CN1827172A - Pig viral infectious disease gene recombined live vaccine using canine II type adenovirus as carrier and preparation process thereof - Google Patents
Pig viral infectious disease gene recombined live vaccine using canine II type adenovirus as carrier and preparation process thereof Download PDFInfo
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Abstract
This invention supplies a series of production techniques of gene recombination live vaccine of swine virus contagion with canine II adenovirus as carrier and finished goods. The viral live vectors vaccine takes swine important virus zymad protective antigens gene as object gene, which are chosen from HCV-E1, E2 gene, FMDV-VP1,VP2,VP3,VP4 gene, TGEV-S,N,M gene, PEDV-S,N,M gene,SIV-HA,NA gene,RV-G,N gene, etc. The produced vaccines contain recombined swine influenza virus HA gene adenovirus carrier live vaccine, swine plague virus E2 gene adenovirus carrier live vaccine and recombination swine AsiaI foot-and-mouth disease virus VPI gene adenovirus carrier live vaccine. Recombination virus has good inheritance stability, and vaccine immunization can induct pig develop differential antiviral neutralization antibody. It has good immune protection effect and has no toxic side effect. The goods are facilitating for preserve and transport;it has long storage life and simple technics, and it fits for commercial manufacture.
Description
Technical field:
It is that the pig virus infectious disease immunity of carrier is with gene recombination living vaccine and production technology with dog II type adenovirus that the present invention aims to provide a series of, be used for the development and the production of pig virus infectious disease vaccine, with the generation of prevention pig virus infectious disease and popular, belong to zoonotic prevention and treatment technology field.
Background technology:
The development of the not only serious harm pig industry of viral infectious of pigs such as swine fever, influenza, rabies, foot and mouth disease and transmissible gastroenteritis of swine, porcine epizootic diarrhea, the annual economic loss that therefore causes is huge, and the generation of zoonosis such as swine flue, rabies and popular, also human life and health in serious threat, therefore the anti-of these eqpidemic diseases is made for the emphasis of veterinary work and studies focus.
For the control of pig virus infectious disease, still follow the principle of " it is auxilliary putting prevention first, treating " at present, can a kind of development of good animal vaccine become the key that effectively prevent making these eqpidemic diseases with using.The vaccine that is used for the anti-system of viral infectious of pig at present mainly still is a traditional vaccine, be weak malicious Seedling of totivirus or inactivated vaccine, as poison or inactivated vaccine etc. a little less than malicious freeze-dried vaccine, swine rabies, foot and mouth disease inactivated vaccine, transmissible gastroenteritis of swine and the epidemic diarrhea a little less than the swine fever.But all there is the shortcoming of traditional vaccine in these vaccines.
Along with the appearance of technique for gene engineering, recombinant vaccine is also come out one after another like the mushrooms after rain.Gene vaccine, subunit vaccine, recombiant vaccine, live vector vaccine etc. become the representative of new generation vaccine gradually.Virus live vector vaccine particularly; because it expresses destination protein antigen; reversion of attenuated vaccine virulence and the untrue shortcoming of dead Seedling deactivation have been overcome; and its expressed proteins is single protective antigen; immune effect is certain; can reach simultaneously the effect of a multiple disease of immunoprophylaxis, therefore extremely research worker favor.
In recent years, adenovirus system has become the research focus as mammalian cell expression vector, recombiant vaccine and gene therapy vector.Adenovirus is in Adenoviridae (Adenoviridae), and is extensive in distributed in nature, and its host comprises people, various mammal and birds.According to the host range difference Adenoviridae is divided into mammalian adenoviruses (Mastadenovirus) and two genus of birds adenovirus (Aviadenovirus), does not have common group antigen between two genus.Viral genome is divided into 4 main early transcription districts and 5 late transcription districts, in the life cycle of virus, each district has different effects, the main effect of E1 district (E1A and E1B) encoding proteins is to activate other gene transcription of virus, and the relevant cell gene transcription is also had tangible regulating and controlling effect; The major function of E2 district (E2A and E2B) encoded protein is to regulate duplicating of viral DNA; It is relevant to the scavenging action of the cell that is infected by the virus that the gene outcome in E3 district and virus are escaped host immune system in vivo; E4 district gene outcome participates in the duplicating of viral DNA, late gene expression, RNA shears with to stop functions such as host cell biomolecule is synthetic relevant; The late gene virus structural protein of mainly encoding.
(Canine adenovirus is pathogenic the strongest a kind of in the mammalian adenoviruses CAV) to hepatitis infectiosa canis virus, is divided into amphitypy, and dog I type adenovirus (CAV-1) is a canine infectious hepatitis virus, causes infectious canine hepatitis and epizootic fox encephalitis; Dog II type adenovirus (CAV-2) is the dog laryngotracheitis virus, causes infectious laryngotracheitis and the enteritis of dog.Artificial vaccination can make Cavia porcellus, racoon and wolf that experimental infection takes place.
Many at present for hepatitis infectiosa canis virus with malicious carrier construction, not only stabilization characteristics of genetics but also fool proof a little less than the CAV-2.CAV-2 has that it can infect somatoblast and also can infect akinete as (1) with the adenovirus hominis confers similar advantages, and the infection cell type is extensive.(2) can directly be transported in the body.(3) carrier is easy to preparation, the titre height, and resistance is strong.(4) has very big clone's ability.
Klonjkowski etc. have made up the recombinant C AV carrier that contains reporter gene LacZ first, and at first with the E1 district gene delection of CAV-2, this has not only removed its potential tumorigenesis ability, and have enlarged the ability of CAV-2 clone exogenous gene.
People such as Mark D.Morrison have made up the proteic recombinant C AV-1 carrier of expression CPV VP2.Usefulness CAV-2 Toronto A26/61 strains such as Kremer have made up a plurality of CAV-2 carriers again in E1 trans-complementation cell line.
What Fischer people such as (2002) had obtained to have replication capacity through vitro recombination is the recombinant virus that carrier contains CDV H and F protein gene expression box respectively with CAV-2.
In a word, adenovirus vector can also can be translated post-treatment to foreign protein by efficiently expressing exogenous gene, as shearing, glycosylation, phosphorylation etc., expressed proteins has the characteristic of native protein, can be used for fields such as pharmacy, recombinant vaccine, gene therapy and oncotherapy, shown good prospects for application.Recombinant adenovirus can carry out immunity by approach such as injection, oral, trachea inoculations, the inoculation animal not only produces humoral immunization and cellular immunization, but also produce the local mucous membrane immunne response, the infection that prevents respiratory tract, digestive tract and the eqpidemic disease that spreads through sex intercourse is extremely important.In recent years, no matter be the further improvement of adenovirus hominis carrier, or the development of animal adenovirus vector, all will control in the humans and animals infectious disease, particularly viral infectious and play a part more and more important in the human gene therapy.
Summary of the invention:
It is the production technology of the pig virus infectious disease gene recombination living vaccine of carrier that the present invention aims to provide with dog II type adenovirus, be used for the development and the production of pig virus infectious disease vaccine, with the generation of prevention pig virus infectious disease and popular, and, disclose its immunoprophylaxis effect by application to correlated product.
The invention also discloses reorganization swine influenza virus HA gene adenovirus vector live vaccine.
The invention also discloses reorganization swine fever virus raq gene adenovirus vector live vaccine.
The invention also discloses reorganization pig AsiaI type foot and mouth disease virus VPI gene adenovirus vector live vaccine.
The pig virus infectious disease gene recombinaton live vector vaccine that the present invention relates to is to be carrier with dog II type adenovirus (CAV-2), and the important viral infectious cause of disease of pig protective antigen gene is the virus live vector vaccine of genes of interest.
The E3 district is the nonessential region of adenovirus growth, and the adenovirus in disappearance or replacement E3 district still can be grown on cell.The adenovirus of reorganization pig virus infectious disease cause of disease protective antigen gene is by deleted adenovirus genome part E3 district, and displacement is simultaneously gone up the destination gene expression box and finished.Wherein the destination gene expression box contains cytomegalovirus (CMV) promoter and poly A (polyA) structure.
This product is faint yellow neutral lyophilized formulations, and production technology may further comprise the steps:
A. the clone of pig virus infectious disease protective antigen gene: obtain the protective antigen gene full-length gene or contain the sequence of epitope by PCR method; as HCV-E1, raq gene; FMDV-VP1, VP2, VP3, VP4 gene; TGEV-S, N, M gene; PEDV-S, N, M gene; SIV-HA, NA gene, RV-G, N gene etc.
B. construction of recombinant plasmid: genes of interest is cloned into contains in the complete genomic vector plasmid of CAV-II, recombiant plasmid has lacked viral genome part E3 district.
A. by PCR method the full genomic clone of CAV-2 is gone among the plasmid pPoly2, acquisition contains the two terminal rescue plasmid pPoly2-UD of CAV-2 genome, behind BstB I+HindIII linearization for enzyme restriction, carry out homologous recombination with the CAV-2 genomic DNA, obtain the full genomic clone plasmid of CAV-2 pPoly2-CAV/2, it is characterized in that containing the full genome of CAV-2.
B. the pig virus infectious disease protective antigen gene (Targetgene that will obtain through steps A; Tg) be cloned among the carrier for expression of eukaryon pVAX through the method for enzyme action and external connection; obtain eukaryon expression plasmid pVAX-Tg; it is characterized in that containing the destination gene expression box, comprise cytomegalovirus (CMV) promoter, genes of interest and poly A (polyA) structure.
C. with eukaryon expression plasmid pVAX-Tg with contain the segmental plasmid pVAX-E3 in CAV-2 genome E3 district enzyme action after be connected external, obtain genes of interest eukaryotic expression transfer vector pVAX Δ E3Tg, it is characterized in that contained CAV-2 genome E3 district portion gene is lacked.
D. eukaryotic expression transfer vector pVAX Δ E3Tg and the full genomic clone plasmid of CAV-2 pPoly2-CAV/2 obtain to contain the recombiant plasmid pCAV-2/Tg of destination gene expression box after enzyme action is connected, it is characterized in that containing the CAV-2 genome in plasmid pPoly2 part fragment and disappearance part E3 district, wherein disappearance part in E3 district is replaced by the destination gene expression box.
C. the acquisition of recombinant virus: recombiant plasmid is transfection sensitive cells (as MDCK, cells such as 293) behind linearization for enzyme restriction, duplicates, packs the acquisition recombinant virus by the cell biological synthesis system.
D. the preparation of vaccine: positive recombinant virus with after the sensitive cells propagation, is added protective agent, and make freeze-dried products by national relevant requirements.
Good effect of the present invention is: the recombinant virus of preparation has good hereditary stability; behind the vaccine immunity pig with this virus preparation; can induce pig to produce special antiviral neutralizing antibody; have the good immune protection effect, have no side effect, goods are preserved easily; be convenient to transportation; the shelf-life limit for length, production technology is simple, is suitable for suitability for industrialized production.
Description of drawings
Fig. 1-1, recombinant cloning vector pMD18-THA construction strategy.
The construction strategy of Fig. 1-2, recombinant expression carrier PVAXHA.
The acquisition and the vaccine production of Fig. 1-3, reorganization swine influenza virus HA gene adenovirus.
Fig. 2-1, contain the plasmid construction policy map of swine fever virus raq gene expression cassette
Fig. 2-2, reorganization swine fever virus raq gene adenovirus obtain and vaccine production.
Fig. 3, reorganization pig AsiaI type foot and mouth disease virus VPI gene adenovirus active carrier vaccine preparation technology flow process.Fig. 4-1, normal mdck cell (10 * 10)
The mdck cell pathological changes (10 * 10) that Fig. 4-2, R-CAV-2/Tg virus cause
The Electronic Speculum ultrastructure (* 40000) of Fig. 4-3, R-CAV-2/Tg viral infection mdck cell
The specific embodiment:
The following example is intended to further describe for example the present invention, rather than limits the present invention by any way.Under the prerequisite that does not deviate from the spirit and principles in the present invention, any change or change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope that awaits the reply of the present invention.
The preparation of embodiment 1 reorganization swine influenza virus HA gene adenovirus vector live vaccine
Prepare the recombinant adenovirus live vector vaccine according to the process route shown in the accompanying drawing 1, have the general features of adenovirus under the recombinant virus Electronic Speculum, behind the infection mdck cell, the specific cell pathological changes can occur.See accompanying drawing 4.Concrete steps are as follows:
1, PCR method obtains to contain the two terminal rescue plasmid pPoly2-UD of CAV-2 genome, carries out homologous recombination with the CAV-2 genomic DNA behind BstB I+Hind III linearization for enzyme restriction, obtains the full genomic clone plasmid of CAV-2 pPoly2-CAV/2.
The PCR primer:
P
1: 5 ' end GCG-TGC-CTA-ACT-ACA-AAC-TCA-AT
5 ' end
G-CCT-GCT-GCT-TGA-TTT-TGT-GAT-C
P
2: 5 ' end
A-CTC-ATA-GAA-GTA-GGC-AGC-TCC-G
5 ' end
CAT-CAT-CAA-TAA-TAT-ACA-GGA-CAA-AG
2, extract test kit with RNA and extract the swine influenza virus geneome RNA, operational approach is seen the test kit description.
3, the RT-PCR method obtains the HA gene, and fragment length is 1707bp.Wherein:
RT: with random primer olig (dT),
PCR: forward primer:
GGATCCCATGGAGAGAATAGTGCTTCTTC
Downstream primer:
CTCGAGTTAAATGCAAATTCTGCATTGTA
Method: in the total RNA that extracts, add 10mM olig (dT) 1ul respectively and mix, 70 ℃ of water-bath 5min, taking-up is put on ice, add 5 * RT-buffer 5ul subsequently, 10mM dNTP 2.5ul, 40U/ul RNA enzyme inhibitor 1ul, 9U/ulAMV reverse transcription 1ul, add no RNA enzyme water to cumulative volume 25ul, carry out reverse transcription, promptly obtain cDNA in 42 ℃ of insulation 1h.70 ℃ of water-bath 10min, the deactivation reverse transcription.Negate transcription product 10 μ L, 10 * PCRbuffer, 4 μ L, 25mmol/L MgCl
2Each 0.5 μ L of 4 μ L, forward primer (25pmol/ μ L), downstream primer (25pmol/ μ L) and 10mmol/L dNTPs adds water 30 μ L, boils degeneration 10min, and ice bath 5min adds Taqplus archaeal dna polymerase 0.25 μ L (5U/ μ L) mixing.Concrete reaction condition: amplification RGP gene is 94 ℃ of degeneration 30sec, 52 ℃ of annealing 40sec, 72 ℃ of extension 70sec, and 25 circulations are extended 10min for back 72 ℃ again, are cooled to 4 ℃ at last, finish reaction.With the analysing amplified effect of 1% agarose gel electrophoresis.Reclaim the purpose fragment, the T carrier of packing into.
4, with restriction endonuclease BamH I and Xho I enzyme action HA gene and carrier for expression of eukaryon pVAX1, electrophoresis reclaims, and 16 ℃ of connections of external use T4 ligase are spent the night, and obtains to contain the eukaryon expression plasmid pVAXHA of swine influenza virus HA.
5, the eukaryon expression plasmid pVAXHA that will contain swine influenza virus HA with contain the segmental plasmid pVAX-E3 in CAV-2 genome E3 district and behind Mlu I and Spe I enzyme action, obtain genes of interest eukaryotic expression transfer vector pVAX Δ E3HA in external the connection.
6, eukaryotic expression transfer vector pVAX Δ E3HA and the full genomic clone plasmid of CAV-2 pPoly2-CAV/2 obtain to contain the recombiant plasmid pCAV-2/HA of destination gene expression box after Asc I+Cla I enzyme action is connected.
7, recombiant plasmid pCAV-2/HA transfection mdck cell behind Asc I+Cla I enzyme action, copy package obtains recombinant virus CAV-2/HA.
8, with recombinant virus inoculation mdck cell, through rolling bottle suspension culture propagation, to viral TCID
50Reach 10
5-6Individual TCID
50/ 0.1mL adds gelatin sucrose as protective agent, (5mL/ bottle) lyophilizing after the packing.
For showing the present invention in the effect aspect the important viral infectious of prevention pig, following test proves:
The outer hereditary stability test of CAV-2/HA virion
CAV-2/HA is inoculated the mdck cell cultivation of going down to posterity, reached for 30 generations.Respectively to the 15th generation of CAV-2/HA, 30 generation culture be template, amplification HA expression cassette nucleic acid fragment (partial sequence that contains the E3 district in both sides) checks order.The result with to the 5th generation culture sequencing result consistent.This shows that CAV-2/HA has a good hereditary stability external.
PCR primer: upstream: 5 '-CAGTTCATTCCTAACTACGAC-3 '
Downstream: 5 ' CAAGTGGAAGTACCAAAGCTG-3 '
The CAV-2/HA vaccine is to the safety testing of pig
Get 5 pigs at random, with CAV-2/HA (1 * 10
5TCID
50/ ml) wherein 3 pigs are carried out counteracting toxic substances, oral 5ml/ head, intramuscular injection 5ml/ head, 10ml/ head altogether; In addition two pig is as negative control, oral 5ml normal saline/head, intramuscular injection 5ml normal saline/head, 10ml/ head altogether.Experiment pig is carried out isolated rearing, observe the clinical change situations such as spirit, appetite, body temperature and feces of pig every day, carried out the total white blood cells counting every 3 days one time.Observe 21d.Do not see during breeding observing that the counteracting toxic substances temperature of pig body raises, appetite reduces, thirsty desire increases, infection symptoms such as depressed, hemafecia, cough, significantly fluctuation does not take place in total white blood cells.
The CAV-2/HA vaccine is to the immunogenicity experiments of pig
With CAV-2/HA immunity 6 three monthly age pigs (the HI antibody titer of anti-CAV-2 and SIV is all less than 1: 2), intramuscular injection, dosage 1 * 10
4TCID
50/ head, immune 1 time of every interval 15d, immunity is 3 times altogether, and immunity is preceding to prepare serum with the 3rd immunity back 15d blood sampling, 56 ℃ of 30min deactivations ,-20 ℃ are frozen to be checked.Detect the HI antibody of anti-CAV-2 and SIV in the porcine blood serum respectively with micro-HI test.Before the immunity, the HI antibody titer of anti-CAV-2 of porcine blood serum and SIV is all less than 1: 2, and after the immunity 3 times, the anti-CAV-2 antibody of porcine blood serum is 1: 64～1: 128, and the HI antibody titer of anti-SIV is 1: 8～1: 16.
The preparation of embodiment 2 reorganization swine fever virus raq gene adenovirus vector live vaccine
According to the preparation of the process route shown in the accompanying drawing 3 preparation recombinant adenovirus live vector vaccine, have the general features of adenovirus under the recombinant virus Electronic Speculum, behind the infection mdck cell, the specific cell pathological changes can appear.See accompanying drawing 4.Concrete steps are as follows:
1, PCR method obtains to contain the two terminal rescue plasmid pPoly2-UD of CAV-2 genome, carries out homologous recombination with the CAV-2 genomic DNA behind PstI+Hind III linearization for enzyme restriction, obtains the full genomic clone plasmid of CAV-2 pPoly2-CAV/2.
The PCR primer:
P
1: 5 ' end GCG-TGC-CTA-ACT-ACA-AAC-TCA-AT
5 ' end
G-CCT-GCT-GCT-TGA-TTT-TGT-GAT-C
P
2: 5 ' end
A-CTC-ATA-GAA-GTA-GGC-AGC-TCC-G
5 ' end
CAT-CAT-CAA-TAA-TAT-ACA-GGA-CAA-AG
2, extract test kit with RNA and extract the swine rabies virus genome RNA, operational approach is seen the test kit description.
3, the RT-PCR method obtains raq gene, and fragment length is 1332bp.Wherein:
PCR: forward primer: 5 '-GGGGAATTCATGCAGCTAGCCTGCAA-3 '
Downstream primer: 5 '-ATTGCGGCCGCTCAACCAGCGGCGAGTTG-3 '
Method: in the total RNA that extracts, add 25mM forward primer 1ul respectively and mix, 70 ℃ of water-bath 5min, taking-up is put on ice, add 5 * RT-buffer 5ul subsequently, 10mM dNTP 2.5ul, 40U/ulRNA enzyme inhibitor 1ul, 9U/ulAMV reverse transcription 1ul, add no RNA enzyme water to cumulative volume 25ul, carry out reverse transcription, promptly obtain cDNA in 42 ℃ of insulation 1h.70 ℃ of water-bath 10min, the deactivation reverse transcription.Negate transcription product 10 μ L, 10 * PCRbuffer, 4 μ L, 25mmol/L MgCl
2Each 0.5 μ L of 4 μ L, forward primer (25pmol/ μ L), downstream primer (25pmol/ μ L) and 10mmol/L dNTPs adds water 30 μ L, boils degeneration 10min, and ice bath 5min adds Taqplus archaeal dna polymerase 0.25 μ L (5U/ μ L) mixing.Concrete reaction condition: amplification RGP gene is 94 ℃ of degeneration 30sec, 57 ℃ of annealing 40sec, 72 ℃ of extension 90sec, and 25 circulations are extended 10min for back 72 ℃ again, are cooled to 4 ℃ at last, finish reaction.With the analysing amplified effect of 1% agarose gel electrophoresis.Reclaim the purpose fragment, the T carrier of packing into.
4, with restriction endonuclease PstI+EcoRI enzyme action HA gene and carrier for expression of eukaryon pVAX1, electrophoresis reclaims, and 16 ℃ of connections of external use T4 ligase are spent the night, and obtains to contain the eukaryon expression plasmid pVE2 of swine fever virus E2.
5, the eukaryon expression plasmid pVE2 that will contain swine fever virus E2 through the MluI+BbeI enzyme action, mend flat with contain the segmental plasmid pVAX in CAV-2 genome E3 district Δ E3 warp
SspIObtain genes of interest eukaryotic expression transfer vector pVAX Δ E3/E2 in external connection after enzyme action, the dephosphorization.
6, eukaryotic expression transfer vector pVAX Δ E3/E2 and the full genomic clone plasmid of CAV-2 pPoly2-CAV2 obtain to contain the recombiant plasmid pCAV-2/E2 of destination gene expression box after the MluI+BbeI enzyme action is connected.
7, recombiant plasmid pCAV-2/E2 transfection mdck cell behind Asc I enzyme action, copy package obtains recombinant virus CAV-2/E2.
8, with recombinant virus inoculation mdck cell, through rolling bottle suspension culture propagation, to viral TCID
50Reach 10
5-6Individual TCID
50/ 0.1mL adds gelatin sucrose as protective agent, (5mL/ bottle) lyophilizing after the packing.
For showing the present invention in the effect aspect the important viral infectious of prevention pig, following test proves:
The outer hereditary stability test of CAV-2/E2 virion
CAV-2/E2 is inoculated the mdck cell cultivation of going down to posterity, reached for 30 generations.Respectively to the 15th generation of CAV-2/E2,30 generation culture be template, amplification E2 expression cassette nucleic acid fragment (partial sequence that contains the E3 district in both sides) checks order.The result with to the 5th generation culture sequencing result consistent.This shows that CAV-2/E2 has a good hereditary stability external.
PCR primer: upstream: 5 '-CAGTTCATTCCTAACTACGAC-3 '
Downstream: 5 ' CAAGTGGAAGTACCAAAGCTG-3 '
The CAV-2/E2 vaccine is to the safety testing of pig
Get 5 pigs at random, with CAV-2/E2 (1 * 10
5TCID
50/ ml) wherein 3 pigs are carried out counteracting toxic substances, oral 5ml/ head, intramuscular injection 5ml/ head, 10ml/ head altogether; In addition two pig is as negative control, oral 5ml normal saline/head, intramuscular injection 5ml normal saline/head, 10ml/ head altogether.Experiment pig is carried out isolated rearing, observe the clinical change situations such as spirit, appetite, body temperature and feces of pig every day, carried out the total white blood cells counting every 3 days one time.Observe 21d.Do not see during breeding observing that the counteracting toxic substances temperature of pig body raises, appetite reduces, thirsty desire increases, infection symptoms such as depressed, hemafecia, cough, significantly fluctuation does not take place in total white blood cells.
The CAV-2/E2 vaccine is to the immunogenicity experiments of pig
Totally 10 of the preceding 27 age in days piglets of wean have been selected for use, intramuscular inoculation swine fever raq gene recombinant adenovirus live vector vaccine 10ml/ head.To the blood sampling of test pig ear vein, separation of serum used swine fever forward indirect agglutination diagnostic kit, measures hog cholera antibody respectively at immunity back 0 day, 8 days, 16 days, 24 days, 32 days, 40 days, 48 days.The result shows, the test swinery is behind the inoculation swine Fever Vaccine, and hog cholera antibody is progressively raising, and in immunity back about 16 days, hog cholera antibody all reached peak 1: 64.Afterwards, be progressively downward trend, about 32-48 after the immunity days, test swinery swine fever average antibody titre still can maintain 1: 50.
The preparation of embodiment 3 reorganization pig AsiaI type foot and mouth disease virus VPI gene adenovirus vector live vaccine
According to the preparation of the process route shown in the accompanying drawing 3 preparation recombinant adenovirus live vector vaccine, have the general features of adenovirus under the recombinant virus Electronic Speculum, behind the infection mdck cell, the specific cell pathological changes can appear.See accompanying drawing 4.Concrete steps are as follows:
1, obtains the full genomic clone plasmid of CAV-2 pPoly2-CAV2 with embodiment 1,2,3 described same procedure.
2, PCR method obtains VPI from the full gene of AsiaI type foot and mouth disease virus PI, and the PCR primer is:
Forward primer: 5 '-CCGGAATTCGCCATGGCTCGCCGGCAGACTACCAC-3 '
Downstream primer: 5 '-CCGCTCGAGTTATAGCCTGTTTCTCGGGTGCAA-3 ' adds restriction enzyme site EcoRI, XhoI respectively in the upstream and downstream primer.
3, with EcoRI and XhoI difference double digestion PCR product and carrier for expression of eukaryon pVAXI, electrophoresis reclaims, and connects, and transforms, and shakes bacterium, and the upgrading grain obtains recombiant plasmid pVAX-VPI after enzyme action is identified.
4, respectively pVAX-VPI, pVAX Δ E3 are carried out double digestion with MluI and SphI, electrophoresis reclaims, and dephosphorization is mended and put down, and connects, and obtains to contain the eukaryotic expression transfer vector pVAX Δ E3-VPI of genes of interest.Need therebetween recon is carried out positive and negative evaluation.
5, with NruI and SalI transfer vector pVAX Δ E3-VPI and pPoly2-CAV2 are carried out double digestion, electrophoresis reclaims, and connects, and obtains recombiant plasmid pPoly2-CAV2/VPI.
6, with recombiant plasmid transfection mdck cell, typical adenovirus pathological changes occurs, thereby obtain recombinant virus CAV-2/VPI.
8, with recombinant virus inoculation mdck cell, through rolling bottle suspension culture propagation, to viral TCID
50Reach 10
5-6Individual TCID
50/ 0.1mL adds gelatin sucrose as protective agent, (5mL/ bottle) lyophilizing after the packing.
For showing the present invention in the effect aspect the important viral infectious of prevention pig, following test proves:
The outer hereditary stability test of CAV-2/VPI virion
CAV-2/VPI is inoculated the mdck cell cultivation of going down to posterity, reached for 30 generations.Respectively to the 15th generation of CAV-2/VPI, 30 generation culture be template, amplification VPI expression cassette nucleic acid fragment (partial sequence that contains the E3 district in both sides) checks order.The result with to the 5th generation culture sequencing result consistent.This shows that CAV-2/VPI has a good hereditary stability external.
PCR primer: upstream: 5 '-CAGTTCATTCCTAACTACGAC-3 '
Downstream: 5 ' CAAGTGGAAGTACCAAAGCTG-3 '
The CAV-2/VPI vaccine is to the safety testing of pig
Get 5 pigs at random, with CAV-2/VPI (1 * 10
5TCID
50/ ml) wherein 3 pigs are carried out counteracting toxic substances, oral 5ml/ head, intramuscular injection 5ml/ head, 10ml/ head altogether; In addition two pig is as negative control, oral 5ml normal saline/head, intramuscular injection 5ml normal saline/head, 10ml/ head altogether.Experiment pig is carried out isolated rearing, observe the clinical change situations such as spirit, appetite, body temperature and feces of pig every day, carried out the total white blood cells counting every 3 days one time.Observe 21d.Do not see during breeding observing that the counteracting toxic substances temperature of pig body raises, appetite reduces, thirsty desire increases, infection symptoms such as depressed, hemafecia, cough, significantly fluctuation does not take place in total white blood cells.
The CAV-2/VPI vaccine is to the immunogenicity experiments of pig
Totally 8 of 55 age in days piglets have been selected for use, intramuscular inoculation Schweineseuche VPI gene recombinant adenovirus live vector vaccine 5ml/ head.To the blood sampling of test pig ear vein, separation of serum used Schweineseuche forward indirect agglutination diagnostic kit, measures Schweineseuche antibody respectively at immunity back 5 days, 15 days, 25 days.The result shows that the test swinery is when 55 ages in days, and the maternal antibody average out to is 1: 12 in the body, behind the inoculation Schweineseuche recombiant vaccine, Schweineseuche antibody progressively raises, and can reach 1: 128 at back 5 days Schweineseuche antibody of immunity, 1: 512 can be reached after 10,1: 1024 can be reached after 15 days.Illustrate Schweineseuche recombiant vaccine immunity 55 age in days piglets after 5 days antibody horizontal can reach more than the average protection titre, have good immunogenicity.
Claims (6)
1, a kind of is the pig virus infectious disease gene recombination living vaccine of carrier with dog II type adenovirus.
2, the reorganization swine influenza virus HA gene adenovirus vector live vaccine of producing according to claim 1 production technology.
3, the reorganization swine fever virus raq gene adenovirus vector live vaccine of producing according to claim 1 production technology.
4, the reorganization pig AsiaI type foot and mouth disease virus VPI gene adenovirus vector live vaccine of producing according to claim 1 production technology.
5, be the production technology of the pig virus infectious disease gene recombination living vaccine of carrier with dog II type adenovirus, may further comprise the steps:
A. the clone of pig virus infectious disease protective antigen gene: obtain the protective antigen gene full-length gene or contain the sequence of epitope by PCR method, described gene is selected from HCV-E1, raq gene, FMDV-VP1, VP2, VP3, VP4 gene, TGEV-S, N, M gene, PEDV-S, N, M gene, SIV-HA, NA gene, RV-G, N gene etc.;
B. construction of recombinant plasmid: genes of interest is cloned into contains in the complete genomic vector plasmid of CAV-II, recombiant plasmid has lacked viral genome part E3 district;
A. by PCR method the full genomic clone of CAV-2 is gone among the plasmid pPoly2, obtain to contain the two terminal rescue plasmid pPoly2-UD of CAV-2 genome, through BstBI+Hind III
Carry out homologous recombination with the CAV-2 genomic DNA behind the linearization for enzyme restriction, obtain the full genomic clone plasmid of CAV-2 pPoly2-CAV/2, contain the full genome of CAV-2;
B. will be cloned among the carrier for expression of eukaryon pVAX through the method for enzyme action and external connection through the pig virus infectious disease protective antigen gene that steps A obtains, obtain eukaryon expression plasmid pVAX-Tg, contain the destination gene expression box, comprise cytomegalovirus (CMV) promoter, genes of interest and poly A (polyA) structure;
C. with eukaryon expression plasmid pVAX-Tg with contain the segmental plasmid pVAX-E3 in CAV-2 genome E3 district enzyme action after be connected external, obtain genes of interest eukaryotic expression transfer vector pVAX Δ E3Tg, contained CAV-2 genome E3 district portion gene is lacked;
D. eukaryotic expression transfer vector pVAX Δ E3Tg and the full genomic clone plasmid of CAV-2 pPoly2-CAV/2 obtain to contain the recombiant plasmid pCAV-2/Tg of destination gene expression box after enzyme action is connected, contain the CAV-2 genome in plasmid pPoly2 part fragment and disappearance part E3 district, wherein disappearance part in E3 district is replaced by the destination gene expression box;
C. the acquisition of recombinant virus: recombiant plasmid is the transfection sensitive cells behind linearization for enzyme restriction, duplicates, packs the acquisition recombinant virus by the cell biological synthesis system;
D. the preparation of vaccine: positive recombinant virus with after the sensitive cells propagation, is added protective agent, make freeze-dried products.
6, the reorganization swine diseases poisonous carrier live vaccine of producing according to claim 5 production technology.
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