTCTCGTTGGATGACCCAGTCAAGTACAAACATACTTGGGGGTTTGAATCGGATACAGCGTATCTGTATGAGTTCACCGGAAACGGTGAGGAT
TGGGAGGATTACAATGATGCGTTTCGTGCGCGCCAGGAAGGGAAAATTTATAAGGCCACTGCCACCAGCATGAAGTTTTATTTTCCCCCGGGCCCTGT
CATTGAACCAACTTTAGGCCTGAATTGATTAATTAATTTAAAAGGCGCGCCAATGAAATGGGGTCCATGCAAAGCCTTTTTGACAAAATTGGTCAACT
TTTTGTGGATGCTTTCACGGAATTCTTGGTGTCCATTGTTGATATCATCATATTTTTGGCCATTTTGTTTGGCTTCACCGTCGCCGGTTGGCTGGTGG
TCTTTTGCATCAGATTGGTTTGCTCCGCGATACTCCGTGCACGCCCTGCCTTTCACTCTGAGCAGTTACAGAAGATCCTATGAGGCCTTTCTTTCCCT
GTGTCAGGTGGACATTCCCACCTGGGGAATCAAACATCCTCTGGGGGTGCTTTGGCACCATAAGGTGTCAACCCTGATTGATGAAATGGTGTCGCGTC
GAATGTACCGCATCATGGAAAAAGCAGGACAGGCTGCCTGGAAACAGGTGGTGAGCGAGGCCACGCTGTCTCGCATTAGTAGTTTGGATGTGGTGGCT
CATTTTCAACATCTTGCCGCCGTTGAAGCCGAGACCTGTAAATATTTGGCCTCTCGGCTACCCATGCTACACAACCTGCGCATGACGGGGTCAAATGT
AACCATAGTATATAATGGTACTTTGAATCAGGTGTTTGCCATTTTCCCGACCCCTGGTTCCCGGCCAAAGCTTCATGATTTTCAGCAATGGCTGATAG
CTGTGCATTCGTCCATATTTTCCTCTGTTGCAGCTTCTTGTACTCTGTTTGTTGTACTGTGGTTGCGGGTCCCAATGCTACGTACTGTTTTTGGTTTC
CGCTGGTTAGGGGCAATTTTTCCTTCGAGCTCTTGGTGAATTACACGGTGTGCCCACCTTGCCTCACCCGGCAAGCAGCCGCACAGCGCTACGAACCT
GGCAAGGCTTTTTGGTGCAGAATTGGGTACGATCGATGTGAGGAGGACGATCACGACGAGCTAGGGTTCGTGATACCGTCTGGCCTCTCCAGCGAAGG
CCACTTGACTAGTGT
TTACGCCTGGCTGGCG
TTCCTGTCCTTCAGCTACACGGCCCAGTTCCATCCCGAGATAT
TTGGGATAGGGAATGTG
AGTCAAG
TTTATGTTGACATCAAGCACCAATTCATCTGCGCTGTTCACGACGGGGATAACGCCACCTTGCCTCGCCATGACAATATTTCAGCCGTATTTCAGACC
TACTACCAACACCAGGTCGACGGCGGCAATTGGTTTCACCTGGAATGGCTGCGTCCTTTCTTTTCCTCTTGGTTGGTTTTAAATGTTTCGTGGTTTCT
CAGGCGTTCGCCTGCAAGCCATGTTTCAGTTCGAGTCTTTCGGACATCAAAACCAACACCACCGCAGCATCAGACTTCGTTGTCCTCCAGGACATCAG
CTGCCTTAGGCATGGCGACTCGTCCTCTCCGACGATTCGCAAAATTTCTCAGTGCCGCACGGCGATAGGGACGCCCGTGTACATCACCATCACTGCCA
ATGTCACAGATGAAAATTATCTACATTCTTCTGATCTCCTCATGCTTTCTTCTTGCCTTTTCTATGCTTCCGAGATGAGTGAAAAGGGATTCAAAGTG
GTGTTTGGCAATGTGTCAGGCACTGTGGCTGTGTGCGTCAACTTTACCAGCTACGTCCAACATGTCAAGGAGTTTACCCAACGCTCCTTAGTGGTCGA
TCATGTGCGACTGCTTCATTTCATGACACCTGAGACCATGAGGTGGGCAACCGTTTTAGCCTGTCTTTTTGCCATCCTACTGGCAATTTGAATGTTCA
AGTATGTTGGGGAAGTGCTTGACCGCGTGCTGTTGCTCGCGATTGCTTTTTTTGTGGTGTATCGTGCCGTTCTATCTTGCTGTGCTCGTCAACGCCAG
CAACAACAACAGCTCTCATATTCAGTTGATTTATAACTTAACGTTATGTGAGCTGAATGGCACAGATTGGCTGGCACAAAAATTTGACTGGGCAGTGG
AGACTTTTGTCATCTTCCCCGTGTTGACTCACATTGTTTCCTATGGGGCACTCACCACCAGCCATTTCCTTGACACAGTTGGTTTGGCCACTGTGTCT
ACCGCCGGATATTATCACGGGCGGTATGTCTTGAGTAGCATTTACGCAGTCTGTGCTCTGGCTGCGCTGATTTGCTTTGTCATTAGGCTTGCGAAGAA
CTGCATGTCCTGGCGCTACTCTTGTACCAGATATACCAACTTCCTTCTGGACACTAAGGGCAGACTCTATCGTTGGCGGTCGCCCGTCATTGTGGAGA
AAGGGGGTAAGGTTGAGGTCGAAGGTCACCTGATCGACCTCAAGAGAGTTGTGCTTGATGGTTCCGCGGCAACCCCTTTAACCAGAGTTTCAGCGGAA
CGATGGGGTCGTCTCTAGACGACTTCTGCAATGATAGCACAGCTCCACAGAAGGTGCTTTTGGCGTTTTCCATTACCTACACGCCAGTGATGATATAT
GCTCTAAAGGTAAGTCGCGGCCGACTGCTAGGGCTTCTGCACCTTTTGATCTTTCTGAATTGTGCTTTTACCTTCGGGTACATGACATTCGTGCACTT
TGAGAGCACAAATAGGGTCGCGCTCACTATGGGAGCAGTAGTTGCACTTCTTTGGGGAGTGTACTCAGCCATAGAAACCTGGAAATTCATCACCTCCA
GATGCCGTTTGTGCTTGCTAGGCCGCAAGTACATTCTGGCCCCTGCCCACCACGTCGAAAGTGCCGCGGGCTTTCATCCGATTGCGGCAAATGATAAC
CACGCATTTGTCGTCCGGCGTCCCGGCTCCACTACGGTCAACGGCACATTGGTGCCCGGGTTGAAAAGCCTCGTGTTGGGTGGCAGAAAAGCTGTTAA
GCAGGGAGTGGTAAACCTTGTTAAATATGCCAAATAACAACGGCAAGCAGCAAAAGAAAAAGAAGGGGAATGGCCAGCCAGTCAATCAGCTGTGCCAA
ATGCTGGGTAAGATCATCGCCCAACAAAACCAGTCCAGAGGCAAGGGACCGGGGAAGAAAAATAGGAAGAAAAACCCGGAGAAGCCCCATTTCCCTCT
AGCGACTGAAGATGACGTCAGGCATCACTTTACCCCTAGTGAGCGGCAATTGTGTCTGTCGTCGATCCAGACTGCCTTCAATCAGGGCGCTGGAACTT
GTGCCCTGTCAGATTCAGGGAGGATAAGTTACACTGTGGAGTTTAGTTTGCCGACGCAACATACTGTGCGTCTGATCCGCGCCACAGCATCACCCTCA
GCATGATGGGCTGGCATTCTTTGGCACCTCAGTGTTAGAATTGGGAGAATGTGTGGTGAATGGCACTGATTGACACTGTGCCTCTAAGTCACCTATTC
AATTAGGGCGACCGTGTGGGGGTAAAGTTTAATTGGCGAGAACCATGCGGCCGTAATTAAAAAAAAAAAA
Claims (4)
1, porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain, after utilizing reverse genetic manipulation and gene recombination technology all nucleotide sequence Delete Alls with the latter half of the GP2 gene of the classical malicious PTK strain of porcine reproductive and respiratory syndrome virus (PRRSV) and GP3～GP5 gene, M protein gene, 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.
2, porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain according to claim 1, it is characterized in that: 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 inserts is 2421bp, the structural protein of the PRRSV that mainly encodes and 3 ' non-coding region.
3, the porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain according to claim 1 and 2 method of producing living vaccine, carry 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 individual layer, standby;
(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 and get product.
4, the detection method of the transgenic microorganism of porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain PC according to claim 1 and 2 strain adopts the RT-PCR method to detect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910062139 CN101603035B (en) | 2009-05-18 | 2009-05-18 | Porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain and method for producing living vaccine with same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910062139 CN101603035B (en) | 2009-05-18 | 2009-05-18 | Porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain and method for producing living vaccine with same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101603035A true CN101603035A (en) | 2009-12-16 |
| CN101603035B CN101603035B (en) | 2011-06-08 |
Family
ID=41468934
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200910062139 Active CN101603035B (en) | 2009-05-18 | 2009-05-18 | Porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain and method for producing living vaccine with same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101603035B (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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