CN101372684A - Method for easily and efficiently constructing recombinant baculovirus - Google Patents

Abstract

The invention discloses a method for conveniently and efficiently constructing a recombinant baculovirus. The method mainly comprises the following steps: a donor plasmid which contains dual-promoter and can directionally and rapidly clone PCR product is constructed; (2) an I-SceI cassette is introduced into the genome of E.coliDH10B, simultaneously two I-Scel sites and a resistance gene are introduced into the baculovirus Bacmid, and the plasmid which can express Red-Gam recombinase is transformed into E.coliDH10B to construct a new receptor strain CTRDH10B/Bacmid/PML104; (3) the recombined donor plasmid is introduced into the CTRDH10B/Bacmid/pML104 to induce the expression of I-SceI endonuclease and the Red-Gam recombinase, and the recombinant Bacmid is obtained by homologous recombination in the bacterium; (4) the recombinant Bacmid DNA transfection insect cells are prepared by the conventional method and analyzed. The method needs no complex PCR verification and fussy plaque purification, and is really efficient, rapid and convenient. The method is especially suitable for constructing high-quality baculovirus cDNA library with large storage capacity.

Description

A kind of method of easily and efficiently constructing recombinant baculovirus

Technical field

The invention belongs to genetically engineered and protein engineering field, specifically is a kind of method of easily and efficiently constructing recombinant baculovirus.

Background technology

Smith etc. (1983) make carrier efficiently expresses the human in fall army worm cell Sf-21 achievement in research with lucerne place three-spotted phytometra many embedding types nuclear polyhedrosis virus (AcMNPV, Autographa californica multiple nucleocapsid nucleopolyhedrovirus) and have opened up the frontier of baculovirus as the vector expression foreign gene.Along with the arrival of genome times afterwards comprehensively, express various eucaryons source cDNA and the functional protein field is full of challenge and opportunity at fast high-flux.To drive that foreign gene efficiently expresses in insect cell, insect cell is cultivated convenient, virus is simple to operate, clone's external source fragment ability big (30-50kb), expression product have characteristics such as correct post-treatment to people and mammalian safe, superpower polyhedron promotor (Ppolh) because have, baculovirus expression system (BES, Baculovirus ExpressonSystem) has become one of outstanding expression system of expressing eukaryotic gene.At present, utilize the source of baculovirus expression system expression alien gene to spread all over various biologies such as virus, bacterium, fungi, animal and plant, its range of application relates to the fundamental research of interaction aspect between protein structure and function, the protein-protein and improvement of production of vaccine, medical diagnosis on disease and viral pesticide etc.Baculovirus is because genome is huge, the clone of foreign gene can not bacteria-like or yeast vector equally cut ways of connecting and directly insert by enzyme, and must be by the mediation of transfer vector.Therefore how efficiently making up recombinant baculovirus apace becomes one step of most critical of utilizing the baculovirus expression foreign gene.Making up at present the recombinant baculovirus method has homologous recombination in traditional insect cell, and swivel base is the Bac-to-Bac system in the bacterium, and direct three kinds of modes such as connection.The homologous recombination method mainly obtains with the screening of many wheel plaque purifying by transfer vector and the wild-type virus DNA homologous recombination in insect cell that will carry foreign gene in the insect cell.The homologous recombination method is lower in traditional insect cell, and even recombination efficiency behind the linearized vector, still can not surpass 25% between 0.1%～1%, needs many wheel plaque purifying, and time and effort consuming seldom uses now.Directly method of attachment, owing to do not need transfer vector, as if rapidly simple, true really not so.Because it is impossible the larger vector about 130kb will being used rare enzyme to carry out abundant complete degestion, and external source fragment and 130kb carrier joint efficiency are very low, therefore cause the background of this method higher, construction of recombinant virus efficient is low, still need loaded down with trivial details plaque purifying, so seldom there is the investigator to use this method construction of recombinant virus to carry out protein expression.For fear of the recombinant virus plaque purge process that wastes time and energy, it is the Bac-to-Bac system that Luckow etc. (1993) have developed the interior reorganization of bacterium, and it is the AcMNPV genomic dna to be transformed into can duplicate in bacterium also and can swivel base take place in bacterium and simultaneously insect cell is kept infective large-scale shuttle vectors with donor plasmid in essence.Its swivel base principle is based on the single-minded site transposon system of Tn7 transposon, it is by being transformed into baculovirus DNA in the Bacmid carrier that can duplicate in coli strain, promptly in the baculovirus genome, contain the F-factor replicon that can duplicate intestinal bacteria, kalamycin resistance gene, Tn7 swivel base contact site and lacZ ' box structure, because the baculovirus genome is the annular closed double chain DNA molecule, therefore this Bacmid can the picture element grain is the same duplicates with low copy form in intestinal bacteria.And in transfer vector the external source gene position under the driving of polyhedron promotor, two ends are respectively the left side of Tn7 transposon, right-hand member swivel base sequence, after recombinant transfer vector being transformed in the intestinal bacteria that contain Bacmid, under the mediation of the transposase that helper plasmid provides, carry out swivel base, to contain expression of exogenous gene box structure swivel base on the recombinant transfer vector in lacZ ' box structure of Bacmid, destroy the α complementation, therefore recombinant virus can be by simple blue hickie method screening, and isolating recombinant virus dna direct transfection insect cell promptly can obtain infective recombinant virus from white colony.This single-minded site swivel base method only needs a step separation and purification and amplification recombinant virus dna, and virus titer just can reach 107pfu/ml, and whole process only needs 7～10 days.

At present most popular is the Bac-to-Bac system, though this system need not carry out homologous recombination in insect cell, but swivel base efficient neither be very high in bacterium, common only about 10% hickie, and the hickie bacterium colony needs further streak culture and PCR verifies to remove false positive, complexity more consuming time.Existing method can satisfy substantially and making up single or a small amount of recombinant virus, in case when needing to make up dozens of even more recombinant virus mass expressing external gene then efficient is too low.And because the efficient of acquisition recombinant virus is less than 90%, the baculovirus cDNA library that improper structure high quality storage capacity is big.

Summary of the invention

The method that the purpose of this invention is to provide a kind of easily and efficiently constructing recombinant baculovirus, can improve the efficient that obtains recombinant baculovirus greatly, avoid complicated PCR checking and loaded down with trivial details plaque purifying, really accomplish efficiently, make up quickly and easily recombinant baculovirus.

The method of a kind of easily and efficiently constructing recombinant baculovirus of the present invention realizes by following step: (1) makes up and contains double-promoter, the donor plasmid of the directed quick clone PCR product of energy; (2) the I-SceI expression cassette of pectinose promotor (pBAD) control is recombinated in the genome of intestinal bacteria E.coliDH10B, simultaneously baculovirus Bacmid is introduced in 2 I-SceI sites and a resistant gene, and can transform by the plasmid of IPTG abduction delivering Red-Gam recombinase among the E.coliDH10B, make up the F-strain CTRDH10B/Bacmid/pML104 that makes new advances; (3) donor plasmid that will carry foreign gene imports and to contain in the acceptor bacterium of Bacmid, induce the expression of playback enzyme I-SceI then through pectinose, the I-SceI enzyme cuts donor plasmid and acceptor Bacmid in recipient bacterium, make its linearizing to improve homologous recombination efficiency and to reduce parent Bacmid background, after IPTG abduction delivering Red-Gam recombinase, homologous recombination obtains reorganization Bacmid in bacterium; (4) ordinary method preparation reorganization Bacmid DNA, transfection insect cell is gathered in the crops the recombinant virus particle and is carried out protein expression and purity analysis after 3～5 days.

Described donor plasmid contains reporter gene, and reporter gene will need expressed exogenous gene to be cloned in the polyhedron promotor downstream (MSC1) by the p10 promoters driven.

Theoretical foundation of the present invention: can realize the transmission of DNA between bacterium easily in conjunction with shifting between bacterium, in conjunction with the reason of transfer from a kind of big plasmid (100kb) that is called F-factor.By F-factor is carried out suitable transformation, disappearance is fallen its DNA zone (oriT) of being responsible for shifting, contain in the goal gene transfer vector at another and to introduce oriT, F-factor is owing to self can't shift because lacking oriT like this, and the transfer vector that the metastasis related protein of its coding just can help to contain oriT is transferred among the acceptor bacterium.In case transfer vector enters in the middle of the acceptor bacterium, can express the playback enzyme with the carrier linearizing by inducing acceptor bacterium, the express recombinant enzyme is with in the middle of the acceptor carrier that it is suitable that the purpose fragment is recombinated simultaneously.Maize Li etc. utilizes this method to make up cover intestinal bacteria and a Yeast expression carrier and a mammalian expression vector, because its employed position high copy number plasmid, reach 100 copies in each cell, therefore also having nearly 10% background, recombination efficiency is about 90%.Baculovirus vector is because its genome is very big, be tens times of conventional plasmid, Bacmid be single plasmid, i.e. 1～2 copy/bacterial cell of copying, therefore most suitable use is in conjunction with shifting and the interior homologous recombination method structure of bacterium recombinant baculovirus, and its reorganization background almost can be ignored.The present invention imports in the acceptor bacterium that contains Bacmid easily by the transfer vector that will carry foreign gene in conjunction with tranfer system between bacterium, expression linearizing donor carrier and acceptor Bacmid by derivable I-sceI playback enzyme, and homologous recombination obtains reorganization Bacmid in the efficient bacterium of derivable Red-Gam mediation, thereby obtains the novel method of recombinant baculovirus.

Beneficial effect:

Can realize the transmission of DNA between bacterium easily in conjunction with shifting between bacterium, after transfer vector enters in the acceptor bacterium, by inducing acceptor bacterium to express the playback enzyme with the carrier linearizing, the express recombinant enzyme is with in the middle of the acceptor carrier that it is suitable that the purpose fragment is recombinated simultaneously.The present invention utilizes between bacterium and makes up recombinant baculovirus in conjunction with transfer and the interior efficiently mode of homologous recombination of bacterium, only need two kinds of bacteriums of simple mixing and resistance screening just can obtain recombinant baculovirus Bacmid, the efficient that obtains recombinant baculovirus reaches very high level, PCR checking and loaded down with trivial details plaque purifying that need not be complicated, use the antibiotic-screening test can obtain to surpass 99% positive recombination fraction, accomplish really efficiently, make up fast, easily recombinant baculovirus.Can transfer among the baculovirus Bacmid by the donor carrier cDNA library that ordinary method is made up, thereby obtain convenient better quality baculovirus cDNA library of preserving.

Description of drawings

Fig. 1 is the donor plasmid structural representation, and this plasmid contains Pp10 and two promotors of Ppolh; The CpoI/NotI site is suitable for directed cloning PCR product with 2 different Bsu36I sites among the MCS2 among the MCS1.

Fig. 2 is the easy mode synoptic diagram of the direct directed cloning PCR of CpoI/NotI product.

Fig. 3 is in conjunction with shifting homologous recombination construction recombinant virus schema in the bacterium.

Fig. 4 utilizes the M13 primer PCR to detect recombinant virus.

Fig. 5 is expression and the purifying that recombinant virus Bacmid-EGFP infects Sf9 cell and EGFP, wherein: (a) be to use the sf9 cell of 488nm Fluirescence observation infection after 72 hours; (b) be that ordinary light is observed the sf9 cell that infects after 72 hours under the same visual field; (c) be Ni ⁺Column purification purpose GFP albumen; (d) be that anti-His tag monoclonal antibody western Blot detects target protein.

Fig. 6 is that recombinant virus Bacmid-red-qk2 infects Sf9 cell and the active detection of anticoagulant enzyme QK2, wherein: be that the 543nm Fluirescence observation infects the sf9 cell after 72 hours (a); (b) be that ordinary light is observed the sf9 cell that infects after 72 hours under the same visual field; (c) be that 10 μ l lysis supernatants add in the blood plasma plate well: 1: normal Sf9 lysis supernatant; 2: the lysis supernatant that recombinant virus Bacmid-red infects; 3:Bacmid-red-qk2 cells infected cracking supernatant.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be noted that, these embodiment only be used to the present invention is described and be not used in the restriction requirement of the present invention protection domain, unreceipted concrete experiment condition and method in the following example, usually according to normal condition as chief editors such as J. Sa nurse Brookers, Science Press, 1992, molecular cloning experiment guide (second edition); The Sun Ming chief editor, Higher Education Publishing House, 2006, genetically engineered (Yao Lunguang writes for the 17 chapter, viral genetic engineering); Li Lulin writes, press of Central China Normal University, and 1996, the rhabdovirus expression vector system, or carry out according to the method that the operational guidance of manufacturer is advised.

Embodiment 1: the method that makes up reorganization AcBacmid

(1) structure of donor plasmid

As shown in Figure 1, this plasmid contains Pp10 and two baculovirus strong promoters of Ppolh, can drive 2 target genes simultaneously and express.The CpoI/NotI site is suitable for directed cloning PCR product with 2 different Bsu36I sites among the MCS2 among the MCS1.R6k γ ori is a condition type replicon, just reproducible breeding in the bacterial strain of expressing pir gene (product is a π albumen), and cis-activating element oriT comes former in F-factor, is responsible for the transfer of donor plasmid.Concrete building process is as follows: Zeocin resistant gene (Em7-ZeoR) is through pcr amplification, and its two sections have added 2 homology arm sequences (HL and HR), 2 I-SceI sites, and BstBI, SacI, pmeI, restriction enzyme sites such as AvrII respectively.Its upstream and downstream primer is respectively: ZeoR-F:5 '-TTCGAATAGGGATAACAGGGTAATACGCATCACTTACAACAGGGGGGACTATGAAA TTATGCATTTGAGGATGCAGCACGTGTTGACAA-3 '; ZeoR-R:5 '-GAGCTCTAGGGATAACAGGGTAAT AAATGCAAATGTATTGTTATAGTATAATCTAATAAATATTTCATTATGTTTAAACC CTAGGAGGTCGACCCCCCTC-3 '.The PCR product is at first cloned the T carrier and is obtained reorganization pSimple-Em7zeo, and the NcoI/SalI double digestion removes the zeocin coding region then, keeps the Em7 promotor.Carrier after the recovery with derive from same enzyme and cut pCohygro product hygromycin coding region and link to each other, obtain the hyg-romycin expression cassette of Em7 promoters driven.Behind the double digestion, this Em7-H ygromycin fragment is connected with the R6kori+oriT product of the same double digestion pMAGIC1 of warp, obtains middle transition carrier pHTdual-pre1.PmeI/AvrII double digestion pFBDM obtains to contain p10 and polh promoter fragment, and the pHTdual-pre1 that this fragment and pmeI/AvrII enzyme are cut links to each other, and obtains middle transition carrier pHTdual-pre2.In order in second multiple clone site MCS2, to introduce 2 Bsu36I sites, the XmaI/NheI site that a Synthetic 2 primer (5 '-CCGGGCCTTAGGATCTC GAGCCATGGTCCTGAGGG-3 ' and 5 '-CTAGCCCTGAGGACATGGCTCGAGATCCTAAGGC-3 '), annealing back insert pHTdual-pre2 obtain the band double-promoter in conjunction with transfer vector.This transfer vector can directed quick clone PCR product as shown in Figure 2.

Carry 6 histidine-tagged egfp genes from template pIRES-egfp (Clontech) amplification, primer be gfp-F (5 '-GTCACCATGGTGCATCACCATCACCATCACATGGTGAGCAAGGGCGAG-3 ', 4 bases of band underscore represent in the primer extra increase be used for that the T4DNA polysaccharase handles can produce and CpoI complementary sticky end, 6 * histidine-tagged sequence is represented with italic) and gfp-R (5 '-GGCCATTACTTGTACAGCTCGT-3 ', underscore is represented to be used for to produce and 5 bases of NotI complementary). gel reclaims the PCR product and handled 45 minutes in 16 ℃ with 0.5U T4 archaeal dna polymerase and 4uM dTTP earlier.Be connected with the pHTdual that cuts through the CpoI/NotI enzyme then, and transform BUN20, obtain recombinant vectors pHTdual-egfp.

(2) structure of recipient bacterium E.coli CTRDH10B

Use at first can induce the I-SceI expression cassette of pectinose promoters driven to introduce in the E.coli DH10B genome in conjunction with the tranfer system construction of recombinant virus.Plasmid pML104 contains recombinase gene red and the gam by semi-lactosi promotor (Plac) control, transforms into DH10B through CaCl2.DH10B/pml104 is cultured to OD600 in 30 ℃ is about 0.2, add IPTG (0.4mM) and continue to cultivate 30 minutes, express to induce recombinase RED and GAM.Plasmid pML294 contains I-sceI expression cassette that inserts umuC gene HindIII site and the card sodium mycin resistant gene (ParaBAD-I-SceI-FRT-npt-FRT:umuC) of being with the FRT flanking sequence, the EcoRI/KpnI double digestion downcuts this fragment, electricity transforms the DH10B/pML104 competent cell that IPTG induced again, and kalamycin resistance filters out positive bacterium colony.Under the recombinase effect, the ParaBAD-I-SceI-FRT-npt-FRT fragment is advanced the genomic umuC of E.coli DH10B position phase by homologous recombination, and positive bacterium colony is knR, spR, and AmpR. then transforms into positive bacterium colony with pcp20, cultivates 2 hours in 30 ℃.Because responsive to temperature type plasmid pcp20 can express the FLP site-specific recombinase, the Flp recombinase of its expression can cut away the npt expression cassette (knR) in the DH10B genome, obtain no kalamycin resistance bacterium colony, remove pcp20 by 42 ℃ of cultivations at last, acquisition can be expressed the purpose bacterial strain CTRDH10B of I-SceI.

(3) transformation of acceptor AcBacmid

Owing to contained the p10 promotor in the donor plasmid, for fear of unexpected reorganization occurs when making up reorganization AcBacmid, the p10 promotor that self exists among the AcBacmid must be removed.Therefore still adopt the bacterial body homologous recombination to remove the p10 gene of AcBacmid and the p74 gene tightly adjacent with it, the AcBacmid electricity is transformed among the CTRDH10B/pML104, the kn/x-gal/IPTG flat screen is selected blue positive bacterium colony.For the template pcr amplification goes out resistant gene GmR, design suitable primer is introduced 50nt homologous recombination arm and 18bp respectively in its both sides I-SecI site with pFBDM.The primer sequence of upstream and downstream is respectively Gm-F:5 '-TACGCATCACTTACAACAGGGGGGACTATGAAATTATGCATTTGAGGATGTAGGGA TAACAGGGTAATCTATTAATATTCCGGAGT-3 '; Gm-R (5 '-AATAAATGCAAATGTATTGTTATAGTATAATCTAATAAATATTTCATTATAGGGAT AACAGGGTAATGGCGTT G TGACAATTTAC-3 '.PCR product electricity transforms in IPTG inductive CTRDH10B/PML104/AcBacmid, and under the effect of recombinase, 2 I-SceI sites of GmR expression cassette and flank thereof replace to fall p10 and the p74 of parent Bacmid through homologous recombination.The blue colonies that GmR resistance and the screening of blue hickie obtain obtains positive bacterium colony CTRDH10B/AcBacmid/pML 104 through PCR checking back.

(4) utilize combination to shift and obtain positive reorganization AcBacmid with the interior homologous recombination of bacterium

As shown in Figure 3, utilize in conjunction with transfer homologous recombination in bacterium and obtain the combination that the recombinant virus process comprises donor bacterium and recipient bacterium, donor plasmid is transferred to recipient bacterium, in the recipient bacterium I-SceI enzyme and red-gam recombinase abduction delivering, impel donor plasmid and acceptor AcBacmid by the linearizing of I-SceI enzyme, the homologous recombination process takes place between donor and the receptor dna homology arm.Detailed process: at first recombinant vectors pHTdual-egfp is transformed into BUN20, BUN20/ donor plasmid in the substratum of Hygromycin that contains 50 μ g/ml and 12.5 μ g/ml tsiklomitsins 30 ℃, 150rpm shaking culture.CTRDH10B/Bacmid/pML 104 is in containing 50 μ g/ml spectinomycins, 30 ℃ of the substratum of 50 μ g/ml kantlex and 0.2%w/v glucose, 150rpm shaking culture.Centrifugal collection bacterium after the overnight incubation, and wash 2 times with substratum, to remove the glucose of disinthibite pectinose promotor and semi-lactosi promoter activity.Use the substratum that contains 0.4mM IPTG respectively with dilution proportion donor and the recipient bacterium of 1:100 and 1:200 then, be cultured to A600 in 30 ℃ and be about 0.2.Press 1:1 mixed donor and recipient bacterium, and in nutrient solution, add pectinose (L-arabinose, 0.2%w/v).Leave standstill earlier and cultivated mixed bacterium about 2 hours, shaking table 150rpm cultivated about 2 hours again, with 1000 times of bacterium liquid dilutions, coated the kantlex that contains 50 μ g/ml at last, on the flat board of the hygromycin of 50 μ g/ml and glucose (0.2%w/v), put 42 ℃ of overnight incubation.Second day, the bacterium colony on the random choose flat board carried out the bacterium colony pcr analysis, and is perhaps streak culture again on gentamicin (10 μ g/ml) flat board, to determine the susceptibility of this bacterium colony to gentamicin.Bacterium colony PCR primer is HL-F (5 '-TACGCATCACTTACAACAGG-3 ') and HR-R (5 '-AATAAATGCAAATGTATTGT-3 '), and this primer can amplify the new segment that homologous recombination is advanced Bacmid.During perhaps because of the generation homologous recombination, the GmR among the former AcBacmid is cut off and is replaced by the external source fragment, therefore only needs simple gentamicin susceptibility to screen and just can eliminate non-positive reorganization bacterium colony.As shown in Figure 4, through these 2 kinds of method validations, positive recombination efficiency reaches 99.5%, and that is to say only has 1 to be false positive in per 200 bacterium colonies, and its background almost can be ignored.Therefore it is efficient that present method makes up reorganization Bacmid ten minutes, do not need the PCR checking, only need the gentamicin sensitivity Detection then can guarantee 100% positive rate, major cause is that Bacmid is that single copy plasmid significantly reduces background, linearizing DNA recombination efficiency is 1000 times of cyclic, and Red-gam recombinase efficient is very high.

(5) generation of recombinant virus

From intestinal bacteria, prepare reorganization Bacmid DNA according to conventional alkaline lysis method, use the Bacmid DNA transfection of will recombinating of liposome or CaPO4 transfection method to advance among the insect cell Sf9 then, promptly can gather in the crops the recombinant virus particle after 3-5 days and carry out protein expression and purity analysis.

(6) expression of recombinant virus in cell

Shown in Fig. 5 (a) and (b), select the positive bacteria of reorganization Bacmid-EGFP to drop into the row cultivation, extract reorganization Bacmid-EGFP DNA, use lipfectin 2000 that its transfection is advanced in the Sf9 cell.After the transfection 3～5 days, place the expression of observation of cell green fluorescence under the inverted fluorescence microscope.The result shows, after the transfection the 4th～5 day, it was green cell that a large amount of cells is arranged.Target protein N end merges 6 Histidine affinity tag, and this is histidine-tagged to have very high avidity to nickel ion, makes expressed albumen easily by the affine resin purification of Ni+-NTA.Centrifugal collecting cell is sent out multiple frozen-thawed cell 3 times, and ultrasonic disruption 2 times uses Ni+ column purification target protein, and SDS-PAGE analyzes purified albumen, and uses the purified albumen of anti-His tag antibody western blot check and analysis.SDS-PAGE result shows that reorganization Bacmid-EGFP transfection dyes the Ni+-NTA purified product of Sf9 cell and single about 30kD purpose band all occurs.Shown in Fig. 5 (c), (d), same western blot result shows that the monoclonal anti physical efficiency of 6 * His and this protein band carry out specific reaction, and further proof is utilized in conjunction with the feasibility that shifts homologous recombination construction recombinant virus method in bacterium.

Embodiment 2: the construction process that carries reporter gene AcBacmid

(1) donor plasmid that carries reporter gene makes up

Obtain donor carrier pHTdual according to (1) step making method among the embodiment 1, it comprises double-promoter (p10 and Ppolh), therefore most suitable a reporter gene is cloned into P10 promotor downstream, the another one goal gene is cloned into polyhedron promotor (Ppolh) downstream, and the expression of the process of the generation that reporter gene can the spike recombinant virus, cells infected and foreign protein is determined the concrete cell harvesting time with convenient like this.With pDsRed2-1 (Clontech) is template, pcr amplification DsRed gene, its primer be respectively Red-F (5 '-TTACCATGGCCTCCTCCGA-3 ', Red-R (5 '-TGACTACAGGAAC AGGTGGT-3 ').The PCR product is cloned into the Bsu36I site of donor carrier pHTdual after T4DNA polysaccharase and dGTP processing, produce recombinant plasmid pHTdual-red, and this red fluorescence gene places under the driving of p10 promotor.With pRset-Qk2 (doctor Yan Junpeng of virusology National Key Laboratory of Wuhan University gives) is template, amplify anticoagulant enzyme gene qk2, primer is Qk2-F (5 '-GTCACCATGCGCAATCTGTTCCTTAC-3 ', Qk2-R (5 '-GGCCACTATTATTGTGAAGCTG-3 ').Similarly, the PCR product is cloned into the CpoI and the NotI site of pHTdual-red plasmid after T4DNA polysaccharase and dTTP processing, obtain recombinant plasmid pHTdual-red-qk2, and this qk2 gene places under the polyhedron promoters driven.

(2) structure of recipient bacterium E.coli CTRDH10B; (3) transformation of acceptor AcBacmid; (4) obtain reorganization AcBacmid; (5) method of recombinant virus generation is made according to (2) among the embodiment 1, (3), (4), (5) step respectively.

(6) expression of recombinant virus in cell

Shown in Fig. 6 (a) and (b), use lipfectin2000 that the Sf9 transit cell is advanced in the recombinant virus transfection and dyed the back 3～5 days, there is the cell about 80% to send red fluorescence, the recombinant virus structure be described and infect successfully.Collect infected cell, 3 ruptured cells of PBS re-suspended cell and multigelation, 3000rpm is centrifugal, and it is standby to keep supernatant.Get the 10ul supernatant and add in the hole of blood plasma flat board, 37 ℃ are spent the night to detect the proteic activity of QK2.Being prepared as follows of blood plasma flat board: prepare 0.8% agar-gel solution 10ml, put zymoplasm (thrombin) solution and an amount of microbiotic that add 0.67ml in the triangular flask of 45 ℃ of water-bath preheatings behind the balance 15min, mixing adds the animal's whole blood of 500 microlitres then.Fall behind the mixing on flat board, thickness get final product for 1～2 centimetre, solidifies the back with the punch tool punching, and the albumen supernatant is dripped in the hole with whether degrade scleroproein in the flat board of detection QK2 albumen.Shown in Fig. 6 (c), the blood plasma treadmill test is the result show, the Sf9 cell lysate supernatant that recombinant virus Bacmid-red-qk2 infected after 5 days can produce the hydrolysis circle effectively in the blood plasma flat board, illustrate that expressed QK2 has the hydrolysis of fibrin activity.

Claims (2)

1. the method for an easily and efficiently constructing recombinant baculovirus, it is characterized in that: this construction process follows these steps to carry out:

(1) structure contains double-promoter, the donor plasmid of the directed quick clone PCR product of energy;

(2) the I-SceI expression cassette of pectinose promotor (pBAD) control is recombinated in the genome of intestinal bacteria E.coliDH10B, simultaneously baculovirus Bacmid is introduced in 2 I-SceI sites and a resistant gene, and can transform by the plasmid of IPTG abduction delivering Red-Gam recombinase among the E.coliDH10B, make up the F-strain CTRDH10B/Bacmid/pML104 that makes new advances;

(3) donor plasmid that will carry foreign gene imports and to contain in the acceptor bacterium of Bacmid, induce the expression of playback enzyme I-SceI then through pectinose, the I-SceI enzyme cuts donor plasmid and acceptor Bacmid in recipient bacterium, make its linearizing to improve homologous recombination efficiency and to reduce parent Bacmid background, after IPTG abduction delivering Red-Gam recombinase, homologous recombination obtains reorganization Bacmid in bacterium;

(4) ordinary method preparation reorganization Bacmid DNA, transfection insect cell is gathered in the crops the recombinant virus particle and is carried out protein expression and purity analysis after 3～5 days.

2. the method for easily and efficiently constructing recombinant baculovirus according to claim 1, it is characterized in that: described donor plasmid contains reporter gene, reporter gene will need expressed exogenous gene to be cloned in the polyhedron promotor downstream (MSC1) by the p10 promoters driven.

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