CN103589754A - Herpes simplex virus type 1 carrier system, and preparation method and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of biology, and relates to a herpes simplex virus type 1 carrier system, and a preparation method thereof. The carrier system comprises a shuttle plasmid by taking DNA (deoxyribonucleic acid) fragment at two sides of an inverted repeat sequence (IR) inside HSV-1 (herpes simplex virus type 1) as homologous recombinant arms, and a bacterial artificial chromosome plasmid pHSV delta IR-BAC (bacterial artificial chromosome) containing IR zone deleted HSV-1 genome. The method for preparing recombinant HSV-1 by using the carrier system comprises the following steps: firstly, cloning a target DNA fragment into pKo5/BN to obtain a recombinant shuttle plasmid; secondly, electro-transforming escherichia coli containing pHSV delta IR-BAC by using the recombinant shuttle plasmid, and screening to obtain recombinant bacteria; finally, extracting the recombinant DNA from the recombinant bacteria, transfecting Vero cells, and purifying to obtain recombinant HSV-1. The carrier system is high in security, can accommodate 30Kb of exogenous DNA, and can construct optional replication type recombinant HSV-1 capable of expressing a plurality of target genes.
Description
Technical field
The invention belongs to biological technical field, the present invention relates to a kind of herpes simplex types 1 virus carrier system and preparation method thereof, also relate to herpes simplex virus vector and carry and express EGFP reporter gene and the application in antitumor.
Background technology
(Herpes simplex virus type 1, is HSV-1) a kind of nervosa virus of having a liking for, and infects very generally in crowd, and clinical manifestation is that mucous membrane or local skin gather the light symptoms such as bleb, once in a while with heating for herpes simplex types 1 virus.HSV-1 is double-stranded DNA virus, and genome 152kb, by unique long segment (U
l) and short-movie section (U
s) form, two ends are inverted terminal repeat sequence TR
land TR
s, two fragment junctions are inner inverted repeats (IR district).The HSV-1 approximately 90 kinds of albumen of encoding, function is mostly clear and definite, and wherein approximately half is essential by virus replication, and other are nonessential for copying, and foreign gene is deleted and replaced to these dispensable genes does not affect virus replication, therefore HSV-1 can transform virus vector as.The advantage of HSV-1 carrier is that its host range is wide, efficiency of infection is high, (genome is free to be existed safety; Anti-herpesvirus medicament can stopped treatment and inadvertent contamination), capacity is large, the sustainable expression of foreign gene that is easy to preparation, carries; The symptom of the zoogenetic infection HSV-1 such as mouse is similar to people, can be used as the animal model of HSV-1 study on the carrier; Genetic background is clear, and recombination method is ripe.At present, HSV-1 is as oncolytic virus and existing basis and the clinical study widely of vaccine carrier.
HSV-1 is longer as the history of oncolytic virus, the HSV-1 oncolytic virus of (first-generation) lacks HSV-1 nucleotide metabolism enzyme gene and obtains at first, comprise disappearance thymidine kinase (Thymidine Kinase, tk) gene and ribonucleoside reductase gene (ribonucleotide reductase, RR) restructuring HSV-1, due to this class, HSV-1 still has neurotoxicity, and tk is anti-herpesvirus medicament Cymevan (Gancyclovir, GCV) or acyclovir (acyclovir, ACV) target molecule, therefore investigator cannot control with medicine the therapeutic process of this class restructuring HSV-1, at present, the oncolytic HSV-1 of tk disappearance is abandoned.S-generation HSV-1 oncolytic virus lacks neural virus gene γ 34.5 and a plurality of early stage (IE) gene immediately simultaneously and obtains, and as G207, HSV1716 and NV1020, they all enter clinical trial.NV1020 has deleted part IR district, a plurality of membrane glycoprotein encoding genes of HSV-2 are inserted to this site simultaneously, the initial object that Roizman builds NV1020 is the vaccine that development prevention HSV-1 and HSV-2 infect, in research, find that its anti-tumor activity is obvious, safe, and transform as the recombinant viruses such as NV1023, NV1042, NV1066, wherein NV1020 has entered the clinical second phase.The ICP47 gene that third generation HSV-1 oncolytic virus disappearance suppresses MHC-1 submission antigen obtains, and as G47 Δ, it can activate the immune clearance of body to tumour, has entered at present preclinical study.The HSV-1 oncolytic virus in (the 4th generation) was in HSV-1, to insert the gene that immuno-stimulating, oncotherapy or tumor-targeting are relevant in recent years, to strengthen it, press down tumoricidal activity, as the restructuring HSV-1 of expression of GM-CSF has entered clinical three phases, the restructuring HSV-1 that expresses IL-12 has also entered preclinical phase experiment.
HSV-1 starts late as the research of vaccine carrier, after Roizman proposition at beginning of the nineties in last century HSV-1 carrier can be used for preventing HSV to infect, investigators have carried out series of studies to HSV-1 as vaccine carrier, confirm that HSV-1 has unique advantage as vaccine carrier, as: after HSV-1 primary infection, hide in body, body is very low for the pre-existing immunity of HSV-1; The restructuring HSV-1 of antigen expressed gene can excite mouse to produce lasting immune response; HSV-1 carrier can be by the oral and comprehensive immunne response of mucosa-immune excitating organism; The inherent immunity reaction of HSV-1 carrier energy excitating organism, has adjuvant function etc.There is investigator that GM-CSF and IL-12 are recombinated to HSV-1 to impel it to activate organism immune response.At present, HSV-1 vaccine carrier mainly obtains by deleting a plurality of immediate early genes, this viroid is that replication defect type or replication are lower, as Knipe etc. by a plurality of gene recombination of SIV to having lacked ICP4, in the replication defect type HSV-1 carrier d106 of ICP22 and ICP27, this recombinant virus can induce stump-tailed macaque to produce neutralizing antibody and the cell immune response for SIV, the virus load of attacking the rear immune group of poison reduces, but the expression level of the SIV gene that d106 carries in cells infected is very low, the immunne response level of induction is not high, part IE gene in d106 is just being set about recovering to improve the replication of virus vector by this study group.
HSV-1 genetic background is clear, and the method for existing several maturations can obtain its recombinant chou.Restructuring HSV-1 the earliest obtains by mutagenesis, and its limitation is that the mutant that mutagenesis produces is mostly unwanted, therefrom filters out required HSV-1 mutant strain very difficult.Twentieth century eighties, homologous recombination method starts for building restructuring HSV-1, be about to a plasmid with marker gene and target gene and wild-type HSV-1 genome cotransfection host cell, in host cell, there is homologous recombination, and from progeny virus, filter out recombinant chou by the marker gene of expressing.Homologous recombination method sudden change direction is clear and definite, and screening easily, has obvious advantage compared with mutagenesis.Nonetheless, the acquisition of restructuring HSV-1 is still a job of wasting time and energy, and builds a strain recombinant virus and often needs 1 year consuming time.And wild-type virus multiplication capacity is while being significantly higher than mutant strain, is just difficult to filter out the virus strain of sudden change; If sudden change disappearance is virus multiplication indispensable gene, need in complementary cell system, breed recombinant virus, this makes again screening of recombinant more loaded down with trivial details.Along with human genome is learned going deep into of research, large capacity vector system is established gradually, and these instruments, not only for the research of mammalian cell gene, are also very easy to large virus genomic research.Wherein, the most frequently used in virusology is bacterial artificial chromosome (bacterial artificial chromosome, BAC).BAC only has 1-2 copy in Host Strains, and heritability is stable, and can pass through Electroporation Host Strains, ordinary method can be from Host Strains separated BAC-DNA, then transfection host cell obtain HSV-1.At present, all members of herpetoviridae have been cloned in BAC, these viral BAC have retained viral infection characterization, after the corresponding cell of its transfection, can copy and discharge progeny virus, and for colibacillary gene engineering method, being also applicable to the operation of viral BAC, this technology has greatly accelerated to obtain the speed of restructuring HSV-1.
Summary of the invention
The present invention has set up a kind of new herpes simplex types 1 virus carrier system, and this herpes simplex types 1 virus vector is safe, can hold 30 Kb exogenous dna fragments.Apply this carrier system selection rf recombinant herpes simplex virus of a plurality of goal gene of construction expression efficiently, particularly build recombinant herpes simplex virus and the herpes simplex virus vector vaccine of gene therapy.
The herpes simplex types 1 virus carrier system that the present invention sets up comprises shuttle plasmid pKo5/BN and HSV-1 bacterial artificial chromosome plasmid pHSVDIR-BAC; Ori, bleomycin (zeocin) resistant gene and the type froctosan saccharase gene of the DNA fragmentation B that described shuttle plasmid pKo5/BN comprises both sides, HSV-1 IR district and N, multiple clone site, temperature sensitivity (
sacB), DNA fragmentation B wherein and N are homologous recombination district; Described pHSVDIR-BAC comprises HSV-1 genomic dna, BAC sequence and the chloramphenicol resistance gene that lacks LiaoIR district.
Shuttle plasmid pKo5/BN provided by the invention is at pKo5(BC Horsburgh; MM Hubinette; and F Tufaro; Genetic manipulation of herpes simplex virus using bacterial artificial chromosomes. Methods Enzymol, 1999; On basis 306:337-352.), adopting conventional Protocols in Molecular Biology transformation to form, is to be mainly cloned into respectively DNA fragmentation B and the N as homologous recombination district at pKo5 multiple clone site two ends.PKo5/BN derives from but is not limited to pKo5 and pKo3 (AJ Link; D Phillips; and GM Church; Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli:application to open reading frame characterization. J. Bact., 1997; 179:6228-6237) etc.
HSV-1 bacterial artificial chromosome plasmid pHSVDIR-BAC provided by the invention transforms and contains pHSV-BAC(BC Horsburgh with above-mentioned pKo5/BN electricity; MM Hubinette; and F Tufaro Genetic manipulation of herpes simplex virus using bacterial artificial chromosomes. Methods Enzymol, 1999; Intestinal bacteria 306:337-352.), screen the genomic bacterial artificial chromosome plasmid of HSV-1 that lacks IR district that comprises obtaining then.PHSVDIR-BAC derives from but is not limited to pHSV-BAC and pBeloBAC (H Shizuya; B Birren; UJ Kim; V Mancino; T Slepak; Y Tachiiri et al., Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector.Proc. Natl. Acad. Sci., 1992; 89:8794-8797) etc.
The present invention has also set up the preparation method of above-mentioned herpes simplex types 1 virus vector, and concrete steps comprise:
1. build shuttle plasmid pKo5/BN
According to HSV-1 genome sequence, (the present invention relates to HSV-1 DNA sequence dna part all with reference to HSV-1 low virulent strain F strain sequence, GenBank:GU734771.1, network address: http://www.ncbi.nlm.nih.gov/nuccore/GU734771) design two pairs of primers respectively pcr amplification be positioned at IR district both sides
bamh I B fragment (be called for short B fragment) and
bamh I N fragment (be called for short N fragment), and add respectively at 5 ' end of the upstream and downstream primer of B fragment
xbai and
spei restriction enzyme site, adds respectively at 5 ' end of the upstream and downstream primer of N fragment
ecor V and
clai restriction enzyme site, B and N fragment that pcr amplification obtains are cloned into pBluescript II KS (+) successively, subsequently the DNA fragmentation that comprises multiple clone site between B and N fragment and two fragments are cloned into pKO5's
xbai and
salbetween I, obtain shuttle plasmid pKo5/BN.
2. build HSV-1 bacterial artificial chromosome plasmid pHSV Δ IR-BAC,
Above-mentioned shuttle plasmid pKo5/BN electricity transforms the intestinal bacteria DH10B competent cell that contains pHSV-BAC, in Bacillus coli cells, in B in pKo5/BN and N fragment and pHSV-BAC there is homologous recombination in the DNA of both sides, HSV-1 genome IR district, then by 43 ℃, cultivate and biochemical and chlorampenicol resistant screening acquisition contains the bacterial strain that homologous recombination occurs, a large amount of extractions, evaluation the final recombinant plasmid pHSV Δ IR-BAC that obtains, the bacterial strain that obtains is the coli strain that contains pHSV Δ IR-BAC.
3. utilize described herpes simplex types 1 virus carrier system to obtain restructuring HSV-1
First, target dna fragment is cloned into B and the intersegmental multiple clone site acquisition recombinant shuttle plasmid of N sheet of pKo5/BN; Subsequently, with the recombinant shuttle plasmid electricity obtaining, transform the intestinal bacteria DH10B competent cell that contains pHSV Δ IR-BAC, in Bacillus coli cells, B in B in recombinant shuttle plasmid and N fragment and pHSV Δ IR-BAC and N fragment generation homologous recombination are also recombinated target DNA to pHSV Δ IR-BAC, then by 43 ℃, cultivate and biochemical and chlorampenicol resistant screening obtains and contains the bacterial strain that homologous recombination occurs, extract, identify and recombinant plasmid that finally acquisition carries target DNA in a large number; Finally, with the Transfected Recombinant Plasmid vero cell that carries target DNA obtaining, by plaque select, purifying and virus identifies the final restructuring HSV-1 that carries target DNA that obtains.
Advantage and the novelty of herpes simplex types 1 virus vector provided by the invention show: the herpes simplex types 1 virus vector that (1) the present invention sets up is deleted IR district and comprised tri-early gene (ICP0 of HSV-1, ICP4 and ICP22), neurotoxicity gene γ 34.5 and this LAT of virus lays dormant associated retroviral are at the DNA fragmentation of interior 15351bp, the open ended exogenous dna fragment of described carrier is more than 30 Kb, the disposable restructuring of target dna fragment containing a plurality of foreign genes can be obtained to restructuring HSV-1 to carrier, therefore utilize HSV-1 carrier system provided by the invention to build restructuring HSV-1, with respect to transform acquisition restructuring HSV-1 at HSV-1 multidigit point for, there is obvious advantage, also be applicable to carry the structure that comprises a plurality of therapeutic genes or comprise the virus vector such as large fragment target DNA of therapeutic gene controlling element.(2) the present invention take HSV-1 low virulent strain F strain to set up herpes simplex types 1 virus vector as prototype, and lacked three early genes and the neurotoxicity gene γ 34.5 that participates in virus replication, therefore a little less than herpes simplex virus vector toxicity provided by the invention, safe.(3) utilize recombinant virus that herpes simplex types 1 virus carrier system that the present invention sets up obtains for the copy choice HSV-1 that recombinates, the sustainable expression in cells infected of its foreign gene carrying, this just makes described carrier be more suitable for gene therapy.(4) the herpes simplex types 1 virus vector that the present invention sets up utilizes bacterial artificial chromosome technology, and the institute of regrouping process can complete intestinal bacteria in steps, therefore the method for the acquisition of setting up restructuring HSV-1 is more efficient, required expense is less.
Accompanying drawing explanation
The collection of illustrative plates of the shuttle plasmid pKo5/BN that Fig. 1 the present invention builds;
The collection of illustrative plates of the HSV-1 bacterial artificial chromosome plasmid pHSV Δ IR-BAC that Fig. 2 the present invention builds ("
" represent that in pHSV Δ IR-BAC, delete in HSV-1 IR district);
Fig. 3 recombinant virus HSV-1/ Δ IR and the growth curve of HSV-1/EGFP in mammalian cell.
Embodiment
Below by specific embodiment, the present invention is further described; these embodiment are only not used in the protection domain of restriction requirement of the present invention for the present invention is described; the unreceipted specific experiment condition of the following example and method; conventionally according to normal experiment method, carry out; if the methods such as molecular biology are all according to molecular cloning experimental technique guide (J. Pehanorm Brooker and D.W. Russell work; Huang Peitang etc. translate; Science Press, the third edition 2002) or the schedule of operation that provides of experiment reagent manufacturer carry out.
According to HSV-1 genome structure, HSV-1's
bamh I B fragment (B) and
bamh I N fragment (N) lays respectively at both sides, IR district, according to the sequence of HSV-1, designs respectively primer, and adds respectively at 5 ' end of the upstream and downstream primer of B fragment
xbai and
spei restriction enzyme site, adds respectively at 5 ' end of the upstream and downstream primer of N fragment
ecor V and
clai restriction enzyme site, pcr amplification obtains the B fragment of 2024bp and the N fragment of 2857bp, B and N fragment are cloned into pBluescript II KS (+) successively, subsequently the DNA fragmentation that comprises multiple clone site between B and N fragment and two fragments are cloned into pKO5's
xbai and
salbetween I, obtain shuttle plasmid pKo5/BN (accompanying drawing 1).
embodiment 2. preparation HSV-1 bacterial artificial chromosome plasmid pHSVΔ
iR-BAC
Alkaline lysis is a large amount of to be extracted and purifying pKo5/BN, get that the pKo5/BN of 2 μ l (1 μ g/ μ l) purifying and 40 μ l DH10B competent cells are mixed is incorporated in precooling in ice-water bath, add in 0.1cm electric shock cup and carry out electricity conversion, electric Transformation Parameters is voltage 2.5 kV, electric capacity 30 μ F and resistance 400 Ω.Competent cell after electricity transforms adds in 1 mLSOC substratum 30 ℃ to hatch 1 h, coats on the LB solid medium that contains zeocin and paraxin 43 ℃ of overnight incubation; Treat that bacterium colony grows to diameter 1mm size, 10 mono-clonals of random choose, mix in the physiological saline of 1 mL, get 40 μ L and coat to contain paraxin and 5 %(v/v) the LB solid medium of sucrose is dull and stereotyped, 30 ℃ of overnight incubation; From each is dull and stereotyped, 10 mono-clonals of random choose (amounting to 100 mono-clonals) line respectively on the solid LB substratum that contains paraxin again, 37 ℃ of overnight incubation, and this step comes again; 100 mono-clonal photocopy of picking, to another solid LB culture medium flat plate that contains paraxin, are treated to bacterium colony grows to diameter 1mm size, and bacterium colony PCR screens recombinant bacterium.A large amount of extractions, evaluation the final recombinant plasmid pHSV Δ IR-BAC that obtains, the bacterial strain that obtains is the coli strain that contains pHSV Δ IR-BAC.
the restructuring HSV-1:HSV-1/ in embodiment 3. preparation disappearance IR districtsΔ
iR
Cultivation of Vero to cell density reaches 70% ~ 80% according to a conventional method, and the recombinant plasmid pHSV Δ IR-BAC(of purifying is established to 2 μ g, 4 μ g and tri-gradients of 8 μ g) with liposome mediated-method transfectional cell.After 2 d, start to observe transfectional cell pathological change (CPE), after 4 d, in cell, occur plaque (meeting such as cell state and transfection reagent affects transfection effect and causes plaque time of occurrence to be stepped back); After occurring, plaque abandons cell culture fluid, with 2% low melting-point agarose and serum-free 2 * 199V substratum equal-volume mixed solution fixed cell, ordinary method picking plaque district's cell also adds in 200 μ L skim-milk solution (the skim-milk solution that is 9% through the autoclaved mass body volume concentrations of routine) and 2 * 199V equal-volume mixed solution, under ice-water bath, ultrasonic disruption cell is with releasing virus, ultrasonic disruption time and interval take that to avoid cytoclasis liquid temp too high be principle, and cell pyrolysis liquid is stored in-80 ℃ of refrigerators standby as viral liquid storage.Virus liquid storage ordinary method superinfection Vero cell 3 times picking plaque, with purified virus, infect and amplicon virus, subsequently in a large number with 25cm
2the full bottle of Tissue Culture Flask infects for example, in 4 ℃ of 3000rpm centrifugal collecting cells to 5mL centrifuge tube, with the PBS of 4mL precooling, rinse cell twice, 4 ℃ of 3000rpm collecting cells, and with lyse-o-lot (the 150mM NaCl of 0.5mL, 10mM Tris, 1.5mM MgCl) re-suspended cell, add 5 μ L 10% NP40, 10 μ L 10% SDS, 5 μ L 0.5M EDTA and 1.8 μ L β-ME, fully mix, twice of ordinary method phenol chloroform extracting, get supernatant, add the dehydrated alcohol of two volumes and the sodium-acetate of 1/10 volume, being placed in-70 ℃ of 2h or-20 ℃ spends the night, 4 ℃ of 12000rpm collect viral DNA.Viral genome is carried out to Molecular Identification, identify that correct recombinant virus is HSV-1/ Δ IR.
embodiment 4. preparations carry the restructuring HSV-1:HSV-1/EGFP of EGFP
Prepare the implementation detail of the restructuring HSV-1 that carries EGFP with reference to embodiment 3, be summarized as follows:
First, reporter gene green fluorescent protein (EGFP, enhanced green fluorescent protein) is gene constructed to shuttle plasmid pKO5/BN, the shuttle plasmid pKO5/BN/ of acquisition Carrying Green Fluorescent Protein gene
eGFP, multiple restriction enzyme digestion is identified shuttle plasmid.Subsequently with correct shuttle plasmid pKO5/BN/
eGFPelectricity transforms the competent escherichia coli cell that contains pHSV Δ IR-BAC, by antibiotics resistance and biochemical screening, obtain the recombinant bacterial strain that homologous recombination occurs, through bacterium colony PCR and Southern blotting, identify the recombinant DNA obtaining containing target gene EGFP, extract in a large number purification of Recombinant plasmid pHSV Δ IR-BAC/EGFP.Finally, after pHSV Δ IR-BAC/EGFP recombinant DNA transfection Vero cell there is obvious cell pathology variation (cell pathological changes in picking, CPE) plaque, ultrasonic treatment cells infected, cell pyrolysis liquid again vero cells infection with purifying and enrichment recombinant virus.Through PCR and Southern, detect the restructuring HSV-1:HSV-1/EGFP that acquisition carries target gene EGFP.
embodiment 5. recombinant virus HSV-1/Δ
the analysis of IR and HSV-1/EGFP multiplication capacity in mammalian cell
First, tri-kinds of viruses of HSV-1/wt, HSV-1/ Δ IR and HSV-1/EGFP are all with 0.5MOI vero cells infection, and respectively at 12h, 18h, 24h, tetra-time point collecting infecting cells of 48h, cell is stored in-80 ℃ of refrigerators after three multigelations and ultrasonic degradation.Subsequently, by virusology ordinary method gradient dilution virus liquid storage vero cells infection, detect the titre of virus in the viral liquid storage that each time point collects.Result shows, titre during tri-kinds of virus infection vero cell 12h of HSV-1/wt, HSV-1/ Δ IR and HSV-1/EGFP is followed successively by 1.00 * 10
7, 1.30 * 10
7with 1.50 * 10
7; Titre while infecting 18h is followed successively by 5.40 * 10
8, 3.05 * 10
8with 1.89 * 10
8; Titre while infecting 24h is followed successively by 2.00 * 10
9, 1.60 * 10
9with 1.20 * 10
9; Titre while infecting 48h is followed successively by 4.00 * 10
8, 2.50 * 10
8with 1.90 * 10
8.Above result shows that wild-type HSV-1 and the recombinant virus HSV-1/ Δ IR building with HSV-1 carrier system provided by the invention and HSV-1/EGFP growth curve and the multiplication capacity in Vero cell approaches, and the competence for added value of HSV-1/ Δ IR and HSV-1/EGFP is slightly lower than wild-type HSV-1.Therefore can be used for structure condition, HSV-1 carrier system provided by the invention selects rf restructuring HSV-1.
Claims (9)
1. a herpes simplex types 1 virus carrier system, is characterized in that, this herpes simplex types 1 virus carrier system comprises the HSV-1 bacterial artificial chromosome plasmid pHSV Δ IR-BAC in shuttle plasmid pKo5/BN and disappearance IR district; Described shuttle plasmid pKo5/BN comprises the inner inverted repeats (IR) of HSV-1 both sides
bamh I B fragment (B) and
bamthe ori of H I N fragment (N), multiple clone site, temperature sensitivity, bleomycin (zeocin) resistant gene and type froctosan saccharase gene (
sacB); Described pHSV Δ IR-BAC comprises HSV-1 genomic dna, BAC sequence and the chloramphenicol resistance gene that lacks LiaoIR district.
2. herpes simplex types 1 virus carrier system according to claim 1, is characterized in that, the DNA fragmentation B in described shuttle plasmid pKo5/BN and N are two homologous recombination arms.
3. herpes simplex types 1 virus carrier system according to claim 1, is characterized in that, the DNA fragmentation B in described shuttle plasmid pKo5/BN is the DNA fragmentation that is positioned at the connected 2024bp in the unique long segment Zhong Yu IR of HSV-1 genome district.
4. herpes simplex types 1 virus carrier system according to claim 1, is characterized in that, the DNA fragmentation N in described shuttle plasmid pKo5/BN is the DNA fragmentation that is positioned at the connected 2857bp in the unique short-movie Duan Zhongyu IR of HSV-1 genome district.
5. herpes simplex types 1 virus carrier system according to claim 1, is characterized in that, the multiple clone site of described shuttle plasmid pKo5/BN comprises several restriction enzyme sites:
bamh I,
smai,
psti,
ecor I and
ecor V.
6. herpes simplex types 1 virus carrier system according to claim 1, it is characterized in that, the HSV-1 genomic deletion IR district that described pHSVDIR-BAC comprises comprises the DNA fragmentation of the 15351bp of tri-early genes of HSV-1 (ICP0, ICP4 and ICP22), neurotoxicity gene γ 34.5 and this LAT of virus lays dormant associated retroviral.
7. the application of herpes simplex types 1 virus carrier system claimed in claim 1 in building restructuring HSV-1.
8. the preparation method of a herpes simplex types 1 virus carrier system claimed in claim 1, it is characterized in that carrying out as follows, first build shuttle plasmid pKo5/BN, B and N fragment are cloned into pBluescript II KS (+) successively, then the DNA fragmentation that comprises multiple clone site between B and N fragment and two fragments are cloned into pKO5's
xbai and
salbetween I, obtain shuttle plasmid pKo5/BN; Secondly, build pHSV Δ IR-BAC, above-mentioned shuttle plasmid pKo5/BN electricity is transformed to the intestinal bacteria that contain pHSV-BAC, screening obtains recombinant bacterial strain, extracts and obtains recombinant plasmid pHSV Δ IR-BAC.
9. the application of the preparation method of herpes simplex types 1 virus carrier system in building restructuring HSV-1 described in claim 8.
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