CN104946678A - Marek's disease virus infectivity recombinant cloning system, and construction method and application thereof - Google Patents
Marek's disease virus infectivity recombinant cloning system, and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a Marek's disease virus (MDV) infectivity recombinant cloning system, and a construction method and application thereof and belongs to the field of biotechnologies. The MDV infectivity recombinant cloning system comprises a plurality of cosmids, wherein an MDV vaccine strain gene segment is cloned in each cosmid; the MDV vaccine strain gene segment contains mutually superposed regions and can be spliced to cover an integral MDV vaccine strain genome; an exogenous gene is inserted into a non-essential region for replication of at least one of the cosmids. The MDV infectivity recombinant cloning system can quickly and efficiently clone an exogenous gene expression cassette into the MDV genome to further realize rescue of the recombinant MDV. The recombinant cloning system can be used for very conveniently inserting different foreign genes into the corresponding regions so as to construct an MDV living-vector multi-vaccine.
Description
Technical field
The present invention relates to a kind of viral infection recombinant clone system, be specifically related to the infectious recombinant clone system of a kind of marek's disease virus and construction process thereof and application, belong to biological technical field.
Background technology
Marek (MD) is the infectivity neoplastic disease of a kind of chicken caused by marek's disease virus (MDV), is formed as feature with lymphadenosis and tumour.Marek is one of principal disease of chicken, and since being also the domestic and international fifties, the most valued chicken is sick.MDV belongs to Alphaherpesviridae Marek sample Tobamovirus, and be a kind of cell-associated simplexvirus, its genome is wire distrand DNA, is about 180kb, and compositional model is TR
l-U
l-IR
l-IR
s-U
s-TR
s.MDV at least encodes 103 protein, but most of albumen is identified not yet.MDV comprises three kinds of serotypes, serum 1 type virus has tumorigenicity, several pathotype can be further divided into, comprise mild MDV (mMDV), strong malicious type MDV (vMDV), ultra-intense virus type MDV (vvMDV) and special ultra-intense virus type MDV (vv+MDV) virus strain.The non-oncogenic virus strain isolated of chicken and herpes turkey virus strain isolated are called serum 2 type and 3 type MDV (Biggs, P.M.The history and biology of Marek ' s disease virus.In:Hirai, K. (Ed.), Marek ' s Disease.Springer-Verlag, Berlin, Heidelberg, New York, 2001, pp.1-24; Calnek, B.W., Witter, R.L.Marek ' s disease.In:Calnek, B.W. (Ed.), Diseases of Poultry.Iowa State University Press, Ames, 1997, pp.369-413.).
Recombinant live-vector vaccine is, with genetic engineering technique, virus or bacterium are built into a carrier, then foreign gene is inserted the living vaccine wherein making it to express.The immunological comparison that such vaccine-induced body produces is extensive, comprises humoral immunization and cellular immunization, even mucosal immunity; Even more important, live vector vaccine can express plurality of antigens simultaneously, makes multivalence or multiple vaccines, plays a pin and prevents many sick effects, is current and one of the Main way researched and developed of future vaccines.Research shows, hsv gene group is comparatively large, can insert for foreign gene or replace to copy dispensable gene many, be a kind of virus vector of desirable structure recombinant live-vector vaccine.Simplexvirus live vector mainly comprises Pseudorabies virus (PRV), I type bovine herpes virus (BHV-1) and marek's disease virus (MDV) etc.At present, the research report of existing a large amount of simplexvirus live vector vaccine, as Guo Wanzhu etc. obtains national novel chiral synthon certificate with TK/gE/gI tri-gene-deleted strain of Min PRV A strain PRV that has been vector construction.Tsukamoto K etc. (2002) construct the recombinant herpesvirus of turkeys of expressing infectious bursal disease virus (IBDV) VP2 gene, immunity SPF chicken metapedes is to resist attack (the Tsukamoto K. of the strong poison of IBDV, Saito S., Saeki S., et al.Complete, Long-Lasting protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens.J Virol.2002, 76 (11): 5637-5645.).As a member of herpetoviridae, MDV is a kind of good live vaccine vectors equally, research finds the VP2 gene inserting IBDV in the US2 district of MDV, the recombinant mdv that the F gene inserting Avian pneumo-encephalitis virus in US10 district obtains to the protected effect of the strong poison of MDV with contrast suitable (the Tsukamoto K. of MDV, Kojima C., Komori Y., et al.Protection of chickens against very virulent infectious bursal Disease virus (IBDV) and Marek's disease virus (MDV) with a recombinant MDV expressing IBDV VP2.Virology 1999, 257:352-362, Sonoda K., Sakaguchi M., Okamura H., et al.Development of an effective polyvalent vaccine against both Marek's and Newcastle diseases based on recombinant Marek's disease virus type 1 in commercial chickens with maternal antibodies.J Virol.2000,74 (7): 3217-3226.).
Current structure MDV recombinant live-vector vaccine mainly uses homologous recombination method and bacterial artificial chromosome (BAC) method.Wherein homologous recombination method be foreign gene to be inserted in the introduction plasmid with homology arm transfected virus cells infected or with viral genome cotransfection permissive cell, obtain recombinant virus by the homologous recombination process in cell.The method building process is complicated time-consuming and efficiency is lower, needs to add riddled basins and carries out the purifying of recombinant virus.Bacterial artificial chromosome (BAC) method is inserted into by complete MDV genome in BAC and transforms it in bacterium.BAC method is based upon on homologous recombination basis, has the shortcoming of building process complexity, cycle length equally; And BAC its own sequence is difficult to remove, the recombinant virus that later stage is obtained is with unnecessary exogenous gene sequence, there is potential safety hazard (Zelnik, V.Marek's disease virus research in the post-sequencing era:new tools for the study of gene functions and virus-host interactions.Avian Pathol.2003,32:323-333.).Set up a kind of quick, efficient, safe MDV live vector vaccine construction platform, current, there is important theory significance and application prospect.In addition, the research of MDV relatively lags behind compared with other simplexviruss, less to the report of external source gene insertion site in its genome.To identify and the foreign gene insertion point of screening in MDV genome contributes to improving the expression level of foreign gene, and then improve the immune efficacy of MDV live vector vaccine.
Fosmid clay is a kind of cloning vector that can hold 30-45kb size foreign DNA, be usually used in the structure (Vinayak of gene library, S., Brooks, C.F., Naumov, A., et al.Genetic manipulation of the Toxoplasma gondii genome by fosmid recombineering.MBio.2014,5 (6): e02021.).This research is incited somebody to action at present widely used MDV attenuated vaccine 814 pnca gene group DNA segmentation now and is inserted in Fosmid clay, constructs the genomic Fosmid library of this vaccine strain, and establishes MDV multiple clips Fosmid rescue system on this basis.Further, enhanced green fluorescence protein (eGFP) gene is inserted into MDV vaccine strain genomic UL41, UL45/46, UL55/LORF10, US2 and US10 five sites by this research, save out the recombinant mdv vaccine strain that eGFP is expressed in 5 strains, thus in MDV vaccine strain genome, identify 5 Nonessencial regions that can insert for foreign gene.
Summary of the invention
As a kind of simplexvirus, the genome that MDV is larger makes it easy to the insertion of foreign gene, and therefore investigators are devoted to the research of MDV live vector vaccine for many years always.The structure of current MDV recombinant virus adopts homologous recombination method mostly.The more time-consuming effort of this method construction of recombinant virus, the later stage needs carry out clone purification to recombinant virus and remove resistant maker gene etc., and process is comparatively complicated.The MDV recombinant virus constructing system setting up a kind of quickness and high efficiency is very necessary for the research of MDV live vector vaccine.The present invention is on the basis of the MDV vaccine strain multiple clips clay rescue system built before, recombinant cosmid is transformed, establish the infectious recombinant clone system of MDV vaccine strain, and apply the recombinant mdv vaccine strain virus that this rescuing system expresses eGFP, the Nonessencial region in MDV genome is studied.
The object of the present invention is to provide the infectious recombinant clone system of a kind of marek's disease virus, this system can quickness and high efficiency exogenous gene expression box is cloned into MDV genome, and then realize the rescue of MDV recombinant virus.
The object of the invention is to be achieved through the following technical solutions:
The infectious recombinant clone system of a kind of marek's disease virus, it is characterized in that, described system comprises multiple clay, in each clay, clone has Marek's disease virus vaccine pnca gene fragment, described Marek's disease virus vaccine pnca gene fragment contains overlapped region and sliceable covering complete Marek's disease virus vaccine pnca gene group, is inserted with foreign gene in the Nonessencial region of wherein clay described at least one.
In the present invention, preferably, described clay is pCC1Fos.
In the present invention, preferably, described marek's disease virus is marek's disease virus attenuated vaccine 814 strain.
In the present invention, preferably, described clay is 5 clays, 5 clays are respectively p814-1, p814-2, p814-3, p814-4, p814-5, and the sequence of the Marek's disease virus vaccine pnca gene fragment of wherein cloning is respectively the 1-47873 position of marek's disease virus 814 strain, 40028-79118 position, 72447-113806 position, 106337-139612 position and 129115-172541 position.
In the present invention, preferably, described Nonessencial region is arranged in the UL41 gene of p814-3, between UL45 and the UL46 gene in p814-3, between UL55 and LORF10 gene in p814-4, in the US2 gene in the US10 gene in p814-5 and in p814-5.
Further, the invention provides the method for the infectious recombinant clone system of marek's disease virus described in structure, comprise the following steps:
(1) cut off the DNA of described marek's disease virus, and the DNA fragmentation of the marek's disease virus after cut-out is connected on clay;
(2) multiple clay is selected to form the infectious recombinant clone system of described marek's disease virus, wherein in each clay, clone has Marek's disease virus vaccine pnca gene fragment, and described Marek's disease virus vaccine pnca gene fragment contains overlapped region and sliceable covering complete Marek's disease virus vaccine pnca gene group;
(3) foreign gene is inserted the Nonessencial region in clay described at least one.
In the present invention, preferably, the concrete steps in step (3), foreign gene being inserted the Nonessencial region in described clay are:
1) Kan-ccdB tolerant gene expression framework is inserted into the Nonessencial region in described clay, builds the recombination mutation clay with Kan-ccdB tolerant gene expression framework; Wherein, the nucleotide sequence of described Kan-ccdB tolerant gene expression framework is as shown in SEQ ID NO:1;
2) build the entry vector pENTR1 of multiple clone site two ends with attL1 and attL2 site-specific recombination sequence, its nucleotide sequence is as shown in SEQ ID NO:2;
3) exogenous gene expression box is inserted into the entry vector pENTR1 of structure, obtain the introduction expression plasmid containing exogenous gene expression box, reacted by LR, replace the Kan-ccdB tolerant gene expression framework in recombination mutation clay with exogenous gene expression box, obtain the recombinant expressed clay inserting foreign gene at Nonessencial region.
In the present invention, preferably, described Kan-ccdB tolerant gene expression framework both sides are with attR1 and attR2 Site-specific recombinase sequence; Described multiple clone site is successively containing BglII-SalI-XbaI-NotI-EcoRI-KpnI-SmaI-SacI-HindIII-BamHI restriction endonuclease sites.
Further, present invention also offers the infectious recombinant clone system of described marek's disease virus for building the application in recombinant marek's disease virus.And
The application of the infectious recombinant clone system of described marek's disease virus in marek's disease virus live vector vaccine.
In one particular embodiment of the present invention, the present invention successfully saves acquisition 5 strain and expresses the recombinant virus of eGFP, thus in MDV vaccine strain genome, identifies 5 copy nonessential district, i.e. foreign gene insertion point, and embodiment is as follows:
1. the present invention constructs 5 recombinant cosmids successively containing marek's disease virus attenuated vaccine 814 pnca gene group 1-47873 position, 40028-79118 position, 72447-113806 position, 106337-139612 position and 129115-172541 position nucleotide sequence, called after p814-1, p814-2, p814-3, p814-4, p814-5 successively, the GeneBank accession number of wherein said marek's disease virus attenuated vaccine 814 strain whole genome sequence is JF742597.Apply this 5 recombinant cosmid transfection chick embryo fibroblast, the virus consistent with former MDV vaccine strain biological character can be saved out.
2. the present invention is to the recombinant cosmid p814-3 described in [1], p814-4 and p814-5 transforms, (the 96441-96620 position nucleotide sequence of 814 pnca gene group sequences is replaced) respectively in the UL41 gene of p814-3, (insert between 103416 of 814 pnca gene group sequences and 103417 nucleotide sequences) between UL45/UL46 gene, (insert between 120336 of 814 pnca gene group sequences and 120337 nucleotide sequences) between the UL55/LORF10 gene of p814-4, (replace the 152042-152583 position nucleotide sequence of 814 pnca gene group sequences) in the US10 gene of p814-5 and (replace the 154040-154655 position nucleotide sequence of 814 pnca gene group sequences) in US2 gene and insert resistant gene Kan-ccdB expression framework (both sides are with attR1 and attR2 Site-specific recombinase sequence), the nucleotide sequence of described Kan-ccdB tolerant gene expression framework is as shown in SEQ ID NO:1, construct 5 recombination mutation clays with the Double gene of kan-ccdB.
3. the present invention constructs the entry vector pENTR1 of multiple clone site two ends with attL1 and attL2 site-specific recombination sequence, its nucleotide sequence is as shown in SEQ ID NO:2, and this multiple clone site is successively containing BglII-SalI-XbaI-NotI-EcoRI-KpnI-SmaI-SacI-HindIII-BamHI restriction endonuclease sites.
4. construct eGFP expression plasmid, eGFP expression cassette is inserted the entry vector pENTR1 that above-mentioned [3] build, obtain eGFP introduction expression plasmid.Reacted by LR, replace the Kan-ccdB tolerant gene expression framework being inserted into each site of MDV vaccine strain genome UL41, UL45/UL46, UL55/LORF10, US10 and US2 (as described in [2]) with eGFP expression cassette respectively, construct 5 and insert in above-mentioned position the recombinant expressed clay that eGFP expresses framework respectively.
5. apply above-mentioned [4] insertion eGFP of building and express other 4 clay cotransfection chick embryo fibroblast in the 5 clay infections clone systems that the recombinant expressed clay of framework and above-mentioned [1] builds, rescue obtains respectively at 5 strain recombinant marek's disease virus of MDV vaccine strain genome UL41, UL45/UL46, UL55/LORF10, US10 and US2 each site successful expression eGFP gene, thus in MDV vaccine strain genome, identify the Nonessencial region that 5 can supply foreign gene insertion and stably express.Research shows, duplication characteristic on cell of the recombinant virus of eGFP is expressed in 5 strains and former MDV vaccine strain does not have notable difference; EGFP gene is followed successively by UL41, UL45/UL46, UL55/LORF10, US2 and US10 from high to low at the expression level in above-mentioned site.
The infectious recombinant clone system of the MDV that the present invention builds comprise 5 respectively in MDV genome UL41 gene, insert the recombination mutation clay of resistant gene Kan-ccdB expression cassette and a universal entry vector pENTR1 between UL45/UL46 gene, between UL55/LORF10 gene, in US10 gene and in US2 gene.In 5 recombination mutation clays, the two ends of Kan-ccdB expression cassette are connected with attR1 and attR2 sequence respectively; In entry vector pENTR1, the two ends of multiple clone site are connected with attL1 and attL2 sequence respectively.Exogenous gene expression box can be inserted above-mentioned 5 positions in MDV genome by application native system easily, and then carry out the rescue of recombinant virus: first, build destination gene expression box, and this expression cassette is connected with entry vector pENTR1 obtains expression plasmid of geting started; Secondly, introduction expression plasmid and corresponding kan-ccdB recombination mutation clay are carried out LR reaction, destination gene expression box can be realized to be cloned into the genomic object of MDV; Finally, by other 4 the recombinant cosmid cotransfection CEF cells in the recombination mutation clay comprising destination gene expression framework of structure and MDV infections clone system, the rescue of MDV recombinant virus can be realized.
Compared with prior art, beneficial effect of the present invention is embodied in:
(1) in MDV recombinant virus structure, the infectious recombinant clone system of the MDV that the present invention sets up has plurality of advantages.First, this process only needs the structure of two step recombinant plasmids and a step LR reaction external source goal gene can be cloned into MDV genome, compared with traditional homologous recombination method, and quickness and high efficiency more.Secondly, this process can not remain other unnecessary gene orders, makes the recombinant virus of structure have good security.What is more important, the MDV genome duplication nonessential region that the present invention screens acquisition is positioned on different recombinant cosmids, therefore apply this recombinant clone system and can insert different foreign genes respectively to corresponding region very easily, and then build the multi-joint live vector vaccine of MDV.
(2) Nonessencial region in MDV genome is the insertion point of foreign gene when building MDV recombinant virus.Research finds, insertion point has a significant impact for the expression level of foreign gene and the immune effect of MDV live vector vaccine.Therefore, the Nonessencial region in MDV genome identified and screen the immune effect contributing to improving MDV live vector vaccine.The present invention constructs the recombination mutation clay inserting foreign gene in US2 and US10 gene, finds that the expression level of foreign gene in US2 gene is higher than US10.Further, the present invention identifies three new Nonessencial regions in MDV vaccine strain genomic UL district, in UL41 gene namely, between UL45/UL46 gene and between UL55/LORF10 gene, research finds, foreign gene group is significantly higher than US2 and US10 region at this trizonal expression level.The research being more than found to be MDV live vector vaccine provides new mentality of designing.
Accompanying drawing explanation
Fig. 1 is MDV genomic DNA fragment pulsed field gel electrophoresis figure; Wherein, M, low scope PFG Marker; 1, MDV genomic dna; 2, genomic dna shears rear fragment; 3,35-48kb DNA after reclaiming;
Fig. 2 is pCC1Fos Vector map;
Fig. 3 is the HindIII restriction enzyme mapping of the malicious rMDV of rescue and former MDV vaccine strain virus genom DNA; Wherein, M1: low scope PFG Marker; M2:15kb DNA molecular amount marks; RMDV:MDV vaccine strain rescue poison; MDV: former MDV vaccine strain virus;
Fig. 4 is the structure schematic diagram of pKS KanccdB plasmid; Wherein A, pMOD-6; B, pDEST22; C, pKS KanccdB;
Fig. 5 is the structure schematic diagram of introduction expression plasmid; Wherein, A, pENTR1; B, pCAGGS-eGFP; C, pENTR1-eGFP;
Fig. 6 is MDV vaccine strain infections clone and transformation clay schematic diagram;
Fig. 7 is the cytopathy figure that recombinant virus and MDV parental virus produce on CEF cell; Wherein, rMDV, MDV vaccine strain rescue poison; MDV, former MDV vaccine strain virus; Mock, does not normally connect poison cell;
Fig. 8 is the prematurity virion subgraph that recombinant virus and MDV parental virus are formed in CEF cell; Wherein, rMDV, MDV vaccine strain rescue poison; MDV, former MDV vaccine strain virus; Mock, does not normally connect poison cell;
Fig. 9 is the expression figure of eGFP gene in recombinant virus; Wherein, A, recombinant virus fluorescence picture; B, western blotting (western blot) detected result; The comparison of C, eGFP gene expression amount in recombinant virus.RMDV, MDV vaccine strain rescue poison; Mock, does not normally connect poison cell;
Figure 10 is the growth curve chart of recombinant virus on CEF cell; Wherein, rMDV, MDV vaccine strain rescue poison; MDV, former MDV vaccine strain virus; Mock, does not normally connect poison cell;
Figure 11 is that PCR detects the stable case figure of eGFP expression cassette in recombinant virus.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The structure in embodiment 1 MDV vaccine 814 pnca gene group Fosmid library
1.1 strain
Marek's disease virus attenuated vaccine 814 strain (Zhang, F., Liu, C.J., Zhang, Y.P., et al.Comparative full-length sequence analysis of Marek's disease virus vaccine strain 814.Arch Virol.2012,157 (1): 177-183.) preserved by Harbin Veterinary Medicine Inst., China Academy of Agriculture, provide.The GeneBank accession number of described marek's disease virus attenuated vaccine 814 strain whole genome sequence is JF742597.
1.2 MDV cultivate and virus genomic extraction
Primary chick embryo fibroblast (CEF) cell is inoculated in marek's disease virus attenuated vaccine 814 strain, when cell until about viral growth to 90% produces pathology, by cell multigelation three times, collecting cell and supernatant.By cell and supernatant in the centrifugal 45min of 32000rpm, abandon supernatant; By precipitation 10mL cell permeabilization liquid (320mM sucrose, 5mM MgCl
2, 10mM Tris-HCl pH 7.5,1%Triton X-100) and resuspended, ice bath 10min; The centrifugal 45min of 32000rpm, abandons supernatant; Precipitation again used cell permeabilization liquid resuspended, ice bath 10min; The centrifugal 45min of 32000rpm, abandons supernatant; By precipitation 5mL made in nuclear buffer (10mM Tris-HCl pH7.5,2mM MgCl
26H
2o, 10% sucrose) resuspended, add 500 μ L 10 × nuclease buffer (NEB company), 50 μ L 100 × BSA (NEB company), 600U micrococcal nuclease (NEB company) and 1 μ L RNase A (100mg/ml, Qiagen), mixing, 37 DEG C of effect 1h; Add 55 μ L 0.5M EDTA, 37 DEG C of effect 5min; Add 22mL lysis buffer (100mM NaCl, 10mM Tris – HCl pH 8.0,25mM EDTA pH 8.0,0.5%SDS) and 135 μ L Proteinase K (20mg/mL, Invitrogen), 50 DEG C of effect 16h; By split product equal-volume phenol-chloroform-isoamyl alcohol extraction twice, once, the centrifugal 5min of each 8000g, gets supernatant liquid to chloroform; Add 1/10 volume 3M sodium-acetate and 2 times of volume dehydrated alcohols ,-20 DEG C of precipitation 2h; The centrifugal 15min of 15000g, abandons supernatant; Add 1mL 70% ethanol to precipitation, the centrifugal 10min of 15000g, abandons supernatant; The dissolving of air-dry rear use 100 μ L Tris-HCl (pH 7.5) will be precipitated, the concentration of the DNA extracted with spectrophotometric determination and purity.
Result obtains the genomic dna that concentration is 1200ng/ μ L, pulsed field gel electrophoresis (BioRad company CHEF
xA Pulsed Field Electrophoresis System system, condition is: electrophoretic buffer 0.5 × TBE, gum concentration 1%, program 5-220kb) show the genomic dna length extracted and be about 170kb (Fig. 1), consistent with the genome length of MDV vaccine 814 strain, show to obtain complete MDV genome.
The structure in 1.3 MDV vaccine strain genome Fosmid libraries
The structure carrying out MDV genome Fosmid library is described according to Epicentre company CopyControlTM Fosmid Library Production Kit test kit, and method is as follows:
1) shearing of MDV genomic dna and end modified
The MDV genome of extraction 200 μ L suction pipette heads are aspirated 50-100 time repeatedly, is analyzed by pulsed field gel electrophoresis, to its fragment length between 30-50kb (Fig. 1).DNA End-Repair Enzyme Mix (Epicentre company) after shearing is carried out flat end and 5 ' phosphorylation modification: mix 5 μ L 10X End-Repair Buffer on ice, 5 μ L 2.5mM dNTP Mix, 5 μ L 10mM ATP, 2 μ L End-Repair Enzyme Mix, DNA after 5 μ g shear, aqua sterilisa supplies 50 μ L; Room temperature effect 45min, 70 DEG C of effect 10min.
Get end modified after DNA carry out pulsed field gel electrophoresis, condition is described above.Cut and comprise the blob of viscose that size is 35-48kb genomic DNA fragment.Join in blob of viscose by 10 × gelase damping fluid, 70 DEG C of effect 15min make blob of viscose melt completely; Add β-gelase I (1U/100mg blob of viscose, NEB company), 42 DEG C of water-bath effect 2h; 70 DEG C of effect 15min deactivation β-gelase I; Add 1/10 volume 3M sodium-acetate, the centrifugal 15min of ice bath 15min, 12000g, gets supernatant; Add 2 times of volume dehydrated alcohols, hatch the centrifugal 15min of 15000g after 2h, abandon supernatant for-20 DEG C; Dissolve with 20 μ L aqua sterilisas after precipitation washes 1 time with 70% ethanol.Result successfully reclaims the DNA fragmentation (Fig. 1) obtained between 35-48kb.
2) connect and pack
Fetch DNA and pCC1Fos carrier (Fig. 2) after receiving to connect, reaction system is: 1 μ l 10X Fast-Link Ligation Buffer, 0.5 μ l 10mM ATP, 1 μ l CopyControl pCC1FOS (0.5 μ g/ μ l), DNA (40ng/ μ l), 1 μ l Fast-Link DNA Ligase after 6.5 μ l reclaim.Room temperature connects 16-18h, 70 DEG C of effect 15min deactivation ligase enzymes.
Product and 25 μ L will be connected pack reagent (MaxPlax Lambda Packaging Extracts, Epicentre company) and mix, hatch 2h for 30 DEG C; Again add in reaction system 25 μ L pack reagent, 30 DEG C hatch 2h after with phage dilution buffer (10mM Tris-HCl pH 8.3,100mM NaCl, 100mM NaCl) supply 1ml; Add 25 μ L chloroforms, mix gently, 4 DEG C of preservations.After packaged mixed solution 10 times of gradient dilutions, get 10 μ L respectively and mix with 100 μ L intestinal bacteria EPI300-T1R, hatch 1h for 37 DEG C.Bacterium liquid is coated overnight incubation on the LB flat board containing 12.5 μ g/ml paraxin, statistics colony counts, calculate the titre (cfu/ml) of cosmid library, result is 1.8 × 10
6cfu/ml.
3) qualification of recombinant cosmid and order-checking
Picking 300 clone from culture plate, alkaline lysis method of extracting clay, uses primer pCC1F:5 '-GGATGTGCTGCAAGGCGATTAAGTTGG-3 ' and pCC1R:5 '-CTCGTATGTTGTGTGGAATTG TGAGC-3 ' to carry out gene order-checking to recombinant cosmid.Result obtains the recombinant cosmid that 273 clones have MDV gene fragment altogether, and Insert Fragment length is between 32-46kb.Calculate with each Insert Fragment 30kb, these 273 clones are 47.6 (273 × 30kb/172kb) to the genomic fraction of coverage of MDV 814 vaccine strain, show that it is enough to cover MDV full-length genome.Above result display, the success of MDV vaccine strain genome Fosmid library construction.
Embodiment 2 virus rescue
2.1 virus rescue
1) for the selection of the clay of Revive virus: according to cosmid end sequencing analysis, 6 group of 5 clay combination is therefrom chosen.5 clays in each combination have all cloned MDV vaccine 814 pnca gene group DNA fragmentation, containing overlapped region and the complete MDV genome of sliceable covering.
2) extraction of recombinant cosmid and linearizing: the cosmid DNA selected by extracting with extraction reagent kit in QIAGEN company.With NotI (NEB company), linearization process is carried out to the clay extracted: NotI restriction endonuclease 100U, clay 10 μ g, 37 DEG C of effect 2h.Digestion products equal-volume phenol-chloroform-isoamyl alcohol extraction twice, once, the centrifugal 5min of each 8000rpm, gets supernatant liquid to chloroform; Add 1/10 volume 3M sodium-acetate and 2 times of volume dehydrated alcohols ,-20 DEG C of precipitation 2h; The centrifugal 15min of 15000g, abandons supernatant; Add 1mL 70% ethanol to precipitation, the centrifugal 10min of 15000g, abandons supernatant; The dissolving of air-dry rear use 40 μ L TE damping fluid (10mM Tris-HCl, 1mM EDTA, pH7.5) will be precipitated.
3) preparation of CEF cell: adopt calcium phosphate transfection method by secondary for 5 sections of MDV genomic DNA fragment cotransfections for CEF cell.The preparation method of CEF cell is as follows: get 9-10 age in days SPF chicken embryo, aseptic taking-up chicken embryo, is placed in the plate filling Hank ' s liquid (purchased from HyClone) and washs, and removes head, four limbs and internal organ, shreds with scissors; Wash twice with Hank ' s liquid, add the pancreatin (4mL/ embryo) of 0.25%, hatch 10min for 37 DEG C; Abandon most pancreatin, add containing 5%FBS, 1% dual anti-DMEM nutrient solution (purchased from HyClone), piping and druming makes cell dispersal repeatedly.8 × 10 are made with after 6 layers of filtered through gauze
5the cell suspension of cell/mL, is sub-packed in Tissue Culture Flask and cultivates in 37 DEG C of incubators.
4) calcium phosphate transfection: 1. preparation is secondary to CEF cell: the primary CEF cells trysinization of cultivating 16-18h got off.By cell with containing 10%FBS, washing twice without dual anti-DMEM, the centrifugal 5min of each 500g room temperature.Cell is resuspended in the DMEM complete culture solution of 1.5 times of volumes, with being dispensed in 60mm culture dish (4 × 10 after 6 layers of filtered through gauze
6cell/ware).2. calcium phospate-DNA precipitate is prepared: the preparation carrying out calcium phospate-DNA precipitate while preparation is time for CEF cell.388 μ l aqua sterilisas, 50 μ l DNA (comprising the MDV genomic DNA fragment that 5 concentration are 200ng/ μ l, the i.e. 2 μ g of each fragment 10 μ l), 62 μ l 2M CaCl are mixed in 1.5ml EP pipe
2; 500 μ l 2 × HBS damping fluid (1.5mM Na are added in another 1.5ml EP pipe
2hPO
4, 10mM KC1,280mM NaCl, 12mM sucrose, 50mM HEPES pH 7.0); By CaCl
2-DNA mixed solution dropwise slowly joins in 2 × HBS damping fluid, then makes it mix at pipe bottom blowing 5-6 bubble; Incubated at room 30min.3. the calcium phospate-DNA precipitate of preparation is dropwise joined the secondary on CEF cell of preparation, slight mixing is placed in 37 DEG C of incubators cultivates.4. suffer a shock: after transfection 4h, discard cell conditioned medium, with DMEM, cell is washed one time; Add 2ml glycerol shock liquid (15% glycerine, 1 × HBS), incubated at room 2min; After washing 3 times with DMEM, the DMEM complete culture solution added containing 10%FBS is cultivated.5. change liquid: after shock 12h, cell culture fluid is changed to and continues to cultivate containing 3%FBS, 1% dual anti-DMEM.
After transfection 4-6 days, the production of observation of cell pathology.Result shows, all can occur MDV typical cells pathology after 6 groups of selected clay combination transfection CEF cells.Choose one group of clay combination that repeatability is best, carry out follow-up test.The DNA fragmentation that inserts of 5 recombinant cosmids in the combination of this clay is respectively the 1-47873 position of (the GenBank JF742597) of MDV vaccine 814 pnca gene group, 40028-79118 position, 72447-113806 position, 106337-139612 position and 129115-172541 position (Fig. 6 A-B), 5 recombinant cosmids called after p814-1, p814-2, p814-3, p814-4, p814-5 respectively.The viral nomenclature this clay combination rescue obtained is rMDV.
The qualification of 2.2 Revive virus
Identify from cytopathy, electron microscopic observation, restriction enzyme mapping and order-checking four aspects to rescue poison.
1) cytopathy: rMDV and former MDV vaccine strain virus are inoculated CEF cell respectively, cultivates the plaque situation that after 4-6 days, observation and comparison virus produces on cells infected.Find the viral no significant difference (Fig. 7) of the plaque form that rMDV produces on CEF cell and size and former MDV vaccine strain.
2) electron microscopic observation: save in malicious rMDV cells infected whether there is typical MDV virus particle with transmission electron microscope observing.Found that, visible a large amount of typical MDV virus particle (majority is exposed prematurity virus) in rMDV cells infected, the virus particle no significant difference (Fig. 8) formed with former MDV vaccine strain virus.
3) restriction enzyme mapping: extract rescue malicious rMDV and former MDV vaccine strain virus genom DNA, carries out after enzyme cuts, analyzing its restriction enzyme mapping with pulsed field gel electrophoresis with HindIII (purchased from NEB company).Result shows, saves the genomic physical map of malicious rMDV and provirus MDV completely the same (Fig. 3).
4) check order: gene order-checking is carried out to this group 5 recombinant cosmids, and compared with former MDV vaccine strain virus genome sequence.Find that gene order and the provirus of Revive virus rMDV are completely the same.
The mensuration of the 2.3 malicious rMDV of rescue and former MDV vaccine strain viral growth curves
The malicious rMDV of rescue and former MDV vaccine strain virus are incubated at the CEF cell in 6 orifice plates with 100PFU dose inoculation, detect virus titer every 24h sampling, until 144h after infecting, draw viral growth curves.Result shows, the growth curve saving malicious rMDV and former MDV does not have notable difference (Figure 10), illustrates that the virus gone out with above-mentioned infections clone rescuing system has identical duplication characteristic with former MDV virus on CEF.Above result shows, MDV vaccine 814 strain multiple clips infections clone rescue system is successfully established.
The sudden change of embodiment 3 recombinant cosmid
Based on the above-mentioned 5 clay infections clone platforms set up, respectively in p814-3 between the genomic UL41 gene internal of MDV, UL45 and UL46 gene, in p814-4 between genomic UL55 and the LORF10 gene of MDV, in p814-5, the genomic US10 gene internal of MDV, US2 gene internal insert eGFP and express framework, build 5 recombination mutation clay p814-3UL41eGFP, p814-3UL45/46eGFP, p814-4UL55/10eGFP, p814-5US10eGFP, p814-5US2eGFP (its forming types can participate in Fig. 6 H-L).Process is summarized as follows:
3.1 the structure of pKS KanccdB
Respectively multiplexed PCR amplification is carried out to pDEST22 plasmid (Invitrogen company), pMOD6 plasmid (Epicentre company) with pair primer of 3 shown in table 1.
The PCR primer of framework expressed by table 1 for cloning Kan-ccdB
| Primer | Sequence (5 '-3 ') |
| R1F | GCG TCTAGAGATGATGAAGATACCCCACCA(XbaI) |
| R1R | GTGTGCGTCGGGTGATGCTGCCAA |
| P6KanF | TTGGCAGCATCACCCGACGCACACATCTCAACCATCATCG |
| P6KanR | ATCTGGCTTTTAGTAAGCCGGATCCACCGAGCTCGAATTCGATGAA |
| ccdBR2F | GGATCCGGCTTACTAAAAGCCAGAT |
| ccdBR2R | GCG AAGCTTCGGCCATCAAACCACTTTGTACAAG(HindIII) |
Its building process is summarized as follows: 1) with R1F and R1R for primer, from pDEST22 amplification obtain attR1 sequence, reaction conditions is: 95 DEG C of 5min, 35 × (94 DEG C of 30s, 65 DEG C of 30s, 72 DEG C of 30s), 72 DEG C of 10min.2) with P6KanF and p6KanR for primer, from pMOD6 amplification obtain kalamycin resistance gene (Kan), reaction conditions is: 95 DEG C of 5min, 35 × (94 DEG C of 30s, 66 DEG C of 30s, 72 DEG C of 1min), 72 DEG C of 10min.3) with ccdBR2F and ccdBR2R for primer, from pDEST22 amplification obtain ccdB-attR2 gene, reaction conditions is: 95 DEG C of 5min, 35 × (94 DEG C of 30s, 64 DEG C of 30s, 72 DEG C of 1min), 72 DEG C of 10min.4) difference purifying 3 DNA fragmentations obtained above, and with these 3 fragments for template, with R1F and ccdBR2R for primer, amplification obtains Kan-ccdB and expresses framework, and its nucleotide sequence is as shown in SEQ ID NO:1, and reaction conditions is: 95 DEG C of 5min, 35 × (94 DEG C of 30s, 66 DEG C of 30s, 72 DEG C of 2min), 72 DEG C of 10min.
The Kan-ccdB obtained is expressed framework utilizes XbaI and HindIII restriction enzyme site to be cloned into pBluescript II KS (+) carrier, obtains pKS KanccdB, as shown in Figure 4.
3.2 with the structure of the recombination mutation clay of Kan-ccdB tolerant gene expression framework
Increase with the Kan-ccdB expression framework of homologous recombination arm from the plasmid pKS KanccdB of above-mentioned structure with the primer with corresponding homology arm shown in table 2, its PCR reaction conditions is: 95 DEG C of 5min, 35 × (94 DEG C of 30s, 66 DEG C of 30s, 72 DEG C of 2min), 72 DEG C of 10min.With the Counter-Selection BAC Modification Kit test kit of Gene Bridges company, increased fragment is cloned into the corresponding position of corresponding clay.Concrete, Kan-ccdB being expressed framework to insert in the UL41 gene that p814-3 comprises between (replacing the 96441-96620 position nucleotide sequence of 814 pnca gene group sequences) and UL45 and UL46 gene (inserting between 103416 of 814 pnca gene group sequences and 103417 nucleotide sequences) respectively, (insert between 120336 of 814 pnca gene group sequences and 120337 nucleotide sequences) between UL55 and the LORF10 gene in p814-4, (replace the 152042-152583 position nucleotide sequence of 814 pnca gene group sequences) in US10 gene in p814-5 and (replace the 154040-154655 position nucleotide sequence of 814 pnca gene group sequences) in US2 gene, obtain recombination mutation clay p814-3UL41KanccdB, p814-3UL45/46KanccdB, p814-4UL55/10KanccdB, p814-5US10 KanccdB, p814-5US2 KanccdB, as shown in Fig. 6 C-G.
Table 2 increases with the PCR primer (underscore part is homologous recombination arm) of the Kan-ccdB expression framework of restructuring arm
3.3 the structure of pENTR1 entry vector
For ease of inserting exogenous gene expression framework in MDV genome, this research is by the gus gene elmination in pENTR-gus plasmid (Invitrogen company), replace with BglII-SalI-XbaI-NotI-EcoRI-KpnI-SmaI-SacI-HindIII-BamHI ten restriction enzyme sites, process is as follows: with ENTR1F and ENTR1R for primer (table 3), take pENTR-gus as template, carry out pcr amplification, reaction conditions is: 95 DEG C of 5min, 35 × (94 DEG C of 30s, 65 DEG C of 30s, 72 DEG C of 2min15s), 72 DEG C of 10min.PCR primer EcoR1 (NEB company) enzyme after purifying is cut.After digestion products purifying, carry out self connecting, transformation of E. coli with T4DNA ligase enzyme (Thermo), obtain entry vector pENTR1 (as shown in Figure 5A), its nucleotide sequence is as shown in SEQ ID NO:2.
Table 3 is for building the PCR primer of pENTR1
The structure of 3.4 pCAGGS-eGFP expression plasmids
Obtain eGFP gene, total length 726bp with primer eGFPF and eGFPR shown in table 4 from pEGFP-C1 carrier (Clontech company) amplification, reaction conditions is 95 DEG C of 5min, 35 × (94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min), 72 DEG C of 10min.Cut by PCR primer EcoR1 after purifying and Cla1 (NEB company) enzyme, be connected into pCAGGS carrier, successfully build pCAGGS-eGFP, as shown in Figure 5 B, its nucleotide sequence is as shown in SEQ ID NO:3.
Table 4 is for building the PCR primer of pCAGGS-eGFP
| Primer | Sequence (5 '-3 ') |
| eGFPF | TTT GAATTCGCCACCATGGTGAGCAAGGGC(EcoRI) |
| eGFPR | TTT ATCGATTTACTTGTACAGCTCGTCCATG(ClaI) |
The structure of 3.5 eGFP introduction expression plasmid pENTR1-eGFP
EGFP expression plasmid SalI and HindIII (NEB company) of above-mentioned structure is carried out double digestion, and obtain eGFP after digestion products reclaims and express framework, its nucleotide sequence is as shown in SEQ ID NO:4.The eGFP of acquisition is expressed framework and is cloned into pENTR1 entry vector by SalI and HindIII restriction enzyme site, obtain eGFP introduction expression plasmid pENTR1-eGFP (as shown in Figure 5 C), its nucleotide sequence is as shown in SEQ ID NO:5.
3.6 contain the structure that eGFP expresses the recombination mutation clay of framework
Get the above-mentioned 3.5 eGFP introduction expression plasmid pENTR1-eGFP built, the recombination mutation clay p814-3UL41KanccdB containing Kan-ccdB tolerant gene expression framework built with above-mentioned 3.2 respectively, p814-3UL45/46KanccdB, p814-4UL55/10KanccdB, p814-5US10KanccdB, p814-5US2KanccdB carries out LR reaction, the eGFP that Kan-ccdB in above-mentioned recombination mutation clay expression framework is replaced with in pENTR1-eGFP plasmid expresses framework, thus obtain respectively in the genomic UL41 gene of MDV, between UL45/UL46 gene, between UL55/LORF10 gene, the recombination mutation clay p814-3UL41eGFP that eGFP expresses framework is inserted in US10 gene and in US2 gene, p814-3UL45/46eGFP, p814-4UL55/10eGFP, p814-5US10eGFP, p814-5US2eGFP (its forming types is as shown in Fig. 6 C-G).
Detailed process is: mixed with the Kan-ccdB recombination mutation clay of 150ng respectively by the introduction expression plasmid pENTR1-eGFP of 100ng, add water to 8 μ L afterwards, add 2 μ L's
lR Clonase
tMiI Enzyme Mix, mixes rear 25 DEG C of effect 1h, adds 1 μ L Proteinase K in 37 DEG C of effect 10min termination reactions.Get 1 μ L transformation of E. coli EPI300 afterwards, PCR is order-checking qualification positive colony also.
The rescue of embodiment 4 recombinant virus and analysis
The rescue of 4.1 recombinant viruses
The recombination mutation cosmid DNA of above-mentioned acquisition is extracted with extraction reagent kit in QIAGEN company.With NotI (purchased from NEB company) by clay linearization process used, after the extracting of phenol chloroform isoamyl alcohol and alcohol settling, prepare transfection DNA.The preparation of CEF cell is carried out with reference to method described in embodiment 2, use one of p814-3UL41eGFP, p814-3UL45/46eGFP, p814-4UL55/10eGFP, p814-5US10eGFP, p814-5US2eGFP five recombination mutation clays and other 4 the clay cotransfections in the MDV infections clone system of above-mentioned structure secondary to CEF cell respectively, calcium phosphate transfection method as described in Example 2.Turn then 4-7 days visible CEF cells occur MDV typical cells pathology and can be observed the expression (as shown in Figure 9 A) of eGFP.Save out the recombinant mdv vaccine strain that eGFP is expressed in five strains respectively, called after successively: r814UL41eGFP, r814UL45/46eGFP, r814UL55/10eGFP, r814US10eGFP, r814US2eGFP.
The qualification of 4.2 recombinant viruses
Above-mentioned five strain recombinant viruses and former MDV vaccine strain virus are inoculated CEF cell respectively, cultivates the plaque situation that after 4-6 days, observation and comparison virus produces on cells infected.Find the viral no significant difference (Fig. 7) of the plaque form that recombinant virus produces on CEF cell and size and former MDV vaccine strain.
Typical MDV virus particle whether is there is with in the above-mentioned five strain recombinant virus-infected cells of transmission electron microscope observing.Found that, all visible a large amount of typical MDV virus particle (majority is exposed prematurity virus) in five strain recombinant virus-infected cells, the virus particle no significant difference (Fig. 8) formed with former MDV vaccine strain virus.
Extract above-mentioned five strain recombinant virus genomes DNA, with pCAGGS carrier upstream sequencing primer 1596U for upstream primer, with homology arm primer corresponding in table 5 for downstream primer, respectively PCR and order-checking qualification are carried out to recombinant virus, occur there are no sudden change.
Table 5 is for the PCR primer of Identification of recombinant baculovirus
| Primer | Sequence (5 '-3 ') |
| 1596U | TTCGGCTTCTGGCGTGTGACCGGC |
| UL41L | TCACATCCCATTAATATCAAATCTGTGTCCGAAGACAATACATATGCGAC |
| UL4546R | GATTATTAAAGAAATAAAGAACCGCTTTAAGAATAGTGTTTATTTTTGTG |
| UL5510L | CTTACTCGATAACAGTTACGTATTATATGTCAATTTTATGATAACAATCTG |
| US10R | TTATAAGTAGGATTCCCCGTCTCCTGTTGGCGATTCCCGAAGATTTGTCA |
| US2R | TGGGTGTGCCCATAATCGCCAGAGCTGCAGACCTATTCCGTTTTGCCAA |
The eGFP expression of 4.3 recombinant viruses
The five strain recombinant viruses above-mentioned rescue obtained and former MDV vaccine strain virus, with the CEF cell of 100PFU inoculation culture in six orifice plates, collect 3 cells infected in hole every 24h, until 120h after infecting.Every hole adds 500 μ L cell pyrolysis liquids (purchased from green skies company), eGFP in 4 DEG C of cracking 30min release cells, the relative expression levels of every hole eGFP is detected with Microplate Fluorescence Reader (exciting light 485nm, absorb light 528nm).As shown in Figure 9 A, in r814UL41eGFP cells infected, the expression amount of eGFP is the highest for result; Secondly be r814UL45/46eGFP and r814UL55/10eGFP, the two is without significant difference; The expression amount of above-mentioned three strain recombinant virus eGFP is all significantly higher than r814US2eGFP; The expression amount of eGFP minimum (Fig. 9 C) in r814US10eGFP cells infected.
The lysis supernatant of 120h after results infect, carries out western blotting (Western blot) and detects.Process is summarized as follows: by 5 × SDS-PAGE sample-loading buffer (the green skies company) process of lysis gained supernatant, with 10%SDS-PAGE separation gel electrophoresis, condition is 80V 30min, 120V 1.5h.By albumin glue transferring film immersion bubble 30min, with half-dried transferring film instrument (Bio-Rad company), albumen is gone to NC film, condition is 15V 1h.After transferring film completes, film is placed in 5% skimming milk and spends the night in room temperature is closed.After film PBST is washed three times, with eGFP monoclonal antibody incubated at room 1.5h, same method washes three times.Anti-(KPL company) incubated at room 1h, the PBST of against murine two film being placed in DyLight-800 mark washs 5 times, observes with Licor company near-infrared fluorescent scanning imaging system (Licor ODYSSEY).Result shows, and five strain recombinant viruses all can successful expression eGFP, and target protein size is about 27kDa, and conform to expection (as Fig. 9 B).EGFP expression amount conforms to fluorescent value detected result.
The growth curve titration of 4.4 recombinant viruses
Above-mentioned five strain recombinant viruses and former MDV vaccine strain virus are incubated at the CEF cell in 6 orifice plates with 100PFU dose inoculation, detect virus titer every 24h sampling, until 144h after infecting, draw viral growth curves.Result shows, the growth curve of five strain recombinant viruses and former MDV does not have notable difference (Figure 10), and 120h reaches the highest titre all after infection.When the highest titre, the growth titre (log10PFU/ml) of MDV, r814UL41eGFP, r81445/46eGFP, r814UL55/10eGFP, r814US10eGFP and r814US2eGFP is respectively 4.95,4.94,4.92,4.97,4.93 and 4.96.
The genetic stability of 4.5 recombinant viruses detects
Above-mentioned five strain recombinant viruses were passed for 20 generations continuously in CEF, extracts virus genom DNA, carry out PCR and order-checking qualification with primer shown in table 5, there are no sudden change or disappearance, (Figure 11) occurs.Five strain recombinant viruses all can express eGFP, cells infected visible green fluorescence, illustrate that recombinant virus has good genetic stability.
Claims (10)
1. the infectious recombinant clone system of marek's disease virus, it is characterized in that, described system comprises multiple clay, in each clay, clone has Marek's disease virus vaccine pnca gene fragment, described Marek's disease virus vaccine pnca gene fragment contains overlapped region and sliceable covering complete Marek's disease virus vaccine pnca gene group, is inserted with foreign gene in the Nonessencial region of wherein clay described at least one.
2. the infectious recombinant clone system of marek's disease virus according to claim 1, it is characterized in that, described clay is pCC1Fos.
3. the infectious recombinant clone system of marek's disease virus according to claim 1, it is characterized in that, described marek's disease virus is marek's disease virus attenuated vaccine 814 strain.
4. the infectious recombinant clone system of marek's disease virus according to claim 1, it is characterized in that, described clay is 5 clays, 5 clays are respectively p814-1, p814-2, p814-3, p814-4, p814-5, and the sequence of the Marek's disease virus vaccine pnca gene fragment of wherein cloning is respectively the 1-47873 position of marek's disease virus 814 strain, 40028-79118 position, 72447-113806 position, 106337-139612 position and 129115-172541 position.
5. the infectious recombinant clone system of marek's disease virus according to claim 1, it is characterized in that, described Nonessencial region is arranged in the UL41 gene of p814-3, between UL45 and UL46 gene in p814-3, between UL55 and LORF10 gene in p814-4, in the US2 gene in the US10 gene in p814-5 and in p814-5.
6. build the method for the infectious recombinant clone system of marek's disease virus described in any one of claim 1-5, it is characterized in that, comprise the following steps:
(1) cut off the DNA of described marek's disease virus, and the DNA fragmentation of the marek's disease virus after cut-out is connected on clay;
(2) multiple clay is selected to form the infectious recombinant clone system of described marek's disease virus, wherein in each clay, clone has Marek's disease virus vaccine pnca gene fragment, and described Marek's disease virus vaccine pnca gene fragment contains overlapped region and sliceable covering complete Marek's disease virus vaccine pnca gene group;
(3) foreign gene is inserted the Nonessencial region in clay described at least one.
7. method according to claim 6, is characterized in that, the concrete steps in step (3), foreign gene being inserted the Nonessencial region in described clay are:
1) Kan-ccdB tolerant gene expression framework is inserted into the Nonessencial region in described clay, builds the recombination mutation clay with Kan-ccdB tolerant gene expression framework; Wherein, the nucleotide sequence of described Kan-ccdB tolerant gene expression framework is as shown in SEQ ID NO:1;
2) build the entry vector pENTR1 of multiple clone site two ends with attL1 and attL2 site-specific recombination sequence, its nucleotide sequence is as shown in SEQ ID NO:2;
3) exogenous gene expression box is inserted into the entry vector pENTR1 of structure, obtain the introduction expression plasmid containing exogenous gene expression box, reacted by LR, replace the Kan-ccdB tolerant gene expression framework in recombination mutation clay with exogenous gene expression box, obtain the recombinant expressed clay inserting foreign gene at Nonessencial region.
8. method according to claim 7, is characterized in that, described Kan-ccdB tolerant gene expression framework both sides are with attR1 and attR2 Site-specific recombinase sequence; Described multiple clone site is successively containing BglII-SalI-XbaI-NotI-EcoRI-KpnI-SmaI-SacI-HindIII-BamHI restriction endonuclease sites.
9. the infectious recombinant clone system of the marek's disease virus described in any one of claim 1-5 is for building the application in recombinant marek's disease virus.
10. the application of the infectious recombinant clone system of the marek's disease virus described in any one of claim 1-5 in marek's disease virus live vector vaccine.
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