CN102628053A - Construction and application of recombinant Chicken Marek's Disease Virus SC9-1 strain and SC9-2 strain - Google Patents
Construction and application of recombinant Chicken Marek's Disease Virus SC9-1 strain and SC9-2 strain Download PDFInfo
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Abstract
The invention relates to construction and application of recombinant chicken Marek's disease virus SC9-1 strain and SC9-2 strain. The construction method of the recombinant virus solves the problem that the continuous secondary adoption of kan containing flp recognition sites at two endsrAfter two specific functional genes on the same virus genome are knocked out, kan cannot be knocked out again by the existing methodrThe technical difficulties of the gene; obtained weightThe group virus MDV SC9-1 strain and MDV SC9-2 are used as vaccines prepared by producing strains of Marek's disease live vaccines, prevent the Marek's disease of chicken induced by super-virulent virus or newly-appeared super-virulent virus MDV, and the protective immune effect of the vaccine is superior to that of CVI988/Rispens strain vaccines which are most widely applied in the current domestic and foreign markets; the antigenicity of the recombinant virus is more similar to that of an epidemic strain in China than that of a similar virus rMd5 delta meq published in the United states, and the recombinant virus does not induce tumor and has no immunosuppressive effect, so the recombinant virus is more practical for China.
Description
Technical field the present invention relates to the to recombinate structure and the application of chicken Marek's disease virus SC9-1 strain and SC9-2 strain belong to the veterinary biologics biology field.
Background technology
From nineteen sixty-eight separate first marek's disease virus (Marek ' s disease virus; Be abbreviated as: MDV); Poultry husbandry adopts vaccine immunity to prevent the history in existing more than 40 year of MD, pathogenic along with MDV in continuous evolution, and the virulence of MDV and pathogenic strengthens gradually; Thereby used vaccine develops into II type SB-1 from III type HVT univalent vaccine and adds the HVT bivalent vaccine, has at last with causing weak but still has the I type CVI988/Rispens vaccine strain of certain tumorigenicity.
MDV's is popular from the chicken crowd; From being main with weak strain (mMDV); To being main with strong virus strain (vMDV), so that superpower virus (vvMDV) strain is more and more general over nearly 20 years, special superpower virus strain (vv+MDV) (Witter RL.Increased virulence of Marek ' s disease virus field isolates.Avian Diseases appearred also before 10 years; 1997,41 (1): 149-163.).Scientist utilizes the whole bag of tricks to make up various MDV candidate strains to attempt to improve the effect of vaccine, but all gets nowhere.For example; Witter and Kreager etc. have compared the protective immunity effect of the new vaccine candidate strain of 10 strains; But the result all unlike the CVI988/Rispens strain good (Witter RL and Kreager KS.Serotype 1 viruses modified by backpassage or insertional mutagenesis:Approaching the threshold of vaccine efficacy in Marek ' s disease.Avian Dis.2004,48:768-782.).It seems,, improve vaccine with ordinary method and as if gone to the end in the face of the pathogenic enhanced trend of MDV street strain.But according to MDV development law in past more than 40 year, its virulence probably can become stronger at a few years from now on.In recent years, in China, even some chicken crowds who has used CV1988/Rispens immunity, the trend that still has the tumour incidence to rise, though domestic also not have to be correlated be separated to the report of special highly virulent strain, the variation of MDV is being carried out always.For example, the isolating GX0101 of street strain of the inventor is though its virulence is littler than Md5 (virulent); But its horizontal transmission ability is far longer than Md5 (Xu Xiaoyun, Sun Aijun, Cui Yanshun; Deng. have the segmental marek's disease virus of REV-LTR reorganization street strain and highly virulent strain pathogenic with the comparison of horizontal transmission property. mikrobe journal (Acta Microbiologica Sinica), 2009,49 (4): 0540-0543; Cui et al., 2009).Obviously, be necessary that new pathogenic stronger MDV street strain in the protective immunity that can break through the CVI988/Rispens strain occurs and comes into vogue before, should develop more effective vaccine with new method as soon as possible.
The inventor advances the BAC carrier with the full genomic clone of GX0101; Made up its infections clone GX0101-BAC,, can save out the bac-GX0101 (Sun Aijun of recombinant virus MDV its transfectional cell; 2009, Chinese Science Bulletin.54:2641-2647).Knock out 2 meq genes among the bac-GX0101 of MDV as follows: with plasmid pKD13 is template, with pcr amplification FRT-kalamycin resistance gene (kanamycin resistance gene, kan
r).In order to pass through homologous recombination with kan
rGene replaces the meq gene among the bac-GX0101, at amplification FRT-kan
r5 of the upstream and downstream primer that gene is used ' end contains the homology arm with the front and back 50bp sequence homology of meq gene respectively.Primer sequence is following:
Δmeq-F：5′-AGAAACATGGGGCATAGACGATGTGCTGCTGAGAGTCACAATGCGGATCAcgtgtaggctggagctgcttc-3′；
Δ meq-R:5 '-CTTGCAGGTGTATACCAGGGAGAAGGCGGGCACGGTACAGGTGTAAAGAGcattcc ggggatccgtcgac-3 ' (represent with capitalization, from FRT-kan by the front and back 50bp homologous sequence of meq gene
rSequence represent with lowercase).
The amplification system of FRT-kalamycin resistance gene is following: 10 * Buffer, 2.5 μ L, template pKD13 (about 100ng/ μ L) 1 μ L, Mg
2+(15mmol/L) 2 μ L, dNTP (2.5mmol/L) 2 μ L, each 1 μ L of upstream and downstream primer (25pmol/ μ L), Taqenzyme (5U/ μ L) 0.1 μ L mends to 25 μ L with distilled water.The pcr amplification program is: 95 ℃ of 10min, and by 95 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, 30 circulations; Last 72 ℃ are extended 10min.Amplified production is identified with gel electrophoresis.
At kan
rTwo ends contain the flp recognition site (flp recognition target, FRT), the flp recombinase can be discerned the FRT site of kalamycin resistance gene both sides 34bp, but the inboard sequence in 2 FRT sites of specificity excision.The PCR product digests 1h to remove circular template plasmid residual in the PCR product with Dpn I enzyme; Postdigestive PCR product utilization electricity conversion instrument transforms (Sun Aijun among the intestinal bacteria EL250 that has into contained GX0101-BAC; 2009, Chinese Science Bulletin.54:2641-2647), electric commentaries on classics condition is 1800V; 100 Ω, 25 μ F.Use the resuspended bacterium of 1ml LB after the conversion immediately, get 100 μ L bacterium liquid behind 32 ℃ of cultivation 2h and be applied in the LB flat board that contains kalamycin resistance.Picking list bacterium colony utilizes PCR to identify kan behind the 16h
rWhether replaced meq gene (Schumacher; D.; B.K.Tischer; W.Fuchs, and N.Osterrieder.2000.Reconstitution of Marek ' s disease virus serotype 1 (MDV-1) from DNA cloned as a bacterial artificial chromosome and characterization of a glycoprotein B-negative MDV-1 mutant.J.Virol.74:11088-11098).Through identifying; After utilizing kalamycin resistance gene to replace a meq gene; Picking colony is inoculated in the LB liquid nutrient medium in 32 ℃ of incubated overnight shakes and cultivates, and treats that its OD value reaches at 0.5 o'clock, adds 10% pectinose and induces 1h; Make the plasmid flp recombinase in the host bacterium activate and express, thereby remove kalamycin resistance gene.Resulting bacterium can generate only containing on the 30 μ g/mL paraxin LB agar plates, grows but can not contain at the same time on the LB agar plate of 30 μ g/mL paraxin and 50 μ g/mL kantlex.Knock out after a meq gene verifies successfully, profit uses the same method and utilizes kan again
rReplace another one meq gene, with the infective cloned plasmids called after GX0101 Δ meq-BAC of the MDV GX0101 of 2 the meq genes of disappearance that obtain, the virus that is produced by its plasmid DNA transfection cell is GX0101 Δ meq.But owing in the process that knocks out second Meq gene, added a Kan again
rGene, according to existing genetic engineered product Biosafety method, must not be with having Kan
rThe live virus of gene is as vaccine.In order to obtain the strain that can be used as vaccine, also must knock out Kan
rGene.
Summary of the invention
The objective of the invention is: utilize the part digestion to having 3 FRT sites of not exclusively inducing generation that the flp recombinase is expressed to knock out the kan that is used to replace the 2nd meq gene in the BAC cloned plasmids of GX0101
rGene; And do not have meq gene and a kan as vaccine through a large amount of bacterium colonies being cultivated in the contrast that contains with not containing on 2 LB flat boards of kantlex, filtered out to can be used for saving out
rThe BAC clone of gene; Obtained the meq genetically deficient virus SC9-1 strain that can be used as vaccine that does not have kalamycin resistance gene with this from the pathogenic MDV GX0101 of street strain of primary, and the SC9-2 strain that has further knocked out most of frame sequence of the BAC carrier that contains chloramphenicol resistance gene (CM+) again.
Technical scheme of the present invention:
It is to utilize the flp-kan+ recombination method to knock out 2 meq genes and do not contain kalamycin resistance gene that genome involved in the present invention makes up thing; First kalamycin resistance gene is to remove with conventional flp enzyme process, and the full flp enzymic digestion of then toing many or too much for use of second kalamycin resistance gene adds the negative sieve method of kalamycin resistance and removes.Obtain to be used for transfectional cell thus and produce the reorganization BAC plasmid pSC9-BAC (see figure 1) of MDVSC9-1 virus and then the genes involved (like chloramphenicol resistance gene, the mini-F factor etc.) of the BAC carrier that the initial BAC of structure of further removal clone process is inserted, obtain to be used for transfectional cell thus and produce the viral reorganization BAC plasmid pSC9-BAC-Kana-Sv-Cre-US2 (see figure 2) of MDV SC9-2 (Fig. 3).
The invention still further relates to the chicken Marek's disease virus MDV SC9-1 strain of reorganization/or MDV SC9-2 strain be used for preventing the purposes of the chicken Marek's disease vaccine of the chicken Marek's disease that virulent or emerging special virulent MDV bring out in preparation.
The present invention also relates to a kind of method of preventing chicken Marek's disease simultaneously, wherein prevents the chicken Marek's disease vaccine of effective dose to chicken, and described vaccine comprises reorganization chicken Marek's disease vaccine virus MDV SC9-1 strain/or MDV SC9-2 strain.
Embodiment
The part digestion to having 3 FRT sites of not exclusively inducing generation that utilization is expressed the flp recombinase knocks out the kan that is used to replace the 2nd meq gene in the BAC cloned plasmids of GX0101
rGene, and do not have meq gene and a kan as vaccine through a large amount of bacterium colonies being cultivated in the contrast that contains with not containing on 2 LB flat boards of kantlex, filtered out to can be used for saving out
rThe BAC clone of gene.(Li Yan roc marek's disease virus meq gene-deleted strain biological characteristics and immune effect research thereof in the research of having delivered early stage; The doctorate paper, 2010, the .And Yanpeng Li of the Chinese Academy of Agricultural Sciences; Aijun Sun; Shuai Su, Peng Zhao, Zhizhong Cui; Hongfei Zhu; Deletion of the Meq gene significantly decreases immunosuppression in chickens caused by pathogenic Marek ' s disease virus.Virology Journal2011,8:2), the BAC cloned plasmids from GX0101 has made up the reorganization BAC cloned plasmids pGX0101 Δ meq-BAC that has knocked out 2 meq genes.
Detailed process is: after utilizing kalamycin resistance gene to replace a meq gene; Picking colony is inoculated in shake cultivation in the LB liquid nutrient medium; Treat that its OD value reaches at 0.5 o'clock; Add 10% pectinose and induce 1h, make the plasmid flp recombinase in the host bacterium, thereby remove kalamycin resistance gene.After inducing with bacterium separate application 32 ℃ of incubated overnight on the LB agar plate that contains 30 μ g/mL paraxin or 50 μ g/mL kantlex; If, show that the reorganization bacterium after inducing still possesses chlorampenicol resistant and no longer possesses kalamycin resistance (Kan only in growth on the LB agar plate that contains 30 μ g/mL paraxin but containing on the LB agar plate of 50 μ g/mL kantlex and do not grow
r), can confirm that this reorganization bacterium is the purpose bacterium.But still keep in the position at first Meq gene place of being removed in this recombinant plasmid a flp recombinase identification the site (flp recognition target, FRT).
After knocking out a Meq gene and empirical tests, profit uses the same method and utilizes kan again
rReplace another one meq gene, with the disappearance that obtains the infective cloned plasmids called after pGX0101 Δ meq-BAC of MDV GX0101 of 2 Meq genes.This clone has inserted a kan again
rGene.In view of knocking out previous kan
rGene is to have stayed a FRT site, on plasmid pGX0101 Δ meq-BAC, has had 3 FRT sites (like Fig. 1).If by same method with inducing the flp recombinase to knock out the 2nd kan
rGene will knock out a part of genome of MDV simultaneously, thereby no longer can save new infections clone virus.On the other hand, GX0101 Δ Meq that we have delivered virus, though do not have pathogenic and possess excellent protective immunizing power, its replication on cell is relatively poor, this characteristic also is not suitable for as commercialized vaccine.
For this reason, the inventor knocks out in the infections clone process of GX0101 of Meq gene at structure, to having carried out relatively screening with pGX0101 Δ meq-BAC with batch 12 plasmid clone bacterium colonies that obtain.These independent bacterium colonies are renumberd respectively be pSC Δ Meq-BAC1 to pSC Δ Meq-BAC12.Respectively the thalline of different bacterium colonies is cultivated back preparation DNA, transfection CEF more in a large number.After the distinctive plaque of MDV appears in cell, be SC1 Δ Meq to SC12 Δ Meq with resulting infections clone viral nomenclature respectively.The levels of replication of these viruses on CEF relatively respectively, it is at most the fastest therefrom to choose the viral plaque that infective cloned plasmids pSC Δ Meq-BAC9 forms on CEF.This infective cloned plasmids pSC Δ Meq-BAC9 is used for further knocking out Kan wherein
rGene.
For this reason; We have designed the novel method of " partly inducing digestion to combine multiplex screening to flp recombinase and FRT "; That is: when microbial culture, reduce inductive condition and time the flp recombinase; Make on a part of pGX0101 Δ meq-BAC plasmid DNA molecule and have only 2 sites by the effect of flp recombinase in 3 FRT sites, thereby make the part molecule have only kan
rThe FRT of gene two sides is discerned and effect by the flp recombinase, so has only kan on this a part of molecule
rGene is fallen by flp recombinase digestion montage, and the MDV genome relevant with another FRT site all keeps.
Respectively with each bacterium colony through repeatedly relatively the screening; Achieve success by following condition at last: the E250 strain intestinal bacteria that infective cloned plasmids pSC Δ Meq-BAC9 transforms are inoculated in shake cultivation in the LB liquid nutrient medium; Treat that its OD value reaches at 0.5 o'clock; The pectinose that adds 5% (the inductive normal concentration is 10% fully) is induced 40min (the inductive time is 60min fully); Plasmid flp recombinase in the host bacterium is only activated by part, thereby make part MDV genomic clone plasmid molecule only remove kalamycin resistance gene but do not influence MDV genome part.
After inducing bacterium is coated on the LB agar plate that contains 30 μ g/mL paraxin 32 ℃ of incubated overnight.All bacterium colonies that possess chlorampenicol resistant are inoculated two types of LB agar plates more respectively: one type only contains 30 μ g/mL paraxin (A); The bacterium colony of inoculating on another kind of 30 μ g/mL paraxin and the 50 μ g/mL kantlex (B) of containing simultaneously, two types of flat boards is checked the number respectively one by one.
The bacterium colony that is chosen in growth on the A plate and on the B plate, does not grow has not promptly possessed the active bacterium colony of anti-kantlex, is inoculated in respectively again on the LB agar plate that contains 30 μ g/mL paraxin,, 32 ℃ of incubated overnight.
The part thalline of getting each bacterium colony extracts genomic dna respectively, uses kan respectively
rGene and MDVpp38 gene-specific primer (pp38-F:5 '-AGGCAGGGCATGGGAAAACAGAAG-3 ', sequence 3 and pp38-R:5 '-ACCGACTAACATACCAGCGAAAAA-3 ', sequence 4) be PCR, be chosen in kan among the PCR
rGene feminine gender but the bacterium colony of MDVpp38 gene masculine is inoculated in the LB meat soup that contains 30 μ g/mL paraxin; Select corresponding several bacterium colonies with plasmid Maxi kit (available from QIAGEN company) large quantity extracting plasmid DNA, the plasmid that successfully is used for transformant generation SC9-1 strain MDV is named the (see figure 1) into pSC9-BAC.Respectively with above each DNA difference transfection CEF individual layer, knocked out Kan then from wherein selecting the distinctive cell plaques of MDV, having saved thus
rThe recombinant virus of resistant gene, called after MDV SC9 Δ Meq Δ Kan
r, be called for short MDV SC9-1.This marek's disease virus (Marek ' s disease virus) strain is deposited in No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms centers, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on December 03rd, 2010, and preserving number is CGMCC No.4399.
Consider of the different standards requirement of some country, when making up SC9-1, made up the SC9-2 strain MDV that has further knocked out the chloramphenicol resistance gene again the Biosafety of recombination product.Earlier by the method for having delivered (Zhao et al.; Self-excision of the BAC sequences from the recombinant Marek ' s disease virus genome increases replication and pathogenicity; Virology Journal; 2008; 5:19), make up one and have respectively middle recombinant plasmid pPartial-F-Kan-Cre-US2-US3 from the mini-F fragment and the genomic US2-US3 of the GX0101 strain MDV joining region sequence of BAC vector plasmid in Kan+ and SV-cre combination gene two sides.With this plasmid and pSC9-BAC DNA cotransformation E250 intestinal bacteria; The bacterium colony that kantlex is had resistance in the culture plate screening that contains kantlex and paraxin; The bacterium colony plasmid is named to pSC9-BAC-Kana-Sv-Cre-US2. prepares heavy dose of DNA with this bacterium colony, and then cultivates with this plasmid DNA transfection CEF cell monolayer.Go down to posterity again after cells transfected digestion being suspended in every 3-5 days, and observe the viral plaque that MDV produces.Because the cre gene in SV-cre is the eukaryotic cell promotor in the SV40 promotor, it can activate the cre expression of gene automatically in the CEF cell.Can be by the enzyme of cre genetic expression on the viral genome that produces in the CEF cell of transfection with 2 the loxP sites digestion on the MDV viral genome in the continuation infected cells in the process that goes down to posterity subsequently that reach that produced in the transfectional cell; Thereby automatically all sequences between 2 loxP sites on the viral genome is cut off, the virus that is produced is SC9-2.The SC9-2 that the structure of its genome associated clip such as the C. in the accompanying drawing obtain has thus knocked out and has contained chloramphenicol resistance gene (CM
r) most of dna sequence dna of BAC carrier framework.In order to mention the ratio in the MDV virus colony of SC9-2 in cells infected, the CEF monolayer culture continuous passage that the pSC9-BAC-Kana-Sv-Cre-US2 plasmid DNA transfection has been produced viral plaque.Connect passed for 8 generations after, detect through PCR, most viral plaques are to have SC9-2 formed, promptly having knocked out BAC carrier and CM+ gene order, but still have minority virus still to have BAC carrier and CM+ gene order.Be inoculated on the 96 porocyte culture plates through limiting dilution assay virus dilution cells infected.The virus infected cell of selecting in the hole of having only 1 viral plaque further goes down to posterity; What obtain purifying thus is the cell culture that has knocked out the SC9-2 strain virus of BAC carrier and CM+ gene order fully; This marek's disease virus (Marek ' s disease virus) strain is deposited in No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms centers, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on November 24th, 2011, and preserving number is CGMCCNo.5502.。
Two, street strain is knocked out the Mareks disease vaccine virus SC9-1 strain of tumour Meq gene and the identification feature of SC9-2
Have three particular sequences that are positioned at specific site on this viral genome and can be used as this viral distinctive sign:
(1), after knocking out the Meq gene, respectively stays the FRT sequence about next about 36bp at original two Meq gene locuss;
(2) inserted the REV-LTR fragment of one section about 539bp at about 370bp place, the upper reaches of virus genomic US/TRS fragment tie point.
(3) difference of MDV SC9-1 and MDV SC9-2 is: MDV SC9-1 has CM
rGene and mini-F plasmid, but MDV SC9-2 does not contain CM
rGene and mini-F plasmid.
Three, MDV SC9-1 strain and MDV SC9-2 strain virus are to the pathogenic of chicken and protective immunity effect
In the research of having delivered, proved that GX0101 Δ meq does not have pathogenic fully to chicken and can bring out 100% protective immunity (Su Shuai, Li Yanpeng; Sun Aijun; Cui Zhizhong. knock out the immanoprotection action of the Mareks disease virus virulent strain of meq, mikrobe journal, 2010 to virulent; 50:380-386.), just because GX0101 Δ meq has Kan
rGene, thereby can not be used as vaccine virus.For this reason, the present invention has adopted and has knocked out Kan fully
rThe new vaccine strain MDV SC9-1 strain of gene and its derived virus MDV SC9-2 press reported method (Su Shuai, Li Yanpeng; Sun Aijun; Cui Zhizhong. knock out the immanoprotection action of the Mareks disease virus virulent strain of meq, mikrobe journal, 2010 to virulent; 50:380-386) use artificial challenge's test with the quadrat method similarity condition the SPF chicken
Concrete grammar is following: with 240 of 1 age in days SPF chickens; Be divided into 8 groups at random, 30 every group, raise respectively in 8 and just have; In the SPF animal rearing shield retaining of press filtration air; During 1 age in days, the 1st group of chicken is with 2000pfu/ dosage abdominal cavity inoculation GX0101 Δ meq only, and the 2nd group of chicken inoculates CVI988/Rispen with 2000pfu/ dosage abdominal cavity inoculation MDV SC9-2, the 4th group of chicken only with 2000pfu/ dosage abdominal cavity only with 2000pfu/ dosage abdominal cavity inoculation SC9-1, the 3rd group of chicken; The 5th group of chicken is for attacking the poison contrast; The 6th group of chicken is blank, and the 7th group of chicken is with 2000pfu/ dosage abdominal cavity inoculation SC9-1 only, and the 8th group of chicken is with 2000pfu/ dosage abdominal cavity inoculation MDV SC9-2 only.After the immunization 5 days, the 1st, 2,3,4 and 5 group of chicken is respectively with 1000pfu/ challenge dose MDV highly virulent strain vv rMd5 only.Observe the growth situation that reaches weekly after attacking poison.Experimental session is organized dead chicken and is cutd open inspection each, and gets the peculiar pathology internal organs of doubtful Marek and do paraffin section, carries out histopathology after the HE dyeing and observes.Attacking poison back 90 days, all survival chickens all are condemned to death and cut open inspection, observe the pathology of chicken, simultaneously, 5 of every group of picked at random, dirty, the spleen of coring respectively, liver are fixed 4 ℃ of preservations in 10% neutral formalin.Paraffin section, HE dyeing carry out histopathology to be observed.
Test-results proves that this MDV SC9-1 and MDV SC9-2 two strain virus do not have pathogenic to the SPF chicken, and virulent vvMDV virus strain Md5 is shown same protective immunity (table 1).Can find out from table, with Kan
rBehind the gene knockout, the SC9-1 strain virus is renderd a service with its parental virus GX0101 Δ meq identical to the pathogenic and protective immunity of SPF chicken.Knocking out Kan
rBehind the gene, the requirement that will meet the Biosafety method can be used as vaccine.MDV SC9-2 strain is further knocking out CM
rAlso identical behind the gene.
The comparison that table 1MDV SC9-1 and MDV SC9-2 and other MDV strains are renderd a service the pathogenic and protective immunity of SPF chicken
* annotate: the different vaccines of 1 age in days SPF chicken difference intraperitoneal inoculation 2000pfu are with viral GX0101 Δ meq, SC9-1, SC9-2 and CVI988/Rispens.The MDV highly virulent strain rMd5. that when 5 ages in days, inoculates 1000pfu respectively observes and writes down the mortality ratio in 13 weeks continuously, dead chicken is all analysed to observe have or not tumour, and makes tissue slice and observe and have or not neoplastic lesion.When 13 weeks, slaughter all chickens, visual inspection has or not tumour and record.MD tumour rate in the table is that dead chicken manifests tumour and slaughters the summation that occurs tumour in the chicken after 13 weeks.The protection index is to calculate according to the prophylactic effect to the tumour incidence.
Four .MDV SC9-1 strains and MDV SC9-2 strain virus had not both had oncogenicity effect to chicken is not had immunosuppressive action yet
Show in the artificial inoculation of SPF chicken test, inoculated the chicken of the malicious MDV bac-GX0101 of original clone strain, growth significantly slowly, and thymus gland is all compared with the fabricius bursa and the ratio of body weight and is shone chicken much lower (P＜0.01).But SC9-1 and SC9-2 strain virus not only do not have oncogenicity effect (seeing table 1), nor can cause immunosuppression.Behind 1 age in days SPF chicken inoculation SC9-1 strain and the SC9-2 strain virus, its body weight and central immune organ thymus gland and the fabricius bursa all do not have significant difference (seeing table 2) with the contrast chicken with the ratio of body weight.This characteristic is important for cause the pass as vaccine.Compare with SC9-2 with SC9-1, the rMd5 Δ meq of the U.S. then also keeps significant immunosuppressive action, therefore is not suitable for as vaccine strain.
SPF chicken bursa and the thymus gland and the weight ratio (n=20) of table 2 inoculation MDV bac-GX0101, SC9-1 and SC9-2 strain virus
All 1 age in days SPF chickens inoculate MDV bac-GX0101 or the SC9-1 strain virus of 1000 PFU respectively through the abdominal cavity.Go the thymus gland and the fabricius bursa to weigh in 6 time-division in age week another name body weight and after slaughtering.Numeral is mean number ± standard deviation in the table.Upper right corner letter is different, shows significant difference highly significant (P＜0.01), and same letter is represented significant difference not remarkable ((P＞0.05).
The structure and the application of chicken Marek's disease virus SC9-1 strain and SC9-2 strain the present invention relates to recombinate.The present invention has following characteristics:
1. having solved continuous quadratic adopts two ends to contain flp recognition site kan
rCan't knock out kan again with current methods behind two specific function genes on the same viral genome of gene knockout
rThe technological difficulties of gene;
2.MDV SC9-1 strain and MDV SC9-2 strain virus are used to prepare vaccine; The chicken Marek's disease that prevention virulent or emerging special virulent MDV bring out, its protective immunity effect is superior to most widely used CVI988/Rispens strain vaccine on the present domestic and international market;
3.MDV parent's poison GX0101 of SC9-1 and MDV SC9-2 virus is the local isolating street strain of China, its antigenicity should more be analogous to the epidemic strain of China than the similar viral rMd5 Δ meq that the U.S. has delivered, thereby more is useful for China;
4.MDV the initial parent's poison MDV GX0101 strain of SC9-1 and MDV SC9-2 virus is one and has inserted the segmental natural recombinant virus of fowl RE hyperplasia virus LTR, its original pathogenic parent's poison Md5 that just significantly is lower than rMd5 Δ meq.Though rMd5 Δ meq protective immunity effect is also fine, can induced tumor yet, still have tangible immunosuppressive action, can not be used as the commercialization vaccine.But SC9-1 strain and SC9-2 strain MDV not only can induced tumors, do not have immunosuppressive action yet, and have the identical protective immunity effect with rMd5 Δ meq, can be used as the production strain of commercialization vaccine.
5.SC9-1 the initial parent's poison GX0101 of strain and SC9-2 strain MDV is one and has inserted the segmental natural recombinant virus of fowl RE hyperplasia virus LTR; Its original pathogenic parent's poison Md5 that just significantly is lower than rMd5 Δ meq, but its horizontal transmission ability is but greater than Md5.Can expect that SC9-1 strain and SC9-2 strain MDV also have the horizontal transmission ability bigger than rMd5 Δ meq.The mass-producing chicken house will be in the limited time a large amount of chickens of immunization, unavoidably some individual take place to leak exempt from.The chicken that this characteristic might make minority leak and exempt from more is prone to by natural immunity, exempts to bring immuning failure to reduce owing to leaking.
Positive effect of the present invention
The structure and the application of SC9-1 strain of chicken Marek's disease vaccine virus and SC9-2 strain the present invention relates to recombinate.Recombinant virus construction process of the present invention has solved continuous quadratic and has adopted two ends to contain flp recognition site kan
rCan't knock out kan again with current methods behind two specific function genes on the same viral genome of gene knockout
rThe technological difficulties of gene; Recombinant virus MDV SC9-1 strain that is obtained and MDV SC9-2 are as the prepared vaccine of production strain of Mareks disease live-vaccine; The chicken Marek's disease that prevention virulent or emerging special virulent MDV bring out, its protective immunity effect is superior to most widely used CVI988/Rispens strain vaccine on the present domestic and international market; The antigenicity of this recombinant virus should more be analogous to the epidemic strain of China than the similar viral rMd5 Δ meq that the U.S. has delivered, not only can induced tumor, and do not have immunosuppressive action yet, thereby more be useful for China.
Mikrobe information involved in the present invention:
(1) marek's disease virus (Marek ' s disease virus) SC9 Δ Meq Δ Kan
rStrain is called for short MDV SC9-1 strain.This virus strain is deposited in No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms centers, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on December 03rd, 2010, and preserving number is CGMCC No.4399.
(2) marek's disease virus (Marek ' s disease virus) SC9-2 strain virus; This virus strain is deposited in No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms centers, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on November 24th, 2011, and preserving number is CGMCC No.5502.
Description of drawings
Fig. 1. be used for the viral complete genomic BAC of reorganization plasmid structural pattern figure that transfectional cell produces SC9-1 strain MDV wherein 2 Δ meq meq gene of being illustrated in this site all be removed.The sequence that in this plasmid, also includes the BAC carrier comprises chloramphenicol resistance gene cm, gpt gene and mini-F sequence.
Fig. 2. the recombinant plasmid pSC9-BAC-Kana-Sv-Cre-US2 structural pattern figure that is used for making up the SC9-2 virus of having removed BAC carrier sequence compares with the pSC9-BAC plasmid of Fig. 1; This plasmid has inserted the Kana-Sv-Cre gene order on the mini-F right side again; The product of the Cre gene that Sv-Cre gene wherein can be expressed after plasmid DNA transfection is gone into eukaryotic cell is the restriction enzyme that can discern 2 loxP sites in one, will remove BAC carrier full sequence.
The full genome mechanic mode figure of Fig. 3 .SC9-2 strain virus wherein BAC carrier sequence that comprises chloramphenicol resistance gene between MDV genome inherent Sort3 and Us3 all removes, only surplus next loxP recognition site.
Claims (8)
1. the genome of the chicken Marek's disease virus of recombinating makes up thing, it is characterized in that having three particular sequences that are positioned at specific site on the said genome structure thing, and this particular sequence also can be used as this viral distinctive sign:
(1), after knocking out the Meq gene, respectively stays sequence 5:FRT:5 '-GAAGTTCCTA TACTTTCTAG AGAATAGGAA CTTC-3 ' of next 34bp at original two Meq gene locuss; After knocking out two Meq genes, MDVSC9-1 strain and MDV SC9-2 strain do not have pathogenic fully; (2) inserted the REV-LTR fragment sequence 6 of one section about 539bp at about 370bp place, the upper reaches of virus genomic US/TRS fragment tie point:
5′-CATTGTGGGAGGGAGCTCCGGGGGAATGTGGGAGGGAGCTCCGGGGGGAATAGCGCTGGCTCGCTAACTGCCATATTAGCTTCTGTAGTCATGCTTGCTTGCCTTAGCCGCCATTGTACTTGATATATTTCGCTGATATCATTTCTCGGAATCGGCATCAAGAGCAGGCTCATAAACCCATAAAAGGAAATGTTTGTTGAAGGCAAGCATCAGACCACTTGCACCATCCAATCACGAACAAACACGAGATCGAACCATCATACTGAGCCAATGGTTGTAAAGGGCAGATGCTATCCTCCAATGAGGGAAAATGTCATGCAACATCCTGTAAGCGGCTATATAAGCCAGGTGCATCTCTTGCTCGGGGTCGCCGTCCTACACATTGTTGTGACGTGCGGCCCAGATTCGAATCTGTAATAAAAGCTTTTTCTTCTATATCCTCAGATTGGCAGTGAGAGGAGATTTTGTTCGTGGTGTTGGCTGGCCTACTGGGTGGGGTAGGGATCCGGACTGAATACCGTAGTATTTCGGTACAACAA-3′。
2. one kind contains the chicken Marek's disease vaccine virus MDV SC9-1 strain that the said genome of claim 1 makes up the reorganization of thing/or MDV SC9-2 strain; It is characterized in that its genome utilizes the flp-kan+ recombination method to knock out 2 meq genes and do not contain kalamycin resistance gene; First kalamycin resistance gene is to remove with conventional flp enzyme process; The full flp enzymic digestion of then toing many or too much for use of second kalamycin resistance gene adds the negative sieve method of kalamycin resistance and removes, and obtains thus being used for the reorganization BAC plasmid pSC9-BAC that transfectional cell produces SC9-1 virus; And then further remove the genes involved that makes up the BAC carrier that inserts in the BAC clone process at first, obtain thus being used for the reorganization BAC plasmid pSC9-BAC-Kana-Sv-Cre-US2 that transfectional cell produces SC9-2 virus like chloramphenicol resistance gene, the mini-F factor;
MDV SC9-1 strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on August 26th, 2010, and preserving number is CGMCC No.4399;
MDV SC9-2 strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 24th, 2011, and preserving number is CGMCC No.5502.
3. one kind comprises the CEF that reorganization chicken Marek's disease as claimed in claim 2 virus MDV SC9-1 strain or SC9-2 strain are infected.
4. a chicken Marek's disease virus vaccine is characterized in that the genome that comprises the described reorganization chicken Marek's disease virus of claim 1 makes up thing.
5. a chicken Marek's disease virus vaccine is characterized in that comprising the chicken Marek's disease vaccine virus MDV SC9-1 strain or the MDV SC9-2 strain of the described reorganization of claim 2.
6. a chicken Marek's disease virus vaccine is characterized in that comprising the described CEF of claim 3.
7. the genome of a reorganization chicken Marek's disease virus according to claim 1 makes up chicken Marek's disease vaccine virus MDV SC9-1 strain or the MDV SC9-2 strain and the purposes of the described CEF of claim 3 in preparation chicken Marek's disease virus vaccine of thing, the described reorganization of claim 2.
8. prevent the method for chicken Marek's disease; Wherein prevent the chicken Marek's disease virus vaccine of effective dose to chicken; Described vaccine comprises the genome that is selected from the described reorganization chicken Marek's disease virus of claim 1 and makes up thing; Comprise the chicken Marek's disease vaccine virus MDV SC9-1 strain or the SC9-2 strain of the described reorganization of claim 2, perhaps comprise the described CEF of claim 3.
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| 李延鹏: "马立克氏病病毒meq基因缺失株生物学特性及其免疫效果研究", 《中国博士学位论文全文数据库》 * |
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| CN103382460A (en) * | 2013-06-17 | 2013-11-06 | 肇庆大华农生物药品有限公司 | Strain of Marek's disease virus vaccine strain, and separation and identification and application thereof |
| CN106397602A (en) * | 2016-08-02 | 2017-02-15 | 青岛明勤生物科技有限公司 | A molecular adjuvant enhanced type protein engineered vaccine for chicken Marek's disease |
| CN106397602B (en) * | 2016-08-02 | 2019-06-18 | 青岛明勤生物科技有限公司 | A kind of reinforced chicken Marek's disease protein engineering vaccine of molecule adjuvant |
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| CN108342367A (en) * | 2018-02-01 | 2018-07-31 | 山东农业大学 | A kind of SCA13 plants of Recombinan t virus strain and its application |
| CN108342367B (en) * | 2018-02-01 | 2021-07-20 | 山东农业大学 | Recombinant Marek's virus strain SCA13 strain and application thereof |
| CN110343671A (en) * | 2019-06-24 | 2019-10-18 | 青岛易邦生物工程有限公司 | A kind of I type Marek's disease virus vaccine strain of recombination for expressing VP2 gene |
| CN113667648A (en) * | 2021-08-23 | 2021-11-19 | 山东农业大学 | Construction and application of recombinant Marek's virus MDLV21 strain |
| CN113667648B (en) * | 2021-08-23 | 2023-02-24 | 山东农业大学 | Construction and application of recombinant Marek's virus MDLV21 strain |
| CN114470160A (en) * | 2022-03-18 | 2022-05-13 | 山东农业大学 | Inhibitors of viral replication and uses thereof |
| CN114470160B (en) * | 2022-03-18 | 2023-08-25 | 山东农业大学 | Virus replication inhibitor and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102628053B (en) | 2013-10-09 |
| FR2968672A1 (en) | 2012-06-15 |
| FR2968672B1 (en) | 2015-10-16 |
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