CN102051379A - Recombinant herpes simplex virus (HSV) genetic operation system and application thereof - Google Patents
Recombinant herpes simplex virus (HSV) genetic operation system and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical fields of medicine and biology, and provides a recombinant herpes simplex virus (HSV) genetic operation system. The recombinant HSV genetic operation system comprises an HSV skeleton vector and a shuttle plasmid, wherein the HSV skeleton vector comprises HSV genomic deoxyribonucleic acid (DNA), a bacterial artificial chromosome (BAC) plasmid skeleton, a multiple cloning site, a eukaryotic resistant gene and a prokaryotic resistant gene, and a site-specific recombinant enzyme system recognition sequence is blended outside the multiple cloning site and the eukaryotic resistant gene; and the shuttle plasmid is a bacterial cloning vector which comprises a multiple cloning site, and a site-specific recombinant enzyme system recognition sequence is blended outside the multiple cloning site. The system effectively improves the convenience and operability of HSV genetic operation, and can be widely applied to the establishment of replication-defective recombinant HSV and conditionally replicating oncolytic HSV.
Description
Technical field
The invention belongs to the medical biotechnology field, be specifically related to a kind of recombinant herpes simplex virus genetic operating system that is applied to gene therapy, this system can be widely used in the replication defect type recombinant herpes simplex virus and select the foundation of rf oncolytic hsv.
Background technology
Gene therapy is the hot fields of medical research over past twenty year.Gene therapy is exactly that people's normal gene or medicative gene are imported defective or the performance therapeutic action of people's somatic target cell to correct gene by certain way, thereby reaches the biomedical new technology of treatment disease purpose.Different with conventional treatments: disease treatment in general sense at be the various symptoms that cause because of gene unconventionality, and gene therapy at be the unusual gene of root one of disease itself.The present gene of human and disease-related of having found has more than 5000 approximately, existing 1/3 separated and affirmation so far.Thereby gene therapy will be the revolution of medical science and pharmaceutical field, will become important medicinal industry of 21 century.The development of gene therapy is extremely rapid, according to statistics, begin from first gene therapy clinical trial of nineteen ninety U.S. FDA official approval, by in March, 2009, existing 1537 each phase gene therapy clinical study schemes (http://www.wiley.co.uk/genmed/clinical/) in the world wide have contained the disease of inherited disease (as hemophilia, cystic fibrosis, family hypercholesterolemia etc.), malignant tumour, cardiovascular disorder, the infectious diseases serious human healths of numerous threats such as (as acquired immune deficiency syndrome (AIDS), similar rheumatisms etc.).Wherein III phase clinical study has 52, and Chinese in addition SFDA has also ratified 2 gene therapy new drug listings in the world first.But generally speaking, gene therapy technology and product also are in the initial stage of its development, still are faced with problems and challenge, and wherein one of topmost challenge is exactly a gene import system inefficiency.Developing new gene import system is one of most important research direction of current gene therapy.
At present, the gene import system mainly comprises virus carrier system and non-virus carrier system, wherein virus carrier system accounts for 68.6% of at present all gene therapy clinical protocol because it natural has a liking for the dominant position that cytoactive makes it to be in the gene therapy import system.Because technical reason, retrovirus (accounting for 21.2%) and adenovirus (accounting for 24.1%) were once becoming topmost gene import system respectively.In recent years, hsv is because its remarkable performance just more and more is subjected to gene therapy researcher's attention.
(Herpes simplex virus, HSV) HSV is divided into I type and II type to hsv, wherein is used as normally I type (HSV-1) of gene therapy vector.HSV-1 is the coating double-stranded DNA virus, and its gene group leader is 152kb, by 2 interconnective long segment (U
LAnd short segment (U
S) form, each sections is terminal for reversing tumor-necrosis factor glycoproteins.84 genes of genome encoding, whether these genes can express in virus replication according to them is divided into indispensable gene and dispensable gene, but dispensable gene is extremely important for virus-host's interaction, as relates to immunologic escape, duplicates or close functions such as host protein is synthetic in Unseparated Cell.HSV-1 life cycle comprises that cracking infects (Lytic infection) and latent period (Latency).In the cracking course of infection, the expression of virogene is that strict space-time order is arranged, be divided into utmost point early gene (immediate-early gene, IE or α), early gene (Early gene, E or β) and late gene (Late gene, L or γ), utmost point early gene comprises 5 infectious cell protein (infected cell protein such as ICPO, ICP4, ICP22, ICP27 and ICP47, ICP) gene, wherein ICP4 and ICP27 are indispensable genes.
HSV-1 be gene therapy commonly used, also be extremely outstanding virus vector, mainly show:
(1) the host cell scope of HSV-l is wide, and the cell of HSV-l enters acceptor HveA and HveC wide expression in the cell surface protein of various unknown function;
(2) efficiency of infection height, even under the very low situation of infection multiplicity (MOI), the HSV of replication defective is at the external cell colony that still can infect about 70%;
(3) can efficiently infect quiescent stage and non-quiescent stage cell, expression alien gene;
(4) capacity is big, can insert the big fragment of external source, is dispensable gene because half is arranged in the gene of HSV-1 genome encoding, can be replaced by the external source therapeutic gene;
(5) can prepare replication defective recombinant chou with the high titre of purifying easily, not have the pollution of wild-type virus body;
(6) can in neuronal cell, set up latent infection, continual and steady expression treatment gene;
(7) the replication defective recombinant chou enters the genetic expression cascade chain (abortive gene expression cascade) that can produce miscarriage behind the cell, foundation is similar to the state of latent infection, only can not be activated, this helps to strengthen the chronic long-term expression (chronic transgene expression) of foreign gene in neuronal cell and non-neuronal cell again.
In general, can be the HSV genome manipulation Vectors in Gene Therapy of non-virulent by three kinds of modes: the one, amplicon (Amplicons) carrier, promptly only the replication orgin of HSV and packaging signal sequence are inserted in the plasmid, when with its transfection to packing cell and after with the superingection of HSV helper virus, just can obtain to contain the pseudovirus of amplicon; The 2nd, recombinant replication-defective type carrier (Replication-defective Vectors) has promptly been rejected and has been duplicated relevant indispensable gene and dispensable gene, to reduce cytotoxicity, is used for the exogenous therapeutic gene of long-term expression in the host neuron cell more; The 3rd, condition replicating vector (Conditionally Replicating Vectors) has promptly been rejected dispensable gene, has been kept and duplicated genes involved, because of it has the characteristic of lysing cell, comes the selective killing tumour cell mainly as oncolytic virus.
The oncolytic hsv is one of oncolytic virus of studying at most at present, and its reason is that the oncolytic hsv has a lot of advantages, except the advantage that the host cell scope is wide, efficiency of infection is high of above-mentioned hsv, also is:
(1) in the host cell that infects, whole replication cycle processes of HSV-1 can finish in 20 hours, discharged the progeny virion of thousands of meters;
(2) the HSV-1 virion can merge by cytolemma and carry out direct cell-cell propagation, also can propagate by extracellular space (extracellular space), this is particularly useful for oncolytic virus, because the virus of low dosage just can realize in solid tumor that virus is invaded (Viral Penetration) efficiently;
(3) have the medicine (as acycloguanosine, Famciclovir etc.) of multiple anti-HSV-1 to be used for the treatment of the infection of HSV-1 clinically, and HSV-1 expresses antitumor suicide gene---and thymidine kinase (herpes simplex virus thymidine kinase, HSV-TK).This just provides a security mechanism for the antitumor action of HSV-TK, because available medicine is closed the replication cycle of HSV-1;
(4) HSV-1 can effectively infect the kinds of experiments animal, helps setting up animal model, also is convenient to the preclinical study result is moved in (translation) clinical trial.
Yet, although HSV has so many advantage, but for adenovirus etc., the HSV gene therapy technology still develops slowlyer, by in March, 2009, the gene therapy clinical protocol that with HSV is carrier has only 51, accounts for 3.3% (http://www.wiley.co.uk/genmed/clinical/) of full gene treatment plan.This major cause wherein is: HSV-1 is the double-stranded DNA virus that genome reaches 152kb, and has a plurality of genes such as ICP0, ICP4, ICP34.5 etc. to be two copies, and it is just much more difficult relatively therefore will to carry out genetic manipulation to HSV-1.But, through nearly 30 years effort, the acquisition of HSV-1 genetic manipulation very big development, the major technique that relates to the genetic manipulation of HSV-1 in general has:
(1) chemomorphosis: this be the earliest HSV-1 is carried out the method for genetic manipulation, adopt chemical mutagen to carry out random mutation and screening expection phenotype, what this method mainly screened is responsive to temperature type mutant (Temperature-sensitive Mutant), cytolysis resistant mutants (Cytolysis-resistant Mutant), drug resistance mutant (Drug-resistant Mutants).
(2) homologous recombination: this also is a kind of early stage HSV-1 Genetic Manipulative Technology, and homologous recombination is directly inserted goal gene in the people HSV skeleton in cell, for ease of the screening mutant, need import marker gene simultaneously.This law also needs to make up simultaneously package cell line if relate to indispensable gene (Essential Gene) sudden change.
(3) amplicon: so-called amplicon is exactly only the dna replication dna initial point oriS of HSV-1 and packaging signal sequence pac to be inserted in the bacterial plasmid, when itself and HSV-1 helper virus cotransfection packing cell, can the concatermer packaged form the pseudovirus that obtains to contain amplicon.But helper virus can bring safety issues such as reorganization.The amplicon vector biggest advantage is to have eliminated basically toxicity or the immunogenicity of HSV-1, and the foreign gene capacity is big (can reach tens of kb, reach as high as 150kb) usually.
(4) COS system: earlier in the COS plasmid that the HSV-1 genome is reprinted respectively at a series of intermeshings (normally 5 or more than), with Protocols in Molecular Biology some specific COS plasmids are wherein done the genetically engineered operation as required then, the COS plasmid of intermeshing is cotransfection permissive cell (Permissive Cell) after linearizing, thereby obtains mutant through homologous recombination.This law biggest advantage is to have only mutant to produce, there is not the pollution of wild-type HSV-1, but shortcoming is that being not used in the suitable restriction endonuclease sites of making sudden change usually in the COS plasmid can use, and some COS plasmid heredity is unstable and cause unnecessary sudden change to occur thus.
(5) BAC technology: bacterial artificial chromosome (Bacterial Artificial Chromosomes, BAC) technology is the HSV-1 genetic manipulation new technology that development in recent years is got up, what mainly use is equipotential exchange (Allelic Exchange) principle, its primary process is: at first make up a BAC plasmid that carries goal gene and homology arm, with viral genome cotransfection cell (as Vero), homologous recombination is inserted the BAC plasmid in the viral genome in cell, extract reproducible recombinant virus genomes cyclic intermediate and Transformed E .coli then, thereby obtain a large amount of HSV-BAC, transfection Vero cell after HSV-BAC being carried out necessary genetic manipulation, the results recombinant virus.BAC technology biggest advantage is its bale capacity big (can pack whole viral genome), need not helper virus, genetic manipulation is simple.The BAC technology is the mainstream technology of present HSV-1 genetic manipulation.
(6) embedded virus (Chimeric Virus) also claims hybrid virus (Hybrid Virus).HSV can not be integrated into host cell chromosome, therefore can only the transient expression foreign gene, finally can cause losing at somatoblast.Embedded virus just can remedy this shortcoming, forms embedded virus normally EBV, AAV and retrovirus etc. with HSV.
Based on this, the present invention is just at the genome of HSV-1 is big, the genetic manipulation difficulty is high problem, design also provides a kind of new recombinant herpes simplex virus genetic operating system, this system is based on the foundation of BAC plasmid skeleton, and site specificity recombinase systems such as Cre-loxP, FLP-FRT have been gone in fusion, effectively reduce the difficulty of hsv genetic manipulation, significantly improved its genetic manipulation convenience and can be handling, can be widely used in the replication defect type recombinant herpes simplex virus and select the foundation of rf oncolytic hsv.
Summary of the invention
Main purpose of the present invention is design and sets up a kind of new recombinant herpes simplex virus genetic operating system.Using this operating system can be convenient, efficiently hsv is carried out various genetic manipulations, is widely used in to set up various replication defect type recombinant herpes simplex virus and select rf oncolytic hsv.
The invention provides a kind of recombinant herpes simplex virus genetic operating system, comprise HSV skeleton carrier and shuttle plasmid; Described HSV skeleton carrier comprises HSV genomic dna, BAC plasmid skeleton, multiple clone site, eucaryon resistant gene and protokaryon resistant gene, incorporates site-specific recombinase system identification sequence in the outside of multiple clone site and eucaryon resistant gene; Described shuttle plasmid is the bacteria clone carrier that comprises multiple clone site, and incorporates site-specific recombinase system identification sequence in the multiple clone site outside.
Described site-specific recombinase system identification sequence is one or several in LoxP sequence, FRT sequence, attP or the attB sequence.
Multiple clone site is generally used for inserting exogenous DNA array.The multiple clone site of HSV skeleton carrier and shuttle plasmid all is that the encoding sequence by a series of excision enzymes is in series, and both multiple clone site can be identical, also can be inequality.Multiple clone site contains following one or several restriction enzyme sites: HindIII, EcoRI, SacI, KpnI, EcoRV, NotI, SphI, NcoI, XhoI, SmaI, PacI, NheI, BamHI, PmeI or SalI.
This recombinant herpes simplex virus genetic operating system also comprises the plasmid of express recombinant enzyme.Described recombinase is Cre enzyme, FRT enzyme, φ C31 or φ BT1 intergrase.The plasmid of expressing these recombinases can be selected from the expression plasmid of commercially available corresponding recombinase.
BAC plasmid skeleton is originated but is not limited to: BAC cloning vector pBeloBACll (New England Biolabs),
BAC (Lucigen) and pBACe3.6 (Frengen et al.1999) etc.[Ref：Frengen，E.et?al.(1999)A?Modular，Positive?Selection?Bacterial?Artificial?Chromosome?Vector?with?Multiple?Cloning?Sites.Genomics?58：250-253.]。
The eucaryon resistant gene includes but not limited to: kalamycin resistance gene (Kanamycin resistance gene, Kan
r), hygromycin gene (Hygromycin resistance gene, Hyg
r), neomycin resistance gene (Neomycin resistance gene, Neo
r), gigohm mycin resistant gene (Zeocin resistance gene, Zeo
r) etc.
The protokaryon resistant gene includes but not limited to: chloramphenicol resistance gene (Chloramphenicol resistance gene, Cm
r), tetracycline resistance gene (Tetracycline resistance gene, Tet
r) etc.
Site-specific recombinase system identification sequence includes but not limited to: the attP of the FRT sequence of the LoxP sequence of Cre enzyme identification, the identification of FLP enzyme, φ C31 and the identification of φ BT1 intergrase and/or attB sequence etc.
Above-mentioned recombinant herpes simplex virus genetic operating system can also comprise target genes such as fluorescence protein gene.Fluorescence protein gene includes but not limited to: red fluorescent protein gene DsRed1, green fluorescence protein gene GFP, yellow fluorescence protein gene YFP and their enhancement type etc.
The present invention also provides the preparation method of above-mentioned recombinant herpes simplex virus genetic operating system: at first, make up homologous recombination precursor plasmid, this plasmid comprises multiple clone site MCS, fluorescence protein gene, eucaryon resistant gene and protokaryon resistant gene, incorporates site-specific recombinase system identification sequence in the outside of MCS and eucaryon resistant gene; Secondly, genomic two the homology arm DNA of HSV have been inserted among the homologous recombination precursor plasmid pRKBAC-MCS; Once more, make up shuttle plasmid, incorporate site-specific recombinase system identification sequence in the multiple clone site MCS2 outside of bacteria clone carrier.
Concrete steps comprise:
1. make up homologous recombination precursor plasmid
The genome that is generally used for the hsv HSV-1 of gene therapy reaches 152kb, and it is very big that its integral body is carried out the genetic manipulation difficulty.Along with the progress of genetic engineering technique, bacterial artificial chromosome BAC technology becomes the mainstream technology of present HSV-1 genetic manipulation.The designed recombinant herpes simplex virus genetic operating system of the present invention is exactly to be based upon on the basis of BAC technology, merges simultaneously to go into site specificity recombinase systems such as Cre-loxP, FLP-FRT.These core features just intactly concentrate design in homologous recombination precursor plasmid.The present invention just uses conventional molecule clone technology, make up homologous recombination precursor plasmid pRKBAC-MCS (Fig. 1), its feature includes but not limited to: set up based on BAC plasmid skeleton, comprise multiple clone site MCS, (fluorescence protein gene) eucaryon resistant gene and protokaryon resistant gene, incorporate site-specific recombinase system identification sequence in the outside of MCS and eucaryon resistant gene.
Wherein, BAC plasmid skeleton is originated but is not limited to: BAC cloning vector pBeloBACl1 (New England Biolabs),
BAC (Lucigen) and pBACe3.6 (Frengen et al.1999) etc.[Ref：Frengen，E.et?al.(1999)A?Modular，Positive?Selection?Bacterial?Artificial?Chromosome?Vector?with?Multiple?Cloning?Sites.Genomics?58：250-253.]
Multiple clone site MCS includes but not limited to: 5 '-BamHI-XhoI-PmeI-NotI-HindIII-SacI-PacI-3 ':
Fluorescence protein gene includes but not limited to: red fluorescent protein gene DsRed1, green fluorescence protein gene GFP, yellow fluorescence protein gene YFP and their enhancement type etc.
The eucaryon resistant gene includes but not limited to: kalamycin resistance gene (Kanamycin resistance gene, Kan
r), hygromycin gene (Hygromycin resistance gene, Hyg
r), neomycin resistance gene (Neomycin resistance gene, Neo
r), gigohm mycin resistant gene (Zeocin resistance gene, Zeo
r) etc.
The protokaryon resistant gene includes but not limited to: chloramphenicol resistance gene (Chloramphenicol resistance gene, Cm
r), tetracycline resistance gene (Tetracycline resistance gene, Tet
r) etc.
Site-specific recombinase system identification sequence includes but not limited to: the attP of the FRT sequence of the LoxP sequence of Cre enzyme identification, the identification of FLP enzyme, φ C31 and the identification of φ BT1 intergrase and/or attB sequence etc.
2. make up the hsv skeleton carrier
The hsv skeleton carrier is core group/one of its genetic operating system, and it is that the hsv genomic dna is inserted in the BAC plasmid, so that in intestinal bacteria the hsv genome is carried out genetic manipulation.Among the present invention, the hsv skeleton carrier is based on that homologous recombination precursor plasmid pRKBAC-MCS sets up.At first from the HSV genome, cut and obtain two homology arm (Homologous Arm through PCR or enzyme, HA) and insert among the homologous recombination precursor plasmid pRKBAC-MCS, be built into homologous recombination plasmid pRKBAC-HA, then homologous recombination plasmid pRKBAC-HA and HSV genomic dna common transfection package cell line Vero and derived cell system thereof, in package cell line, carry out homologous recombination, and therefore set up HSV skeleton carrier pHsvEasy-X.
The feature of HSV skeleton carrier pHsvEasy-X includes but not limited to: inserted the HSV genomic dna in homologous recombination precursor plasmid pRKBAC-MCS.Wherein, the HSV genomic dna that is inserted can be the HSV genomic dna of total length, also can be the HSV genomic dna of disappearance partial sequence, and the HSV genome is wild-type both, also mutant.
Above-mentioned provide genomic HSV include but not limited to 1 type HSV (HSV type 1, HSV-1) and 2 type HSV (HSV-2).
3. structure shuttle plasmid
Shuttle plasmid also is core group/one of its genetic operating system, and its major function is to import foreign gene or gene fragment in HSV skeleton carrier pHsvEasy-X.The present invention uses conventional molecule clone technology and sets up shuttle plasmid pHsvShuttle-MCS (+) (Fig. 2), its feature includes but not limited to: set up based on conventional bacteria clone carrier, comprise multiple clone site MCS, incorporate site-specific recombinase system identification sequence in the MCS outside.
Wherein, conventional bacteria clone carrier includes but not limited to: pORF-MCS, pTransfer, pUC18 etc.
Multiple clone site MCS but be not limited to: 5 '-SalI-HindIII-EcoRI-SacI-KpnI-EcoRV-NotI-SphI-NcoI-XhoI-S maI-PacI-NheI-BamHI-3 '.This MCS direction also can be inverted, then carrier called after pHsvShuttle-MCS (-).
Site-specific recombinase system identification sequence includes but not limited to: the attP of the FRT sequence of the LoxP sequence of Cre enzyme identification, the identification of FLP enzyme, φ C31 and the identification of φ BT1 intergrase and/or attB sequence etc.And this site-specific recombinase system identification sequence is consistent with the site-specific recombinase system identification sequence among the homologous recombination precursor plasmid pRKBAC-MCS.
As required, shuttle plasmid pHsvShuttle-MCS portability or do not carry external source oncotherapy gene.
The present invention also provides the application of above-mentioned recombinant herpes simplex virus genetic operating system, is about to the recombinant herpes simplex virus that this recombinant herpes simplex virus genetic operating system is used to make up replication defect type.
Also can, this recombinant herpes simplex virus genetic operating system is used to make up the oncolytic hsv of selecting rf.
Above-mentioned homologous recombination precursor plasmid pRKBAC-MCS, HSV skeleton carrier pHsvEasy-X and shuttle plasmid pHsvShuttle-MCS have constituted multi-functional, universal recombinant herpes simplex virus genetic operating system jointly.This system is based on the BAC technology, and incorporated site specificity recombinase systems such as Cre-loxP, FLP-FRT, effectively improved the hsv genetic manipulation convenience and can be handling.Use basic Protocols in Molecular Biology, just can be widely used in this recombinant herpes simplex virus genetic operating system and set up various replication defect type recombinant herpes simplex virus and select rf oncolytic hsv, and be further used for therapy of tumor research.
In the embodiments of the invention, the electricity conversion E.coli competent cell that at first comprises HSV skeleton carrier pHsvEasy-X with the ordinary method preparation, E.coli electricity method for transformation according to routine, shuttle plasmid pHsvShuttle-MCS (+) and the above-mentioned E.coli competent cell that contains HSV skeleton carrier pHsvEasy-X of the common importing of the expression plasmid pET-FTP of intergrase (integrates) FTP, E.coli cell resistance screening after the electric shock, overnight incubation.Identify and obtain the FTP-FRT locus specificity resulting HSV-BAC plasmid of recombinating.
Then with the above-mentioned HSV-BAC plasmid DNA that obtains of conventional test kit mass preparation.The expression plasmid cotransfection Vero cell of HSV-BAC plasmid and intergrase Cre.After the conventional screening and culturing, difference harvested cell and supernatant liquor, multigelation cell-released virus particle.
Get a frozen viral liquid diluted sample, vero cells infection was cultivated 3-5 days, selected the plaque of 6-10 redfree fluorescence under fluorescent microscope, respectively the multigelation releasing virus.Go above-mentioned viral liquid inductance to dye and be incubated at the Vero cell, and a large amount of amplification cultivation, viral liquid gathered in the crops.Above-mentioned virus is identified with conventional Protocols in Molecular Biologies such as Southern Blot, PCR final resulting recombinant virus meets expection.
The recombinant herpes simplex virus genetic operating system that contains fluorescence protein gene of member is identical with expection too according to the method described above, illustrate that recombinant herpes simplex virus genetic operating system of the present invention is not only the efficient tool of setting up the replication defect type recombinant herpes simplex virus and selecting rf oncolytic hsv, and can effectively carry and express the external source goal gene.
The invention provides a kind of recombinant herpes simplex virus genetic operating system.This system is based on the foundation of bacterial artificial chromosome BAC plasmid skeleton, merge and go into site specificity recombinase systems such as Cre-loxP, FLP-FRT, comprise parts such as homologous recombination precursor plasmid pRKBAC-MCS, HSV skeleton carrier pHsvEasy-X and shuttle plasmid pHsvShuttle-MCS, effectively improve the convenience and the operability of hsv genetic manipulation, can be widely used in the foundation of replication defect type recombinant herpes simplex virus and selection rf oncolytic hsv.
Compare with other recombinant herpes simplex virus genetic operating system, the invention provides the recombinant herpes simplex virus genetic operating system and have more superiority and novelty, specifically mainly show: the whole genetic recombination operating process of (1) hsv mainly concentrates in the intestinal bacteria to be finished, convenient, fast and expense is cheap; (2) all the recombinant herpes simplex virus genetic operating system only comprises three plasmids, and component is simple, efficient.Wherein be associated with the HSV skeleton carrier with the source precursor plasmid and can conveniently clone the HSV of different sources, shuttle plasmid is associated with the HSV skeleton carrier and is convenient to the foreign gene insertion; (3) specially designed multiple clone site is particularly suitable for the clone of various HSV genomes and foreign gene in homologous recombination precursor plasmid, the shuttle plasmid; (4) this genetic operating system organically combines bacterial artificial chromosome technology and site-specific recombinase system, has solved big, the difficult problem of operating of HSV genome capacity; (5) this genetic operating system can be applicable to the foundation of replication defect type recombinant herpes simplex virus and selection rf oncolytic hsv simultaneously, does not need the independent system of apportion.
Description of drawings
Fig. 1 homologous recombination precursor plasmid pRKBAC-MCS physical map.
Fig. 2 shuttle plasmid pHsvShuttle-MCS physical map.
Embodiment
Embodiment 1
The foundation of homologous recombination precursor plasmid
Homologous recombination precursor plasmid is the basis of setting up the hsv skeleton carrier.The homologous recombination precursor plasmid pRKBAC-MCS of the embodiment of the invention is based on BAC plasmid cloning vector pBeloBACl1 (New England Biolabs) foundation.The dna replication dna initial point Ori2, the prokaryotic organism selectable marker gene Cm that comprise intestinal bacteria (E.coli) F-factor among the pBeloBACl1
r, it exists with single copy form in E.coli, can clone the dna fragmentation of 300kb and keep its stability.Adopt conventional molecule clone technology, in advance at replication origin Ori2 and selective marker Cm
rBetween insert red fluorescence marker gene RFP (Red Fluorescent Protein among the plasmid pDsRed1-N1 (Clontech), RFP), use artificial synthetic multiple clone site district (Mutiple Cloning Site then, MCS) replace cloning site, clonal selection marker gene lacZ, COS site and LoxP site among the pBeloBACl1,5 ' the end of this MCS carries the LoxP site, 3 ' end carries the FRT site, thus interstitial granules pRBAC-MCS in constituting.Middle interstitial granules pRBAC-MCS inserts eucaryon resistant gene Kan after the PacI enzyme is cut
rThereby, set up homologous recombination precursor plasmid pRKBAC-MCS (accompanying drawing 1 and SEQ No.1).
Embodiment 2
The foundation of hsv skeleton carrier
The hsv skeleton carrier is based on homologous recombination precursor plasmid pRKBAC-MCS, technology such as applying gene clone and homologous recombination import to the HSV genomic dna in the BAC plasmid, so are convenient to the HSV genome and preserve in E.coli, go down to posterity and carry out genetic manipulation.In the embodiment of the invention, with HSV-1 mutantion line G47 Δ (oncolytic HSV) is object, use homology arm haL and haR that conventional round pcr amplification is about 1.0-2.0kb, and, be built into homologous recombination plasmid pRKBAC-HA through the multiple clone site district that enzyme is cut insertion homologous recombination precursor plasmid pRKBAC-MCS.Homologous recombination plasmid pRKBAC-HA is mixed common transfection African green monkey kidney cell line Vero with G47 Δ genomic dna, carry out homologous recombination, gather in the crops progeny virus after 3 days.Then use the progeny virus vero cells infection, and cover agarose upper strata, select with the G418 resistance simultaneously with 0.75%, proceed conventional cell cultures 5-7 days after, under fluorescent microscope, select red G418 resistance plaque.Infecting multiplication once in the Vero cell after, these red resistance plaques continuation infect HEK293 then, the full cell DNA of results after 2 hours, and electricity commentaries on classics E.coli DHlOB (Invitrogen), and coat the agar plate that contains Cm (15 μ g/ml).Select some Cm resistance bacterium colonies, extracting BAC plasmid DNA is cut and pcr amplification evaluation recon with the HindIII enzyme.The genomic reorganization of the resulting HSV of comprising BAC plasmid promptly is HSV skeleton carrier pHsvEasy-X.
Embodiment 3
The foundation of shuttle plasmid
Shuttle plasmid is the carrier that imports foreign gene or gene fragment in HSV skeleton carrier pHsvEasy-X.In the embodiment of the invention, the shuttle plasmid skeleton derives from plasmid pORF-MCS (Invivogen).The method that connects by flush end replaces whole open reading frame (mend and put down end) among the pORF-MCS with artificial synthetic multiple clone site district MCS behind NdeI and PacI double digestion, comprise LoxP and FRT sequence at the two ends of MCS.Recombinant plasmid is according to direction of insertion difference called after pHsvShuttle-MCS (+) (accompanying drawing 2 and SEQNo.2) or the pHsvShuttle-MCS (-) of MCS.
Shuttle plasmid pHsvShuttle-MCS (+) can be further used for inserting external source oncotherapy gene, reporter gene or other need be cloned into dna fragmentation (as tumour-specific gene promoter etc.) in the HSV genome.The embodiment of the invention is further inserted green fluorescent protein reporter gene EGFP in pHsvShuttle-MCS (+) (Enhanced Green Fluorescent Protein, EGFP), complete EGFP expression cassette derives from plasmid pFUGW (Addgene).Recombinant plasmid called after pHsvShuttle-EGFP.
Embodiment 4
The foundation of the recombinant herpes simplex virus of replication defect type
The HSV skeleton carrier pHsvEasy-X of above-mentioned foundation and shuttle plasmid pHsvShuttle-MCS (+) or its plasmid of deriving can be directly used in the recombinant herpes simplex virus that makes up replication defect type.In the embodiment of the invention, the electricity conversion E.coli competent cell that at first comprises HSV skeleton carrier pHsvEasy-X with the ordinary method preparation, E.coli electricity method for transformation according to routine, the above-mentioned E.coli competent cell that contains HSV skeleton carrier pHsvEasy-X of the common importing of the expression plasmid pET-FTP of shuttle plasmid pHsvShuttle-MCS (+) and reporter gene pHsvShuttle-EGFP and intergrase (integrates) FTP, E.coli cell after the electric shock at first changes in the test tube that adds 500 lSOC contain tsiklomitsin (10 μ g/ml) 30 ℃ of shaking culture 1 hour over to, gets wherein 100 μ l then and changes over to and contain tsiklomitsin (10 μ g/ml), continued shaking culture 3 hours down at 30 ℃ among the 900 μ l SOC of ammonia benzyl (50 μ g/ml) and paraxin (15 μ g/ml).Get the above-mentioned culture of 50-250 μ l and coat the LB plate that contains ammonia benzyl (50 μ g/ml) and paraxin (15 μ g/ml), in 43 ℃ of following overnight incubation.From plate, select 6-10 single bacterium colony small-scale volume (5ml) overnight incubation under 37 ℃.Extract the DNA of above-mentioned BAC with conventional alkaline process, and cut with usual manner such as pcr amplification with the HindIII enzyme and to identify its reorganization result.Through above-mentioned FTP-FRT locus specificity recombinate resulting HSV-BAC plasmid called after pHsvEasy-nul1 and pHsvEasy-EGFP respectively.
Then use conventional test kit mass preparation (～250ml) pHsvEasy-nul1 and pHsvEasy-EGFP plasmid DNA.The expression plasmid pcDNA3-nCre of HSV-BAC plasmid pHsvEasy-nul1 and pHsvEasy-EGFP and intergrase Cre cotransfection Vero cell according to a conventional method, and in the DMEM that contains 6%FBS and G418 (300mg/ml), continue conventional the cultivation 60 hours.Difference harvested cell and supernatant liquor, multigelation cell-released virus particle, 4 ℃ of following 3500rpm removed cell debris in centrifugal 15 minutes, supernatant liquor were merged change in the above-mentioned viral liquid, were divided into some five equilibriums, and are frozen down in-80 ℃.Get a frozen viral liquid sample respectively with the DMEM dilution 10 that contains G418 (300mg/ml) and 2%FBS
3, 10
4, 10
5With 10
6Doubly, the 100 μ l that each extent of dilution is got wherein are used for infecting the Vero cell that is incubated at 96 orifice plates, 37 ℃ were descended conventional cell cultures 3-5 days, under fluorescent microscope, select the plaque of 6-10 redfree fluorescence, multigelation releasing virus respectively changes in the EP pipe viral supernatant liquor frozen down in-80 ℃ after centrifugal.Get above-mentioned frozen viral liquid 50 μ l steps infection and be incubated at the Vero cell, and a large amount of amplification cultivation, viral liquid gathered in the crops.Above-mentioned virus is identified that final resulting recombinant virus is called after oHSV-nul1 and oHSV-EGFP respectively with conventional Protocols in Molecular Biologies such as Southern Blot, PCR.
SEQUENCE?LISTING
＜110〉Fudan University
＜120〉a kind of recombinant herpes simplex virus genetic operating system and application thereof
<130>76
<160>2
<170>PatentIn?version?3.1
<210>1
<211>9725
<212>DNA
<213>Artificial
<400>1
agatctataa?cttcgtataa?tgtatgctat?acgaagttat?gtaccaggag?taggtaggat 60
ccctcgaggt?ttaaacgcgg?ccgcaagctt?gagctcttaa?ttaattcaaa?tatgtatccg 120
ctcatgagac?aataaccctg?ataaatgctt?caataatatt?gaaaaaggaa?gagtcctgag 180
gcggaaagaa?ccagctgtgg?aatgtgtgtc?agttagggtg?tggaaagtcc?ccaggctccc 240
cagcaggcag?aagtatgcaa?agcatgcatc?tcaattagtc?agcaaccagg?tgtggaaagt 300
ccccaggctc?cccagcaggc?agaagtatgc?aaagcatgca?tctcaattag?tcagcaacca 360
tagtcccgcc?cctaactccg?cccatcccgc?ccctaactcc?gcccagttcc?gcccattctc 420
cgccccatgg?ctgactaatt?ttttttattt?atgcagaggc?cgaggccgcc?tcggcctctg 480
agctattcca?gaagtagtga?ggaggctttt?ttggaggcct?aggcttttgc?aaagatcgat 540
caagagacag?gatgaggatc?gtttcgcatg?attgaacaag?atggattgca?cgcaggttct 600
ccggccgctt?gggtggagag?gctattcggc?tatgactggg?cacaacagac?aatcggctgc 660
tctgatgccg?ccgtgttccg?gctgtcagcg?caggggcgcc?cggttctttt?tgtcaagacc 720
gacctgtccg?gtgccctgaa?tgaactgcaa?gacgaggcag?cgcggctatc?gtggctggcc 780
acgacgggcg?ttccttgcgc?agctgtgctc?gacgttgtca?ctgaagcggg?aagggactgg 840
ctgctattgg?gcgaagtgcc?ggggcaggat?ctcctgtcat?ctcaccttgc?tcctgccgag 900
aaagtatcca?tcatggctga?tgcaatgcgg?cggctgcata?cgcttgatcc?ggctacctgc 960
ccattcgacc?accaagcgaa?acatcgcatc?gagcgagcac?gtactcggat?ggaagccggt 1020
cttgtcgatc?aggatgatct?ggacgaagag?catcaggggc?tcgcgccagc?cgaactgttc 1080
gccaggctca?aggcgagcat?gcccgacggc?gaggatctcg?tcgtgaccca?tggcgatgcc 1140
tgcttgccga?atatcatggt?ggaaaatggc?cgcttttctg?gattcatcga?ctgtggccgg 1200
ctgggtgtgg?cggaccgcta?tcaggacata?gcgttggcta?cccgtgatat?tgctgaagag 1260
cttggcggcg?aatgggctga?ccgcttcctc?gtgctttacg?gtatcgccgc?tcccgattcg 1320
cagcgcatcg?ccttctatcg?ccttcttgac?gagttcttct?gagaattcag?ctcgctgatc 1380
agcctcgact?gtgccttcta?gttgccagcc?atctgttgtt?tgcccctccc?ccgtgccttc 1440
cttgaccctg?gaaggtgcca?ctcccactgt?cctttcctaa?taaaatgagg?aaattgcatc 1500
gcattgtctg?agtaggtgtc?attctattct?ggggggtggg?gtggggcagg?acagcaaggg 1560
ggaggattgg?gaagacaata?gcaggcatgc?tggggatgcg?gtgggctcta?tggcttctga 1620
ggcggaaaga?accagctggg?gctcgattaa?ttaacgggtt?tcgatgaatt?gatccgaagt 1680
tcctattctc?tagaaagtat?aggaacttcg?aattgtcgac?caattctcat?gtttgacagc 1740
ttatcatcga?atttctgcca?ttcatccgct?tattatcact?tattcaggcg?tagcaaccag 1800
gcgtttaagg?gcaccaataa?ctgccttaaa?aaaattacgc?cccgccctgc?cactcatcgc 1860
agtactgttg?taattcatta?agcattctgc?cgacatggaa?gccatcacaa?acggcatgat 1920
gaacctgaat?cgccagcggc?atcagcacct?tgtcgccttg?cgtataatat?ttgcccatgg 1980
tgaaaacggg?ggcgaagaag?ttgtccatat?tggccacgtt?taaatcaaaa?ctggtgaaac 2040
tcacccaggg?attggctgag?acgaaaaaca?tattctcaat?aaacccttta?gggaaatagg 2100
ccaggttttc?accgtaacac?gccacatctt?gcgaatatat?gtgtagaaac?tgccggaaat 2160
cgtcgtggta?ttcactccag?agcgatgaaa?acgtttcagt?ttgctcatgg?aaaacggtgt 2220
aacaagggtg?aacactatcc?catatcacca?gctcaccgtc?tttcattgcc?atacggaatt 2280
ccggatgagc?attcatcagg?cgggcaagaa?tgtgaataaa?ggccggataa?aacttgtgct 2340
tatttttctt?tacggtcttt?aaaaaggccg?taatatccag?ctgaacggtc?tggttatagg 2400
tacattgagc?aactgactga?aatgcctcaa?aatgttcttt?acgatgccat?tgggatatat 2460
caacggtggt?atatccagtg?atttttttct?ccattttagc?ttccttagct?cctgaaaatc 2520
tcgataactc?aaaaaatacg?cccggtagtg?atcttatttc?attatggtga?aagttggaac 2580
ctcttacgtg?ccgatcaacg?tctcattttc?gccaaaagtt?ggcccagggc?ttcccggtat 2640
caacagggac?accaggattt?atttattctg?cgaagtgatc?ttccgtcaca?ggtatttatt 2700
cgcgataagc?tcatggagcg?gcgtaaccgt?cgcacaggaa?ggacagagaa?agcgcggatc 2760
tgggaagtga?cggacagaac?ggtcaggacc?tggattgggg?aggcggttgc?cgccgctgct 2820
gctgacggtg?tgacgttctc?tgttccggtc?acaccacata?cgttccgcca?ttcctatgcg 2880
atgcacatgc?tgtatgccgg?tataccgctg?aaagttctgc?aaagcctgat?gggacataag 2940
tccatcagtt?caacggaagt?ctacacgaag?gtttttgcgc?tggatgtggc?tgcccggcac 3000
cgggtgcagt?ttgcgatgcc?ggagtctgat?gcggttgcga?tgctgaaaca?attatcctga 3060
gaataaatgc?cttggccttt?atatggaaat?gtggaactga?gtggatatgc?tgtttttgtc 3120
tgttaaacag?agaagctggc?tgttatccac?tgagaagcga?acgaaacagt?cgggaaaatc 3180
tcccattatc?gtagagatcc?gcattattaa?tctcaggagc?ctgtgtagcg?tttataggaa 3240
gtagtgttct?gtcatgatgc?ctgcaagcgg?taacgaaaac?gatttgaata?tgccttcagg 3300
aacaatagaa?atcttcgtgc?ggtgttacgt?tgaagtggag?cggattatgt?cagcaatgga 3360
cagaacaacc?taatgaacac?agaaccatga?tgtggtctgt?ccttttacag?ccagtagtgc 3420
tcgccgcagt?cgagcgacag?ggcgaagccc?tcgattaaga?tacattgatg?agtttggaca 3480
aaccacaact?agaatgcagt?gaaaaaaatg?ctttatttgt?gaaatttgtg?atgctattgc 3540
tttatttgta?accattataa?gctgcaataa?acaagttaac?aacaacaatt?gcattcattt 3600
tatgtttcag?gttcaggggg?aggtgtggga?ggttttttaa?agcaagtaaa?acctctacaa 3660
atgtggtatg?gctgattatg?atctagagtc?gcggccggcc?gctacaggaa?caggtggtgg 3720
cggccctcgg?tgcgctcgta?ctgctccacg?atggtgtagt?cctcgttgtg?ggaggtgatg 3780
tccagcttgg?agtccacgta?gtagtagccg?ggcagctgca?cgggcttctt?ggccatgtag 3840
atggacttga?actccaccag?gtagtggccg?ccgtccttca?gcttcagggc?cttgtggatc 3900
tcgcccttca?gcacgccgtc?gcgggggtac?aggcgctcgg?tggaggcctc?ccagcccatg 3960
gtcttcttct?gcattacggg?gccgtcggag?gggaagttca?cgccgatgaa?cttcaccttg 4020
tagatgaagc?agccgtcctg?cagggaggag?tcttgggtca?cggtcaccac?gccgccgtcc 4080
tcgaagttca?tcacgcgctc?ccacttgaag?ccctcgggga?aggacagctt?cttgtagtcg 4140
gggatgtcgg?cggggtgctt?cacgtacacc?ttggagccgt?actggaactg?gggggacagg 4200
atgtcccagg?cgaagggcag?ggggccgccc?ttggtcacct?tcagcttcac?ggtgttgtgg 4260
ccctcgtagg?ggcggccctc?gccctcgccc?tcgatctcga?actcgtggcc?gttcacggtg 4320
ccctccatgc?gcaccttgaa?gcgcatgaac?tccttgatga?cgttcttgga?ggagcgcacc 4380
atggtggcga?ccggtggatc?tgagtccggt?agcgctagcg?gatctgacgg?ttcactaaac 4440
cagctctgct?tatatagacc?tcccaccgta?cacgcctacc?gcccatttgc?gtcaatgggg 4500
cggagttgtt?acgacatttt?ggaaagtccc?gttgattttg?gtgccaaaac?aaactcccat 4560
tgacgtcaat?ggggtggaga?cttggaaatc?cccgtgagtc?aaaccgctat?ccacgcccat 4620
tgatgtactg?ccaaaaccgc?atcaccatgg?taatagcgat?gactaatacg?tagatgtact 4680
gccaagtagg?aaagtcccat?aaggtcatgt?actgggcata?atgccaggcg?ggccatttac 4740
cgtcattgac?gtcaataggg?ggcgtacttg?gcatatgata?cacttgatgt?actgccaagt 4800
gggcagttta?ccgtaaatac?tccacccatt?gacgtcaatg?gaaagtccct?attggcgtta 4860
ctatgggaac?atacgtcatt?attgacgtca?atgggcgggg?gtcgttgggc?ggtcagccag 4920
gcgggccatt?taccgtaagt?tatgtaacgc?ggaactccat?atatgggcta?tgaactaatg 4980
accccgtaat?tgattactat?taataactaa?tgcatggcgg?taatacggtt?atccacagaa 5040
tcaggggata?acgcaggaaa?gaacatgtcg?agtgagcgag?gaagcaccag?ggaacagcac 5100
ttatatattc?tgcttacaca?cgatgcctga?aaaaacttcc?cttggggtta?tccacttatc 5160
cacggggata?tttttataat?tatttttttt?atagttttta?gatcttcttt?tttagagcgc 5220
cttgtaggcc?tttatccatg?ctggttctag?agaaggtgtt?gtgacaaatt?gccctttcag 5280
tgtgacaaat?caccctcaaa?tgacagtcct?gtctgtgaca?aattgccctt?aaccctgtga 5340
caaattgccc?tcagaagaag?ctgttttttc?acaaagttat?ccctgcttat?tgactctttt 5400
ttatttagtg?tgacaatcta?aaaacttgtc?acacttcaca?tggatctgtc?atggcggaaa 5460
cagcggttat?caatcacaag?aaacgtaaaa?atagcccgcg?aatcgtccag?tcaaacgacc 5520
tcactgaggc?ggcatatagt?ctctcccggg?atcaaaaacg?tatgctgtat?ctgttcgttg 5580
accagatcag?aaaatctgat?ggcaccctac?aggaacatga?cggtatctgc?gagatccatg 5640
ttgctaaata?tgctgaaata?ttcggattga?cctctgcgga?agccagtaag?gatatacggc 5700
aggcattgaa?gagtttcgcg?gggaaggaag?tggtttttta?tcgccctgaa?gaggatgccg 5760
gcgatgaaaa?aggctatgaa?tcttttcctt?ggtttatcaa?acgtgcgcac?agtccatcca 5820
gagggcttta?cagtgtacat?atcaacccat?atctcattcc?cttctttatc?gggttacaga 5880
accggtttac?gcagtttcgg?cttagtgaaa?caaaagaaat?caccaatccg?tatgccatgc 5940
gtttatacga?atccctgtgt?cagtatcgta?agccggatgg?ctcaggcatc?gtctctctga 6000
aaatcgactg?gatcatagag?cgttaccagc?tgcctcaaag?ttaccagcgt?atgcctgact 6060
tccgccgccg?cttcctgcag?gtctgtgtta?atgagatcaa?cagcagaact?ccaatgcgcc 6120
tctcatacat?tgagaaaaag?aaaggccgcc?agacgactca?tatcgtattt?tccttccgcg 6180
atatcacttc?catgacgaca?ggatagtctg?agggttatct?gtcacagatt?tgagggtggt 6240
tcgtcacatt?tgttctgacc?tactgagggt?aatttgtcac?agttttgctg?tttccttcag 6300
cctgcatgga?ttttctcata?ctttttgaac?tgtaattttt?aaggaagcca?aatttgaggg 6360
cagtttgtca?cagttgattt?ccttctcttt?cccttcgtca?tgtgacctga?tatcgggggt 6420
tagttcgtca?tcattgatga?gggttgatta?tcacagttta?ttactctgaa?ttggctatcc 6480
gcgtgtgtac?ctctacctgg?agtttttccc?acggtggata?tttcttcttg?cgctgagcgt 6540
aagagctatc?tgacagaaca?gttcttcttt?gcttcctcgc?cagttcgctc?gctatgctcg 6600
gttacacggc?tgcggcgagc?gctagtgata?ataagtgact?gaggtatgtg?ctcttcttat 6660
ctccttttgt?agtgttgctc?ttattttaaa?caactttgcg?gttttttgat?gactttgcga 6720
ttttgttgtt?gctttgcagt?aaattgcaag?atttaataaa?aaaacgcaaa?gcaatgatta 6780
aaggatgttc?agaatgaaac?tcatggaaac?acttaaccag?tgcataaacg?ctggtcatga 6840
aatgacgaag?gctatcgcca?ttgcacagtt?taatgatgac?agcccggaag?cgaggaaaat 6900
aacccggcgc?tggagaatag?gtgaagcagc?ggatttagtt?ggggtttctt?ctcaggctat 6960
cagagatgcc?gagaaagcag?ggcgactacc?gcacccggat?atggaaattc?gaggacgggt 7020
tgagcaacgt?gttggttata?caattgaaca?aattaatcat?atgcgtgatg?tgtttggtac 7080
gcgattgcga?cgtgctgaag?acgtatttcc?accggtgatc?ggggttgctg?cccataaagg 7140
tggcgtttac?aaaacctcag?tttctgttca?tcttgctcag?gatctggctc?tgaaggggct 7200
acgtgttttg?ctcgtggaag?gtaacgaccc?ccagggaaca?gcctcaatgt?atcacggatg 7260
ggtaccagat?cttcatattc?atgcagaaga?cactctcctg?cctttctatc?ttggggaaaa 7320
ggacgatgtc?acttatgcaa?taaagcccac?ttgctggccg?gggcttgaca?ttattccttc 7380
ctgtctggct?ctgcaccgta?ttgaaactga?gttaatgggc?aaatttgatg?aaggtaaact 7440
gcccaccgat?ccacacctga?tgctccgact?ggccattgaa?actgttgctc?atgactatga 7500
tgtcatagtt?attgacagcg?cgcctaacct?gggtatcggc?acgattaatg?tcgtatgtgc 7560
tgctgatgtg?ctgattgttc?ccacgcctgc?tgagttgttt?gactacacct?ccgcactgca 7620
gtttttcgat?atgcttcgtg?atctgctcaa?gaacgttgat?cttaaagggt?tcgagcctga 7680
tgtacgtatt?ttgcttacca?aatacagcaa?tagtaatggc?tctcagtccc?cgtggatgga 7740
ggagcaaatt?cgggatgcct?ggggaagcat?ggttctaaaa?aatgttgtac?gtgaaacgga 7800
tgaagttggt?aaaggtcaga?tccggatgag?aactgttttt?gaacaggcca?ttgatcaacg 7860
ctcttcaact?ggtgcctgga?gaaatgctct?ttctatttgg?gaacctgtct?gcaatgaaat 7920
tttcgatcgt?ctgattaaac?cacgctggga?gattagataa?tgaagcgtgc?gcctgttatt 7980
ccaaaacata?cgctcaatac?tcaaccggtt?gaagatactt?cgttatcgac?accagctgcc 8040
ccgatggtgg?attcgttaat?tgcgcgcgta?ggagtaatgg?ctcgcggtaa?tgccattact 8100
ttgcctgtat?gtggtcggga?tgtgaagttt?actcttgaag?tgctccgggg?tgatagtgtt 8160
gagaagacct?ctcgggtatg?gtcaggtaat?gaacgtgacc?aggagctgct?tactgaggac 8220
gcactggatg?atctcatccc?ttcttttcta?ctgactggtc?aacagacacc?ggcgttcggt 8280
cgaagagtat?ctggtgtcat?agaaattgcc?gatgggagtc?gccgtcgtaa?agctgctgca 8340
cttaccgaaa?gtgattatcg?tgttctggtt?ggcgagctgg?atgatgagca?gatggctgca 8400
ttatccagat?tgggtaacga?ttatcgccca?acaagtgctt?atgaacgtgg?tcagcgttat 8460
gcaagccgat?tgcagaatga?atttgctgga?aatatttctg?cgctggctga?tgcggaaaat 8520
atttcacgta?agattattac?ccgctgtatc?aacaccgcca?aattgcctaa?atcagttgtt 8580
gctctttttt?ctcaccccgg?tgaactatct?gcccggtcag?gtgatgcact?tcaaaaagcc 8640
tttacagata?aagaggaatt?acttaagcag?caggcatcta?accttcatga?gcagaaaaaa 8700
gctggggtga?tatttgaagc?tgaagaagtt?atcactcttt?taacttctgt?gcttaaaacg 8760
tcatctgcat?caagaactag?tttaagctca?cgacatcagt?ttgctcctgg?agcgacagta 8820
ttgtataagg?gcgataaaat?ggtgcttaac?ctggacaggt?ctcgtgttcc?aactgagtgt 8880
atagagaaaa?ttgaggccat?tcttaaggaa?cttgaaaagc?cagcaccctg?atgcgaccac 8940
gttttagtct?acgtttatct?gtctttactt?aatgtccttt?gttacaggcc?agaaagcata 9000
actggcctga?atattctctc?tgggcccact?gttccacttg?tatcgtcggt?ctgataatca 9060
gactgggacc?acggtcccac?tcgtatcgtc?ggtctgatta?ttagtctggg?accacggtcc 9120
cactcgtatc?gtcggtctga?ttattagtct?gggaccacgg?tcccactcgt?atcgtcggtc 9180
tgataatcag?actgggacca?cggtcccact?cgtatcgtcg?gtctgattat?tagtctggga 9240
ccatggtccc?actcgtatcg?tcggtctgat?tattagtctg?ggaccacggt?cccactcgta 9300
tcgtcggtct?gattattagt?ctggaaccac?ggtcccactc?gtatcgtcgg?tctgattatt 9360
agtctgggac?cacggtccca?ctcgtatcgt?cggtctgatt?attagtctgg?gaccacgatc 9420
ccactcgtgt?tgtcggtctg?attatcggtc?tgggaccacg?gtcccacttg?tattgtcgat 9480
cagactatca?gcgtgagact?acgattccat?caatgcctgt?caagggcaag?tattgacatg 9540
tcgtcgtaac?ctgtagaacg?gagtaacctc?ggtgtgcggt?tgtatgcctg?ctgtggattg 9600
ctgctgtgtc?ctgcttatcc?acaacatttt?gcgcacggtt?atgtggacaa?aatacctggt 9660
tacccaggcc?gtgccggcac?gttaaccggg?ctgcatccga?tgcaagtgtg?tcgctgtcga 9720
<210>2
<211>230l
<212>DNA
<213>Artificial
<400>2
gatccgaagt?tcctattctc?tagaaagtat?aggaacttcg?aattgtcgac?aagcttgatc 60
tggcttatcg?aaattaatac?gactcactat?agggagaccg?gaattcgagc?tcggtaccga 120
tatcgcggcc?gcgcatgctc?catggctcga?gcccgggtta?attaagctag?cggatccgga 180
acccttaata?taacttcgta?taatgtatgc?tatacgaagt?tattaggtcc?aagaacatgt 240
gagcaaaagg?ccagcaaaag?gccaggaacc?gtaaaaaggc?cgcgttgctg?gcgtttttcc 300
ataggctccg?cccccctgac?gagcatcaca?aaaatcgacg?ctcaagtcag?aggtggcgaa 360
acccgacagg?actataaaga?taccaggcgt?ttccccctgg?aagctccctc?gtgcgctctc 420
ctgttccgac?cctgccgctt?accggatacc?tgtccgcctt?tctcccttcg?ggaagcgtgg 480
cgctttctca?tagctcacgc?tgtaggtatc?tcagttcggt?gtaggtcgtt?cgctccaagc 540
tgggctgtgt?gcacgaaccc?cccgttcagc?ccgaccgctg?cgccttatcc?ggtaactatc 600
gtcttgagtc?caacccggta?agacacgact?tatcgccact?ggcagcagcc?actggtaaca 660
ggattagcag?agcgaggtat?gtaggcggtg?ctacagagtt?cttgaagtgg?tggcctaact 720
acggctacac?tagaagaaca?gtatttggta?tctgcgctct?gctgaagcca?gttaccttcg 780
gaaaaagagt?tggtagctct?tgatccggca?aacaaaccac?cgctggtagc?ggtggttttt 840
ttgtttgcaa?gcagcagatt?acgcgcagaa?aaaaaggatc?tcaagaagat?cctttgatct 900
tttctacggg?gtctgacgct?cagtggaacg?aaaactcacg?ttaagggatt?ttggtcatga 960
gattatcaaa?aaggatcttc?acctagatcc?ttttaaatta?aaaatgaagt?tttaaatcaa 1020
tctaaagtat?atatgagtaa?acttggtctg?acagttacca?atgcttaatc?agtgaggcac 1080
ctatctcagc?gatctgtcta?tttcgttcat?ccatagttgc?ctgactcccc?gtcgtgtaga 1140
taactacgat?acgggagggc?ttaccatctg?gccccagtgc?tgcaatgata?ccgcgagacc 1200
cacgctcacc?ggctccagat?ttatcagcaa?taaaccagcc?agccggaagg?gccgagcgca 1260
gaagtggtcc?tgcaacttta?tccgcctcca?tccagtctat?taattgttgc?cgggaagcta 1320
gagtaagtag?ttcgccagtt?aatagtttgc?gcaacgttgt?tgccattgct?acaggcatcg 1380
tggtgtcacg?ctcgtcgttt?ggtatggctt?cattcagctc?cggttcccaa?cgatcaaggc 1440
gagttacatg?atcccccatg?ttgtgcaaaa?aagcggttag?ctccttcggt?cctccgatcg 1500
ttgtcagaag?taagttggcc?gcagtgttat?cactcatggt?tatggcagca?ctgcataatt 1560
ctcttactgt?catgccatcc?gtaagatgct?tttctgtgac?tggtgagtac?tcaaccaagt 1620
cattctgaga?atagtgtatg?cggcgaccga?gttgctcttg?cccggcgtca?atacgggata 1680
ataccgcgcc?acatagcaga?actttaaaag?tgctcatcat?tggaaaacgt?tcttcggggc 1740
gaaaactctc?aaggatctta?ccgctgttga?gatccagttc?gatgtaaccc?actcgtgcac 1800
ccaactgatc?ttcagcatct?tttactttca?ccagcgtttc?tgggtgagca?aaaacaggaa 1860
ggcaaaatgc?cgcaaaaaag?ggaataaggg?cgacacggaa?atgttgaata?ctcatactct 1920
tcctttttca?atattattga?agcatttatc?agggttattg?tctcatgagc?ggatacatat 1980
ttgaatgtat?ttagaaaaat?aaacaaatag?gggttccgcg?cacatttccc?cgaaaagtgc 2040
cacctgacgt?ctaagaaacc?attattatca?tgacattaac?ctataaaaat?aggcgtatca 2100
cgaggccctt?tcgtctcgcg?cgtttcggtg?atgacggtga?aaacctctga?cacatgcagc 2160
tcccggagac?ggtcacagct?tgtctgtaag?cggatgccgg?gagcagacaa?gcccgtcagg 2220
gcgcgtcagc?gggtgttggc?gggtgtcggg?gctggcttaa?ctatgcggca?tcagagcaga 2280
ttgtactgag?agtgcaccat?a 2301
Claims (10)
1. a recombinant herpes simplex virus genetic operating system is characterized in that, this recombinant herpes simplex virus genetic operating system comprises HSV skeleton carrier and shuttle plasmid; Described HSV skeleton carrier comprises HSV genomic dna, BAC plasmid skeleton, multiple clone site, eucaryon resistant gene and protokaryon resistant gene, incorporates site-specific recombinase system identification sequence in the outside of multiple clone site and eucaryon resistant gene; Described shuttle plasmid is the bacteria clone carrier that comprises multiple clone site, and incorporates site-specific recombinase system identification sequence in the multiple clone site outside.
2. recombinant herpes simplex virus genetic operating system according to claim 1 is characterized in that, described site-specific recombinase system identification sequence is one or several in LoxP sequence, FRT sequence, attP or the attB sequence.
3. recombinant herpes simplex virus genetic operating system according to claim 1, it is characterized in that described multiple clone site contains following one or several restriction enzyme sites: HindIII, EcoRI, SacI, KpnI, EcoRV, NotI, SphI, NcoI, XhoI, SmaI, PacI, NheI, BamHI, PmeI or SalI.
4. recombinant herpes simplex virus genetic operating system according to claim 1 is characterized in that, described protokaryon resistant gene is chloramphenicol resistance gene or tetracycline resistance gene.
5. recombinant herpes simplex virus genetic operating system according to claim 1, it is characterized in that described eucaryon resistant gene is one or several in kalamycin resistance gene, hygromycin gene, neomycin resistance gene or the gigohm mycin resistant gene.
6. recombinant herpes simplex virus genetic operating system according to claim 1 is characterized in that, this recombinant herpes simplex virus genetic operating system also comprises recombinase.
7. recombinant herpes simplex virus genetic operating system according to claim 6 is characterized in that, described recombinase is Cre enzyme, FRT enzyme, C31 or BTl intergrase.
8. the preparation method of recombinant herpes simplex virus genetic operating system according to claim 1, it is characterized in that, at first, make up homologous recombination precursor plasmid, this plasmid comprises multiple clone site MCS, fluorescence protein gene, eucaryon resistant gene and protokaryon resistant gene, incorporates site-specific recombinase system identification sequence in the outside of MCS and eucaryon resistant gene; Secondly, genomic two the homology arm DNA of HSV have been inserted among the homologous recombination precursor plasmid pRKBAC-MCS; Once more, make up shuttle plasmid, incorporate site-specific recombinase system identification sequence in the multiple clone site MCS2 outside of bacteria clone carrier.
9. the application of the described recombinant herpes simplex virus genetic operating system of claim 1 in the recombinant herpes simplex virus that makes up replication defect type.
10. the application of the described recombinant herpes simplex virus genetic operating system of claim 1 in making up the oncolytic hsv of selecting rf.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019149288A1 (en) * | 2018-02-02 | 2019-08-08 | 杭州菁因康生物科技有限公司 | Efficient genetic engineering vector |
| WO2020109389A1 (en) | 2018-11-28 | 2020-06-04 | Innovative Molecules Gmbh | Helicase primase inhibitors for treating cancer in a combination therapy with oncolytic viruses |
| WO2024001845A1 (en) * | 2022-06-28 | 2024-01-04 | 中国科学院上海药物研究所 | Preparation method and use of defective filovirus |
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2009
- 2009-11-10 CN CN2009101985762A patent/CN102051379A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019149288A1 (en) * | 2018-02-02 | 2019-08-08 | 杭州菁因康生物科技有限公司 | Efficient genetic engineering vector |
| WO2020109389A1 (en) | 2018-11-28 | 2020-06-04 | Innovative Molecules Gmbh | Helicase primase inhibitors for treating cancer in a combination therapy with oncolytic viruses |
| WO2024001845A1 (en) * | 2022-06-28 | 2024-01-04 | 中国科学院上海药物研究所 | Preparation method and use of defective filovirus |
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Application publication date: 20110511 |