CN1477192A - Recombinant pseudo-rabies virus expressing swine parvovirus VP2 gene and vacine and its preparation method - Google Patents
Recombinant pseudo-rabies virus expressing swine parvovirus VP2 gene and vacine and its preparation method Download PDFInfo
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Abstract
The present invention relates to main structure gene VP2 of artificial constructed pseudorabies virus, PrV and porcine parvovirus, PPV. In the pseudorabies virus genome in which the main toxicity gene (TK) and virus generation nonessential gene (gG) are deleted the VP2 gene of porcine parvovirus can be site-specifically inserted to make it be positioned in strong late promoter downstream of pseudorabies virus, and the inserted exogenous gene coded protein has good immunogenicity, and can stimulate swine to produce protective immune reaction for resisting two virulent challenges of porcine parvovirus and pseudorabies virus. Said invention also includes recombinant pseudorabies virus, Hzau AVL-PRppvV-VP2, vaccine prepared by using it and its preparation method.
Description
Technical field
The invention belongs to the virusology technical field, be specifically related to a kind of artificial constructed Pseudorabies virus and pig parvoviral genetically engineered bivalent vaccine strain (PrV TK
-/ gG
-/ VP
2 +), there is the transgenation of two places in its genome, causes TK and gG gene inactivation, and utilizes the DNA recombination method, has inserted a foreign gene in gG promotor downstream location, and this foreign gene is the primary structure gene VP of pig parvoviral
2
The invention still further relates to the preparation method of described recombination engineered vaccine and this vaccine thereof.
Background technology
Pseudoabies is that (pig is the storage person and the circulator of this virus for pseudorabies virus, the PrV) acute infectious disease that causes of multiple domestic animal such as infected pigs, ox, sheep, dog, cat, rabbit, small white mouse, mink, fox and wildlife by Pseudorabies virus.(14:151-163) animal of other except that pig is the form of distributing for Mettenleiter et al., Comp Immu Microbiol Infect Dis.1991, has heating after the morbidity usually, very itches and pattern of fever symptom such as encephalomyelitis, is lethal infection.This disease is eruption and prevalence to pig, and harm mainly shows: the miscarriage of (1) sow, produce breeding difficulty syndromes such as stillborn foetus, mummy tire, and clinical in to produce stillborn foetus.(2) newborn piglet mass mortality, 15 ages in days are 100% with interior piglet mortality ratio.(3) weanling pig morbidity is dead, shows as and has loose bowels, and strike sample or circus movement are spat out white foams, spasm and symptom such as scratch where it itches.(4) cause that boar is sterile.Testis swelling, atrophy take place in boar, lose the ability of using of planting.That sow shows as is out of heat, return feelings, join repeatly infertile etc.(5) growing and fattening pigs show as the chronic respiratory symptom, weightening finish is slow, the price of deed reduces, the time of postponement listing, and nervous symptoms even death condition appear in the minority case.
PrV is generally through the pharynx nasalis infection animal, and infecting partial mucous membrane internal breeding, divide a word with a hyphen at the end of a line through lymphokinesis or nerve fiber then, it is generally acknowledged that virus arrives brain and causes the nervus centralis dysfunction through nerve pathway, or, set up latent infection in the trigeminal nerve at olfactory bulb.(321-322) strain than strong virus force also can spread whole body for Nauwynck etc., In proceeding of PRRVS and Aujeszky ' sDisease.1999, and tissue characteristics, for example reproductive tract are had a liking in performance.PrV is except having the neural preferendum of intensive, and latent infection also is its key character.Reported first such as Stevens the latent infection (Science.1971 of HSV in rat, 2669-2673), Sabo and Rajcajm have reported that PrV in the gasserian ganglion of pig latent infection takes place, and detect and in other tissue of pig, also have this phenomenon (Acta Virol.1976,20:208-214).During the latent infection, have infective virus particle and do not exist, but a virus genomic part still transcribes active, transcribing of taking place in the latent infection also has proteinic translation, this protein is regulated neuronic survival, so influence latent infection and activate again between balance.Osorio thinks behind MDBK (Main-Darby Bovine Kidney) the cell infection PrV of tissue culture, can produce typical apoptosis Osorio (Osorio.In proceeding of PRRVS and Aujeszky ' s Disease.1999,323).Utilize intranasal inoculation PrV to cause the acute infection of pig, in the gasserian ganglion tissue, typical apoptosis takes place in the lymphocyte that soaks into, and apoptosis does not take place in the nervous tissue cell, show that PrV can induce or suppress apoptotic generation in dissimilar cells, in the nervous tissue of the animal of acute infection PrV, the apoptosis of cell is suppressed, this is PrV escape immunity and a kind of important mechanisms (the Aleman et al. that sets up latent infection just, JVirol.2001,25:469-479).
The genome of Pseudorabies virus is the wire double-stranded DNA, be about 150kbp, G+C content is up to 74%, whole genome 70-100 the albumen of encoding at least, wherein thymidine kinase (Thymidine Kinase, TK) gene is positioned at UL district, total length 963bp, 320 amino acid of encoding, catalytic deoxidation thymidine phosphorylation.The TK gene is relevant with the virulence of PrV, be the main virulence gene of Pseudorabies virus, relevant with the latent infection of virus, show on evidence it to virus at very important (the Kit S etc. of the propagation of nervus centralis, Am J Vet Res.1985,46:1359-1367; Nunberg et al., JVirol.1989,63:3240-3249).The TK gene is that virus multiplication institute is nonessential, disappearance virus strain and wild malicious the same can normally propagation, and do not exist virulence return strong danger (Yokyama etc., Virol.1991,185:55-65).GG glycoprotein is not present on the virus envelope; but be secreted into outside the cells infected; this proteic gG gene of encoding also is the dispensable gene of Pseudorabies virus propagation; after the disappearance; do not influence the propagation of virus; the mutated viruses strain has good immunogenicity; can produce protective immunological reaction behind the inoculation animal; but do not produce the antibody of anti-gG; therefore can be used as a sign distinguishing immune animal and wild virus infection animal; help setting up the swinery that no pseudoabies infects, and finally provide useful instrument for eradicating this disease.
As everyone knows, Mayr and Mahnel found pig parvoviral (porcine parvovirus first when carrying out the Pestivirus suis tissue culture in 1966, PPV), begin to think the virus of latent infection in the culturing cell, Cartwright (1967) is in the Study of Etiology to the breeding difficulty of pig, from pathological material of disease, isolated pig parvoviral, thus confirmed first should virus pathogenic effects.Pig parvoviral is one of member of Parvoviridae parvovirus genus, it also is one of main pathogen of pig breeding dysfunction, worldwide be widely current, its harm mainly shows as infects sow, especially first farrowing sow miscarriage, product stillborn foetus, mummy tire etc., and the pig at other age does not show tangible clinical symptom after infecting.In tissue culture, there are empty capsid and complete virus particle simultaneously.Press different ratios mixing back inoculation pig testis cell Deng with empty capsid and complete PPV virus particle, found that, no matter be in the cell or the sophisticated virus particle quantity in extracellular all reduces, hemagglutinative titer also reduces greatly simultaneously, and directly related (the Choil et al. of the hollow capsid number of particles of inhibition degree and PPV, Arch Virol.1987,96:75-87).In addition, handle cell with PPV empty capsid particle with different concentration, use the complete virus particle cells infected again, the same propagation of virus of finding is subjected to severe inhibition.
PPV belongs to from the principal mode parvovirus, and genome is the strand linear DNA, has two open reading frames, a coding Nonstructural Protein, another coding structure albumen (VP1, VP2), wherein VP
2Be the main capsid protein that constitutes virus particle, account for 80% of viral capsid proteins total amount, the major antigen determinant of parvovirus is positioned at VP
2On.Serological test confirms, VP
2Can induce and produce hemagglutination inhibition antibody and neutralizing antibody.In addition, it is active that VP2 albumen also has unique biological, and it not only has good antigenicity behind vivoexpression, and can the oneself be assembled into virus like particle (Virus-Like Patricles VLP), have the feature similar to totivirus, for example: its form is the three-dimensional symmetrical structure of icosahedro, about diameter 23nm, can the aggegation guinea-pig red blood cell, produce the intensive immunne response with inducing behind its inoculation animal, can resist the attack of the strong poison of PPV, the VP of vivoexpression
2The research that the self-characteristic that be packaged into virus particle of polypeptide energy is the reorganization multivalent subunit vaccine lays a solid foundation.Respectively with PPV VP
2Link to each other with the antigenic determinant district of 118-132 amino acids that comprises lymphocyte choroid plexus encephalitis (LCMV) and hepatitis B virus HBSAg, utilize this recombinant protein immunized mice can induce the intensive ctl response, time length reaches 9 months in vivo, and can resist the corresponding virus attack of lethal quantity.These presentation of results PPV VP
2Be not only the main immunogenic albumen of PPV, and can be used as the protein transduction vehicle that carries exogenous antigen determinant polypeptide, for good condition (Sedlik etc., Proc Natl Acad Sci USA have been created in the research of polyvalent recombinant vaccine, 1997,94:7503-7508; Sedliket etc., J Virol.1999,73:2739-2744).
Pseudoabies and porcine parvovirus have caused enormous economic loss all over the world, seriously endangered the development of pig industry, discover that vaccine immunization is the generation that prevents and control these two kinds of diseases, the key that reduces financial loss.That succeeds in developing at present has separately attenuated live vaccines and an inactivated vaccine, and being extensive use of of vaccine not only can reduce morbidity, and can make the wild malicious latent infection of PrV again activation phenomena reduce greatly.Wherein attenuated live vaccines is easy to produce, and is with low cost, is fit to very much the demand of large scale of pig farm.Be that chemical factors is induced and gone down to posterity in a large number continuously on culturing cell and produce attenuated virus in the method that adopts often of development attenuated live vaccines in the past; these all are out of contior virus mutation; causing vaccine to be formed is non-homologous mixture; the variant viral that wherein contains unknown virulence and unknown protection, and the virus a little less than causing so also exists virulence to return strong danger.
Develop rapidly along with biotechnology, in the development that the further investigation and the genetic engineering technique of aspects such as virus structure and levels of replication is widely used in biological products, by the Special Areas in the viral genome is changed, cause pathogenic the weakening of virus, overcome the defective in the former acquisition attenuated vaccine process, easier fast, have purpose ground and obtain accurate and stable attenuated vaccine.
In order to adapt to large scale of pig farm, reduce production costs, reduce immunization to the stress reaction of pig (stress reaction: can cause Abwehrkraft des Koepers to descend, poor growth, and can bring out the generation of multiple disease.), need develop polyvalent vaccine energetically, reach the purpose of the anti-multiple disease of a pin.Traditional polyvalent vaccine mostly is inactivated vaccine, with the cause of disease or the mixing of cause of disease composition of multiple deactivation, makes polyvalent vaccine then.Yan Zhi polyvalent vaccine must cause the raising of dosage of inoculation in order to guarantee the antigen amount of every kind of cause of disease like this, is unfavorable for clinical application.
Research about virus vector concentrates on vaccinia virus, adenovirus and simplexvirus (Moss etc., Ann RevImmunol.1987,5:305-324 always; Adv Vieus Res.1988 such as Piccini, 34:43-64; Berker.Biotech.1988,6:6003-6020; Shih etc., Proc Natl Acad Sci USA.1984,81:5867-5870; Van Zijl etc., J Virol.1991,65:2761-2765; Kit etc., Vaccine, 1991,9:564-572).Because the PrV genome is huge, have more propagation dispensable gene and non-coding region, can not influence duplicating of PrV in these zone disappearances or insertion foreign gene, for example TK, gG gene, and PrV has host range widely, with PrV is that carrier development reorganization divalence or polyvalent vaccine prevent that the multiple disease of animal from becoming possibility, guaranteed of the present invention succeeding in developing.
Summary of the invention:
Task of the present invention is to overcome the defective that prior art exists, and its first purpose just provides the dna sequence dna of coding pig parvoviral main immunogenic gene VP2.
The recombinant pseudorabies virus strain that another object of the present invention provides a kind of artificial constructed expression pig parvoviral VP2 gene (for example: TK-/gG-/VP2+) Zhi Bei genetically engineered bivalent vaccine, to overcome the shortcoming of conventional polyvalent vaccine, solve repeatedly the drawback of immunization.Adopt vaccine of the present invention can prevent system porcine parvovirus and pseudoabies viral disease simultaneously.
Another object of the present invention provides the method for this Pseudorabies virus of preparation and pig parvoviral recombination engineered vaccine.The present invention is achieved through the following technical solutions:
A kind of recombinant vaccine strain that derives from PRV (Pseudorabies virus) and pig parvoviral, this strain are the PRV (Pseudorabies virus) of reorganization: Pseudorabies virus, HzauAVL-PRppvV-VP2; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), preservation date: on August 16th, 2002, deposit number: CGMCC:0788-2.
Derive from the coding main immunogenic albumen VP of pig parvoviral
2DNA,SEQ IDNo：1：atgtggaaca acacaaccct attaatgcag gcactgaatt gtctgcaaca ggaaatgaat 60ctgggggtgg gggcggcggt ggcgggggta ggggtgctgg gggggttggt gtgtctacag 120gtactttcaa taatcaaaca gaatttcaat acttggggga gggcttggtt agaatcactg 180cacacgcatc aagactcata catctaaata tgccagaaca cgaaacatac aaaagaatac 240atgtactaaa ttcagaatca ggggtggcgg gacaaatggt acaagacgat gcacacacac 300aaatggtaac accttggtca ctaatagatg ctaacgcatg gggagtgtgg ttcaatccag 360cggactggca gttaatatcc aacaacatga cagaaataaa cttagttagt tttgaacaag 420caatattcaa tgtagtactt aaaacaatta cagaatcagc aacctcacca ccaaccaaaa 480tatataataa tgatctaact gcaagcttaa tggtcgcact agacaccaat aacacacttc 540catacacacc agcagcacct agaagtgaaa cacttggttt ttatccatgg ttacctacaa 600aaccaactca atacagatat tacctatcat gcatcagaaa cctaaatcca ccaacataca 660ctggacaatc acaacaaata acagactcaa tacaaacagg actacacagt gacattatgt 720tctacacaat agaaaatgca gtaccaattc atcttctaag aacaggagat gaattctcca 780caggaatata tcactttgac acaaaaccac taaaattaac tcactcatgg caaacaaaca 840gatctctagg actgcctcca aaactactaa ctgaacctac cacagaagga gaccaacacc 900caggaacact accagcagct aacacaagaa aaggttatca ccaaacaatt aataatagct 960acacagaagc aacagcaatt aggccagctc aggtaggata taatacacca tacatgaatt 1020ttgaatactc caatggtgga ccatttctaa ctcctatagt accaacagca aacacacaat 1080ataatgatga tgaaccaaat ggtgctataa gatttacaat ggattaccaa catggacact 1140taaccacatc ttcacaagag ctagaaagat acacattcaa tccacaaagt aaatgtggaa 1200gagctccaaa gcaacaattt aatcaacagg caccactaaa cctagaaaat acaaataatg 1260gaacactttt accttcagat ccaataggag ggaaatctaa catgcatttc atgaatacac 1320tcaatacata tggaccatta acagcactaa acaatactgc acctgtattt ccaaatggtc 1380aaatatggga taaagaactt gatacagatc taaaacctag actacatgtt acagctccat 1440ttgtttgtaa aaacaatcca ccaggacaac tatttgtaaa aatagcacca aacctaacag 1500atgatttcaa tgctgactct cctcaacaac ctagaataat aacttattca aacttttggt 1560ggaaaggaac actaacattc acagcaaaaa tgagatccag taatatgtgg aaccctattc 1620aacaacacac aacaacagca gaaaacattg gtaactatat tcctacaaat attggtggca 1680taagaatgtt tccagaatat tcacaactta taccaagaaa attatactag aaataactct 1740gtaaataaaa actcagttac ttggttaatc atgtactact atcattgtat acttcaataa 1800aaataaattg taaaatcaat aaaactaagt tacttagttt ctgtatacct atactag 1857
A kind of recombinant pseudorabies virus genetically engineered bivalent vaccine of expressing pig parvoviral VP2 gene, it contains aforesaid recombinant porcine pseudorabies poison Pseudorabies virus, HzauAVL-PRppvV-VP2; Be deposited in CGMCC, preservation date: on August 16th, 2002, deposit number: CGMCC:0788-2.
A kind of method for preparing the recombinant pseudorabies virus genetically engineered bivalent vaccine of expressing pig parvoviral VP2 gene, it contains described recombinant porcine pseudorabies poison: Pseudorabies virus, HzauAVL-PRppvV-VP2; Be deposited in CGMCC, preservation date: on August 16th, 2002, deposit number: CGMCC:0788-2, described method also comprises: utilize the round pcr amplification to obtain the VP2 gene of pig parvoviral, respectively introduce a BamH I restriction enzyme site simultaneously at its two ends, thereby be inserted into and obtain the pUSK-VP2 transfer vector among the transfer vector pUSK, by liposome-mediated transfection, genomic dna cotransfection PK-15 cell with pUSK-VP2 carrier and Pseudorabies virus TK-/gG-/LacZ+ parent plant, wait to occur to collect viral liquid after the cytopathy, the plaque purifying is identified the recombinant virus that obtains with the PCR detection method simultaneously.After the recombinant virus that obtains is carried out the evaluation of biological characteristics, be prepared into freeze-dried live vaccine, and then estimate its security and immunogenicity, the final recombinant pseudorabies virus vaccine that obtains to express pig parvoviral VP2 gene by animal experiment.
The TK that the present invention relates to
-/ gG
-/ LacZ
+Parent plant (is the strain Pseudorabies virus of preservation of the present invention, the parent of HzauAVL-PRppvV-VP2) derives from PRV (Pseudorabies virus) Hubei Province A strain (Pseudorabies virus), this strain separates, identifies (Chen Huanchun etc. by Hua Zhong Agriculture University animal virus research department, the isolation identification of porcine pseudorabies virus Hubei Province A strain, journal of animal science and veterinary medicine, 1998,29 (2), 151-156), it is that a strain that is separated to from the pig farm of breaking out pseudoabies is bred titre height, the virulent strain that immunogenicity is strong at cell culture.TK
-/ gG
-/ LacZ
+The TK genetically deficient of recombinant virus living vaccine 205bp, big fragment takes place and inserts disappearance in the gG gene, virulence reduces greatly, can in noble cells, duplicate, but duplicating in undifferentiated neurocyte is prevented from or limits, even latent infection takes place in the inoculation back, also is difficult for being activated.Even this vaccine strain not only effectively also fool proof to newborn piglet, can produce strong protection, and can be used for the leading immunity of piglet growing and fattening pigs, piglet are fool proof, has excellent prevention and result of treatment.In addition, PrV Fa TK
-/ gE
-/ gI
-/ LacZ
+Fool proof to animals such as sheep, ox, dogs.
New departure of polyvalent vaccine of the present invention research be with the attenuated live vaccines strain as virus live vector, express the former albumen of protective immunity of other cause of disease.Behind live recombinant vectors virus inoculation animal, the foreign protein of its expression is similar to that natural infection produces, and can induce to produce body fluid and cellullar immunologic response, can reach the purpose that multiple disease is resisted in once inoculation simultaneously.In order to prevent the diseases such as sow breeding difficulty that system Pseudorabies virus and pig parvoviral cause simultaneously, utilize the main immunogenic gene VP of genetic engineering technique with PPV
2, be inserted into Pseudorabies virus TK
-/ gG
-/ LacZ
+In the genome of vaccine strain, be located at the downstream of gG promotor, express the back a few amino acids formation fusion rotein initial with gG albumen.Because the VP that inserts
2Gene itself has termination codon and poly (A) sequence, and does not merge with gG albumen end sequence, so the VP that expresses
2Proteic biological characteristics does not change, and gG albumen still can not get expressing simultaneously.This vaccine can be used as the marker vaccine (animal of marker vaccine immunity can utilize corresponding differential diagnosis method, distinguishes mutually with the animal of wild virus infection) of eradicating Pseudorabies virus and pig parvoviral.Referring to shown in Figure 1.
Sequence table, accompanying drawing and explanation thereof:
Sequence table SEQ ID No:1 has shown the dna sequence dna of pig parvoviral VP2 gene.
Fig. 1: the genome synoptic diagram that is the A strain of Pseudorabies virus Hubei Province.
Fig. 2:
Fig. 3: be the Southern-blotting analytical results of expressing the recombinant pseudorabies virus TK-/gG-/VP2+ strain of pig parvoviral VP2 gene.
Among the figure: A:Southern-blotting analyzes; B: the electrophoresis on 0.3% sepharose; 1, the BamHI enzyme is cut the genome of TK-/gG-/VP2+ strain; 2, BamH I enzyme is cut the genome of TK-/gG-/LacZ+ parent plant; 3, DL15000 DNA Marker
Fig. 4: recombinant pseudorabies virus TK
-/ gG
-/ VP
2 +Pig parvoviral VP is expressed in strain
2Proteic SDS-PAGE, Western-blotting analytical results.
Among the figure: the A:TK-/gG-/VP2+ strain is expressed the proteic SDS-PAGE of VP2 and is analyzed; The B:TK-/gG-/VP2+ strain is expressed the proteic Western-blotting of VP2 and is analyzed; M: protein Marker; 1: the PK-15 cell that infects the TK-/gG-/VP2+ strain; 2: the PK-15 cell 3. normal PK-15 cells that infect the TK-/gG-/LacZ+ strain
Fig. 5: recombinant pseudorabies virus particle enlarged view.
Among the figure: A: the enlarged view of recombinant pseudorabies virus particle; The virus-like particle enlarged view that B:VP2 albumen forms.
The black arrow indication of solid line among the figure is the recombinant pseudorabies virus particle;
The virus-like particle that the white arrow indication forms for VP2 albumen; The black arrow indication of dotted line is the nuclear membrane of PK-15 cell.
(recombinate in the experiment code name of pseudo-rabies strain of the present invention is: TK to further describe recombinant pseudorabies virus HzauAVL-PRppvV-VP2 below by embodiment and concrete operation steps
-/ gG
-/ VP
2, down together) and the structure of vaccine and the biological characteristics of strain;
Embodiment:
Clone, the order-checking of embodiment 1VP2 gene:
Go up propagation PPV at pig kidney passage cell (IBRS-2), adopt synchronous inoculation method, promptly in cell dispersion, press 1/10 inoculation PPV kind poison of nutrient solution, 30～36h stops to cultivate after connecing poison, and centrifugal collecting infecting cell extracts pig parvoviral RF-DNA then and (draws the W from Moliter T, Joo H S, Coliett M S.Virol, 1984,137:241-254).In order to obtain the full gene of PPV VP2, the PPV genome sequence according to reports such as Bergeron has designed the one couple of PCR primers oligonucleotide, and its sequence is as follows:
Upstream primer: 5 '-TTAGGATCCCAATGAGTGAAAATGTGGAAC-3 '
Downstream primer: comprise a BamH I restriction endonuclease sites in 5 '-TACAGGATCCGTAAACACATGAGAGCTTG-3 ' upstream and downstream primer respectively, and the BamH I site in the upstream primer helps the VP2 gene is inserted in the transfer vector accurately, is located at gG promotor downstream and forms the amalgamation and expression box.50 μ L reaction systems are adopted in the PCR reaction, and its component is as follows:
10 * PCR damping fluid, 5 μ L
10μM?P
1 2μL
10μM?P
2 2μL
25mM?MgCl
2 7μL
10mM?dNTPs 1μL
5U/ μ L Taq enzyme 0.5 μ L
RF-DNA template 5 μ L
Sterilization distilled water 27.5 μ L increase by following program: behind 95 ℃ of 3min, carry out 95 ℃ of 1min → 58 ℃ 1min → 72 ℃ of 2min again, and 30 circulations, last 72 ℃ are extended the 10min termination reaction.Amplified production in the pMD-18 carrier, adopts the terminal cessation method of two deoxidations to record its sequence the VP2 gene clone that obtains after 0.8% agarose gel electrophoresis is determined.Referring to shown in Figure 1.
The structure of embodiment 2pUSK-VP2 transfer vector:
Carrier construction according to a conventional method.Utilize BamH I enzyme to cut the pUSK carrier, treat agarose gel electrophoresis determine enzyme cut complete after, the dehydrated alcohol precipitation, 75% washing with alcohol treats that the ethanol volatilization is dissolved in water fully, uses the CIAP dephosphorylation, reclaims behind 1% agarose gel electrophoresis.BamH I enzyme is cut the PCR product of VP2 gene, reclaim the back and cut through enzyme, dephosphorylized pUSK carrier mixes, at T
4The dna ligase effect connects down, will connect product then and transform DH
5α intestinal bacteria, a small amount of prepare plasmid, enzyme is cut evaluation and determined that it is connected into direction, thereby obtain the pUSK-VP2 transfer vector.As shown in Figure 2.
Propagation PrV TK on the IBRS-2 cell
-/ gG
-/ LacZ
+Parent plant extracts its genomic dna owing to only have an EcoR I restriction enzyme site in the genomic dna of PrV, and is arranged in the gG gene.With EcoR I with PrVTK
-/ gG
-/ LacZ
+The genomic dna enzyme be cut into two sections, use liposome-mediated rotaring dyeing technology with itself and pUSK-VP
2Transfer vector cotransfection IBRS-2 cell is transferred to the full gene of VP2 in the genomic dna of the dual-gene disappearance strain of PrV TK, gG by homologous recombination takes place, and has displaced the LacZ gene simultaneously, receives poison after waiting to produce cytopathy.Viral liquid is inoculated in the PK-15 cell, keeps liquid with the DMEM (for example U.S. GIBCO company product) that contains 1% low melting-point agarose and cover, in 37 ℃ of 5%CO
2Condition under cultivate, when just cytopathy occurring, toluylene red dyeing 1h with 0.01%, because sick cell can not be painted, and normal cell is dyed redness, thereby the round point shape plaque occurs, the single plaque inoculation of picking PK-15 cell enlarged culturing, utilize the PCR detection method of PPV VP2 gene to identify positive recombinant virus, so carry out purifying three times, utilize the PCR detection method of LacZ gene can not detect PrV TK at last
-/ gG
-/ LacZ
+The existence of strain, thus determine only to contain recombinant pseudorabies virus TK in the viral liquid
-/ gG
-/ VP
2 +
Embodiment 4
Recombinant pseudorabies virus HzauAVL-PRppvV-VP2 (TK
-/ gG
-/ VP
2 +) biological characteristics and the evaluation thereof of strain: with reference to (Sa Mubulu I, not Ritchie E and Manny A Disi T. are outstanding, second edition. Jin Dongyan, Li Mengfeng, Hou Yunde etc. translate the school, " molecular cloning experiment guide ", Beijing, Science Press, 1998) in method carry out Southern-blotting, SDS-PAGE and Western-blotting identifies, and under transmission electron microscope, observe the form and the foreign protein VP of recombinant virus in the same cell
2The virus-like particle that forms voluntarily after obtaining to express.
Southern-blotting identifies: extract recombinant pseudorabies virus TK
-/ gG
-/ VP
2 +After the genomic dna of strain is cut with the BamHI enzyme, after 6 hours, transfer to according to a conventional method on the cellulose acetate membrane at 30 volts of electrophoresis on 0.3% the sepharose, standby behind 80 ℃ of roasting films.Pcr amplification VP
2After the dna fragmentation of one section about 445bp in the gene, DNA return the test kit recovery, with digoxigenin labeled and detection kit mark, as VP
2The dna probe of gene places-20 ℃ of preservations standby.At last according to a conventional method, with VP
2The dna probe of gene and the hybridization of standby cellulose acetate membrane, the result as shown in Figure 3.Test-results shows, the TK that BamH I enzyme is cut
-/ gG
-/ VP
2 +Pnca gene group DNA swimming lane has tangible hybrid belt, and the TK that BamH I enzyme is cut
-/ gG
-/ LacZ
+Pnca gene group DNA swimming lane does not then have, and confirms VP
2Gene has been inserted in the genome of parent plant.
SDS-PAGE and Western-blotting identify: with recombinant pseudorabies virus TK
-/ gG
-/ VP
2 +Strain is inoculated in the PK-15 cell that just grows up to individual layer, collects sick cell after 16 hours.Prepare sample according to a conventional method, carry out SDS-PAGE and Western-blotting, the result as shown in Figure 4.Analyze through SDS-PAGE, infect TK
-/ gG
-/ VP
2 +The PK-15 cell swimming lane of strain, find a specificity band, Western-blotting to analyze discovery it can react with the pig parvoviral positive serum at about 64kD place, a hybrid belt occurs, does not then have in two contrast swimming lanes, shows pig parvoviral VP
2Albumen has obtained expression.
Electron microscopic observation: infect recombinant pseudorabies virus TK
-/ gG
-/ VP
2 +The PK-15 cell of strain, wait to occur collecting cell after the pathology, prepare ultra-thin frozen section according to a conventional method, utilize H7000 transmission electron microscope observe and take a picture (Fig. 5), the result shows, has occurred two kinds of virions that difference in size is very big in the same cell.Big virion has cyst membrane, is distributed in endochylema mostly, and its form size is consistent with Pseudorabies virus.Little virion is a ghost, is the three-dimensional symmetry of icosahedro, and no cyst membrane is lattice-like and arranges, with baculovirus expression pig parvoviral VP in nucleus
2The empty particle that albumen forms is similar.These results effectively confirm recombinant pseudorabies virus TK
-/ gG
-/ VP
2 +Pig parvoviral VP has been expressed in strain in the PK-15 cell
2Albumen.
Recombinant pseudorabies virus strain HzauAVL-PRppvV-VP2 be the present invention constructed with the dual-gene disappearance strain of Pseudorabies virus TK
-/ gG
-/ LacZ is a virus vector, with the main immunogenic gene VP of pig parvoviral
2Be inserted into the dual-gene disappearance strain of Pseudorabies virus TK
-/ gG
-In the genome of/LacZ, the artificial constructed divalence attenuated vaccine strain that obtains.Its genetic stability and biological characteristics and the A strain of parent's strain Hubei Province do not have difference.It is double-stranded DNA that recombinant virus has cyst membrane, nucleic acid, and the virion diameter is 120-180nm, to ether, chloroform and trypsinase sensitivity, and can complete inactivation in 56 ℃ of 30min.TCID on the PK-15 cell
50Still can reach 10
-8.0/ mL.Recombinant virus was uploaded for 30 generations at the PK-15 cell continuously, and its propagation titre does not have obvious variation, expression pig parvoviral VP2 albumen that can be stable.
This recombinant virus need be gone up propagation and detect survival condition at pig kidney subculture cell (PK-15), the substratum of PK-15 cell is Dulbecco ' the s Modified Eagle Medium (DMEM) (U.S. GIBCO company product) that contains 10% calf serum, the pH scope is 6.8-7.2, in 37 ℃ of cultivations.Can adopt very low temperature to freeze and/or lyophil preservation.
The preparation of embodiment 5 Pseudorabies virus and pig parvoviral genetically engineered bivalent vaccine:
With the recombinant pseudorabies virus TK that obtains
-/ gG
-/ VP
2 +Identify that its genetic stabilities are found in the back of going down to posterity for 30 times, foreign protein can stably express, and has good biologic activity.The propagation titre of this vaccine strain on pig kidney passage cell reaches 10
8.0TCID
50, through screening, adopting the easy chick embryo fibroblast propagation vaccine virus of preparation, its propagation titre reaches 10
7.0TCID
50Vaccine kind poison by 1/10 ratio inoculated into chick embryo inoblast, is collected qualified viral liquid after the cytopathy, adding gelatin protective material is (in every 100ml deionized water with sucrose 40g; gelatin 8g; after fully melting, put autoclaving), vaccine is made in freeze-drying according to a conventional method.
The security of embodiment 6 Pseudorabies virus and pig parvoviral genetically engineered bivalent vaccine:
Healthy Balb/C small white mouse with 30 pseudoabies serology feminine genders is divided into three groups at random, wherein inoculates TK respectively for two groups
-/ gG
-/ VP
2 +Vaccine and PrV Hubei Province A strain (parent plant), another is organized as the blank group, observes 14 days.Small white mouse spirit appetite vaccinated and control group is all normal, no abnormal performance; And the small white mouse of the strong poison of inoculation (the A strain of PrV Hubei Province) is dead from inoculating beginning in back 72 hours, and is all dead by 144 hours, shows that this genetic vaccine is safe (table 1) to small white mouse.Table 1 Pseudorabies virus and pig parvoviral genetically engineered bivalent vaccine to safety testing group number of animals (only) death toll (only) mean time to death of Balb/C small white mouse (hour) inoculation TK
-/ gG
-/ VP
2 +Organize 10 00 inoculation PrV Hubei Province A strain groups, 10 10 116.2 ± 45.9 blank groups 10 00
With TK
-/ gG
-/ VP
2 +Vaccine is inoculated the piglet of 15 ages in days, and slight exothermic reaction appears in part, recovers normal two days later, and the spiritual appetite of piglet is normal during this period, and no abnormality seen changes, and can detect Pseudorabies virus neutralizing antibody and pig parvoviral hemagglutination inhibition antibody.The piglet of PrV Hubei Province A strain (virulent strain) inoculation all presented exothermic reaction in second day, continues a week, and a death is arranged, and analyse and find to have typical pseudoabies pathological change, and control group had not both had fervescence, did not have unusual clinical manifestation yet.
TK
-/ gG
-/ VP
2 +Vaccine inoculation and nonvaccinated pregnant sow, the nest litter size is suitable substantially, stillborn foetus, mummy tire etc. all do not occur, confirms that this vaccine is safe to pregnant sow.
The immunogenicity test of embodiment 7 Pseudorabies virus and pig parvoviral genetically engineered bivalent vaccine:
At replacement gilt breed preceding 15 days inoculations Pseudorabies virus and pig parvoviral genetically engineered bivalent vaccine (TK
-/ gG
-/ VP
2 +), inoculated the strong poison of PPV (pig parvoviral) Chinese strain and PrV (Pseudorabies virus) Hubei Province A virulent strain in 42 days, 60 days respectively in gestation then, tangible clinical symptom does not all appear in pregnant sow, spirit appetite is all normal, and can detect high-caliber serum Pseudorabies virus neutralizing antibody and pig parvoviral hemagglutination inhibition antibody, and the horizontal transmission phenomenon does not appear.Immune swine stillborn foetus, mummy tire the time do not occur in childbirth, and the institute pig that farrow all health survives.The contrast pregnant sow of the strong poison of inoculation PPV Chinese strain, part fetus occurs and heavily absorbs, and abdominal circumference reduces termination of pregnancy, and remaining gives birth to the mummy tire and minority is weak young.The contrast pregnant sow of inoculation PrV Hubei Province A virulent strain, though given birth to son alive, piglet is suffered from diarrhoea subsequently, vomiting and nervous symptoms etc., poor growth and with death.These results show, this Pseudorabies virus and pig parvoviral genetically engineered bivalent vaccine (TK
-/ gG
-/ VP
2 +), can protect pregnant sow to resist the attack of the strong poison of PPV Chinese strain and PrV Hubei Province A virulent strain simultaneously, thus anti-these two kinds of diseases of system.
＜110〉＜120〉VP2＜160〉1＜170〉PatentIn Version 3.1＜210〉1＜211〉1857＜212〉DNA＜213〉 ( Pseudorabies virus )＜220〉＜220〉＜221〉gene＜222〉 ( 1 ) ... ( 1857 )＜220〉＜221〉CDS＜222〉 ( 1 ) ... ( 1857 )＜400〉1atgtggaaca acacaaccct attaatgcag gcactgaatt gtctgcaaca ggaaatgaat 60ctgggggtgg gggcggcggt ggcgggggta ggggtgctgg gggggttggt gtgtctacag 120gtactttcaa taatcaaaca gaatttcaat acttggggga gggcttggtt agaatcactg 180cacacgcatc aagactcata catctaaata tgccagaaca cgaaacatac aaaagaatac 240atgtactaaa ttcagaatca ggggtggcgg gacaaatggt acaagacgat gcacacacac 300aaatggtaac accttggtca ctaatagatg ctaacgcatg gggagtgtgg ttcaatccag 360cggactggca gttaatatcc aacaacatga cagaaataaa cttagttagt tttgaacaag 420caatattcaa tgtagtactt aaaacaatta cagaatcagc aacctcacca ccaaccaaaa 480tatataataa tgatctaact gcaagcttaa tggtcgcact agacaccaat aacacacttc 540catacacacc agcagcacct agaagtgaaa cacttggttt ttatccatgg ttacctacaa 600aaccaactca atacagatat tacctatcat gcatcagaaa cctaaatcca ccaacataca 660ctggacaatc acaacaaata acagactcaa tacaaacagg actacacagt gacattatgt 720tctacacaat agaaaatgca gtaccaattc atcttctaag aacaggagat gaattctcca 780caggaatata tcactttgac acaaaaccac taaaattaac tcactcatgg caaacaaaca 840gatctctagg actgcctcca aaactactaa ctgaacctac cacagaagga gaccaacacc 900caggaacact accagcagct aacacaagaa aaggttatca ccaaacaatt aataatagct 960acacagaagc aacagcaatt aggccagctc aggtaggata taatacacca tacatgaatt 1020ttgaatactc caatggtgga ccatttctaa ctcctatagt accaacagca aacacacaat 1080ataatgatga tgaaccaaat ggtgctataa gatttacaat ggattaccaa catggacact 1140taaccacatc ttcacaagag ctagaaagat acacattcaa tccacaaagt aaatgtggaa 1200gagctccaaa gcaacaattt aatcaacagg caccactaaa cctagaaaat acaaataatg 1260gaacactttt accttcagat ccaataggag ggaaatctaa catgcatttc atgaatacac 1320tcaatacata tggaccatta acagcactaa acaatactgc acctgtattt ccaaatggtc 1380aaatatggga taaagaactt gatacagatc taaaacctag actacatgtt acagctccat 1440ttgtttgtaa aaacaatcca ccaggacaac tatttgtaaa aatagcacca aacctaacag 1500atgatttcaa tgctgactct cctcaacaac ctagaataat aacttattca aacttttggt 1560ggaaaggaac actaacattc acagcaaaaa tgagatccag taatatgtgg aaccctattc 1620aacaacacac aacaacagca gaaaacattg gtaactatat tcctacaaat attggtggca 1680taagaatgtt tccagaatat tcacaactta taccaagaaa attatactag aaataactct 1740gtaaataaaa actcagttac ttggttaatc atgtactact atcattgtat acttcaataa 1800aaataaattg taaaatcaat aaaactaagt tacttagttt ctgtatacct atactag 1857SEQ ID NO.1
Claims (4)
1, a kind of recombinant vaccine strain that derives from PRV (Pseudorabies virus) and pig parvoviral is characterized in that, this strain is the recombinant porcine pseudorabies poison: Pseudorabies virus, HzauAVL-PRppvV-VP2; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), preservation date: on August 16th, 2002, deposit number: CGMCC:0788-2.
2、VP2DNA,SEQID No：1：atgtggaaca acacaaccct attaatgcag gcactgaatt gtctgcaaca ggaaatgaat 60ctgggggtgg gggcggcggt ggcgggggta ggggtgctgg gggggttggt gtgtctacag 120gtactttcaa taatcaaaca gaatttcaat acttggggga gggcttggtt agaatcactg 180cacacgcatc aagactcata catctaaata tgccagaaca cgaaacatac aaaagaatac 240atgtactaaa ttcagaatca ggggtggcgg gacaaatggt acaagacgat gcacacacac 300aaatggtaac accttggtca ctaatagatg ctaacgcatg gggagtgtgg ttcaatccag 360cggactggca gttaatatcc aacaacatga cagaaataaa cttagttagt tttgaacaag 420caatattcaa tgtagtactt aaaacaatta cagaatcagc aacctcacca ccaaccaaaa 480tatataataa tgatctaact gcaagcttaa tggtcgcact agacaccaat aacacacttc 540catacacacc agcagcacct agaagtgaaa cacttggttt ttatccatgg ttacctacaa 600aaccaactca atacagatat tacctatcat gcatcagaaa cctaaatcca ccaacataca 660ctggacaatc acaacaaata acagactcaa tacaaacagg actacacagt gacattatgt 720tctacacaat agaaaatgca gtaccaattc atcttctaag aacaggagat gaattctcca 780caggaatata tcactttgac acaaaaccac taaaattaac tcactcatgg caaacaaaca 840gatctctagg actgcctcca aaactactaa ctgaacctac cacagaagga gaccaacacc 900caggaacact accagcagct aacacaagaa aaggttatca ccaaacaatt aataatagct 960acacagaagc aacagcaatt aggccagctc aggtaggata taatacacca tacatgaatt 1020ttgaatactc caatggtgga ccatttctaa ctcctatagt accaacagca aacacacaat 1080ataatgatga tgaaccaaat ggtgctataa gatttacaat ggattaccaa catggacact 1140taaccacatc ttcacaagag ctagaaagat acacattcaa tccacaaagt aaatgtggaa 1200gagctccaaa gcaacaattt aatcaacagg caccactaaa cctagaaaat acaaataatg 1260gaacactttt accttcagat ccaataggag ggaaatctaa catgcatttc atgaatacac 1320tcaatacata tggaccatta acagcactaa acaatactgc acctgtattt ccaaatggtc 1380aaatatggga taaagaactt gatacagatc taaaacctag actacatgtt acagctccat 1440ttgtttgtaa aaacaatcca ccaggacaac tatttgtaaa aatagcacca aacctaacag 1500atgatttcaa tgctgactct cctcaacaac ctagaataat aacttattca aacttttggt 1560ggaaaggaac actaacattc acagcaaaaa tgagatccag taatatgtgg aaccctattc 1620aacaacacac aacaacagca gaaaacattg gtaactatat tcctacaaat attggtggca 1680taagaatgtt tccagaatat tcacaactta taccaagaaa attatactag aaataactct 1740gtaaataaaa actcagttac ttggttaatc atgtactact atcattgtat acttcaataa 1800aaataaattg taaaatcaat aaaactaagt tacttagttt ctgtatacct atactag 1857
3, a kind of recombinant pseudorabies virus genetically engineered bivalent vaccine of expressing pig parvoviral VP2 gene is characterized in that, it contains the described recombinant porcine pseudorabies poison of claim 1 Pseudorabies virus, HzauAVL-PRppvV-VP2; Be deposited in CGMCC, preservation date: on August 16th, 2002, deposit number: CGMCC:0788-2.
4, a kind of method for preparing the recombinant pseudorabies virus genetically engineered bivalent vaccine of expressing pig parvoviral VP2 gene, it is characterized in that, it contains recombinant porcine pseudorabies poison as claimed in claim 1: Pseudorabies virus, HzauAVL-PRppvV-VP2; Be deposited in CGMCC, preservation date: on August 16th, 2002, deposit number: CGMCC:0788-2, described method also comprises: utilize the round pcr amplification to obtain the VP2 gene of pig parvoviral, respectively introduce a BamH I restriction enzyme site simultaneously at its two ends, thereby be inserted into and obtain the pUSK-VP2 transfer vector among the transfer vector pUSK, by liposome-mediated transfection, genomic dna cotransfection PK-15 cell with pUSK-VP2 carrier and Pseudorabies virus TK-/gG-/LacZ+ parent plant, wait to occur to collect viral liquid after the cytopathy, the plaque purifying is identified the recombinant virus that obtains with the PCR detection method simultaneously.After the recombinant virus that obtains is carried out the evaluation of biological characteristics, be prepared into freeze-dried live vaccine, and then estimate its security and immunogenicity, the final recombinant pseudorabies virus vaccine that obtains to express pig parvoviral VP2 gene by animal experiment.
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| CN105368797A (en) * | 2015-09-09 | 2016-03-02 | 河南农业大学 | Recombinant porcine pseudorabies virus strain expressing porcine parvovirus VP2 gene |
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| CN109609468B (en) * | 2018-12-10 | 2020-06-30 | 畜科生物工程有限公司 | Six-gene-deleted porcine pseudorabies virus, porcine pseudorabies vaccine and preparation method thereof |
| CN111705040A (en) * | 2020-06-17 | 2020-09-25 | 国药集团动物保健股份有限公司 | Method for removing mycoplasma in pseudorabies virus liquid |
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