CN1109754C - Process for preparing full-function helper virus used for production of recombinant adeno-associated virus and its usage - Google Patents
Process for preparing full-function helper virus used for production of recombinant adeno-associated virus and its usage Download PDFInfo
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Abstract
The present invention comprises a method for producing a recombinant herpes simplex virus (HSV1-rc) loading 2 type adeno-associated virus (AAV-2) rep-cap gene and the application of the method in the production of the recombinant adeno-associated virus (rAAV). The recombinant herpes simplex virus can provide all the desired auxiliary functions of copying and packaging plasmids into rAAV virions in cells and can be used for the large-scale preparation of rAAV. HSC1-rc is generated on the basis of a set of cosmids (SetC cosmids comprising cos 6, cos 14, cos 28, cos 48 and cos 56) containing HSV1 virus complete genomes. First, the AAV-2 rep-cap gene is inserted into an HSV-1 genome of one cosmid in a base by using a recombinant DNA technique, for example, the AAV-2 rep-cap gene is inserted into the HSV1UL2 gene of cos 6 to construct cos 6-rcdelta UL2 and inserted into the HSV1UL44 gene of cos 56 to construct cos 56-rcdelta UL 44; then, skeleton parts of the recombinant cosmid in which the rep-cap is inserted and other four corresponding cosmids are cut by enzymic cutting, a liposome method is used for the cotransfection of HSV11 sensitive cells such as BHK-21, and 5 HSV1 fragments carries out homologous recombinant in the cells to generate HSV1-rc. HSV1-rc is used for infecting the cells transfected by rAAV vector plasmids or stably carrying cell strains of the rAAV vector plasmids, which can produce a large amount of rAAV virions with infection. The rAAV produced by using the method can lead the exogenous gene in mammalian cells and express the exogenous gene.
Description
(1) invention field:
The invention belongs to the viral genetic engineering field, be specifically related to required the duplicating and the packaging function system of mass production recombinant adeno-associated virus (rAAV).The present invention relates to a kind of production method and purposes in recombinant adeno-associated virus (rAAV) is produced thereof of having loaded the recombinant herpes simplex virus (HSV1-rc) of 2 type adeno associated virus (AAV-2) rep-cap genes (4.3kb).This recombinant herpes simplex virus can provide the rAAV plasmid in time multiplexed cell system and be packaged into the required whole subsidiary functions of rAAV virion, and can be used for a large amount of preparations of rAAV.The generation of HSV1-rc be a cover is contained the complete genomic cosmid of HSV1 virus (the SetC clay comprises cos6, cos14, cos28, cos48 realizes on the basis of cos56) transforming.Infect rAAV vector plasmid cells transfected or the stable cell strain that carries the rAAV vector plasmid with HSV1-rc, just can produce has infective rAAV virion in a large number.The rAAV of Chan Shenging can import foreign gene in the mammalian cell and express in this way.
(2) background technology:
(adeno-associated virus AAV) is Parvoviridae (parvovirus) member to adeno associated virus, and its genome is the single stranded DNA that 4682 Nucleotide are formed.AAV is a dependovirus, needs other virus as adenovirus or hsv, or cofactor provides just reproducible of subsidiary function.When not having helper virus to exist, its genome will be incorporated into behind the AAV cells infected becomes latent state in the cell chromosome, and does not produce progeny virus.
The full-length gene group of AAV-2 is cloned in the escherichia coli plasmid.Wherein contain the long reversing terminal repeat of 2 each 145bp (Inverted terminal repeat, ITR).They are the genomic replication orgin of AAV, and duplicate, integrate with AAV or function such as packing relevant.Its genome rest part can be divided into 2 functional zone, rep gene regions and cap gene regions.The rep gene has the product of 4 kinds of different forms: Rep78, Rep68, Rep52, Rep40.They duplicate and necessary adjusting albumen such as viral gene expression for AAV.3 kinds of structural protein of cap genes encoding, VP1, VP2, VP3, mutual group is dressed up the shell of AAV virus.The albumen of rep and cap genes encoding all is trans-acting albumen in the AAV toxigenicity is duplicated.
AAV is considered to one of gene therapy ideal candidate carrier.Many laboratories have all made up can be with the rAAV virus of foreign gene transfered cell.The primary structure feature of AAV vector plasmid is that the rep-cap gene is excised from viral genome, replaces required dna fragmentation.
The classical way that produces rAAV virus is for importing rAAV vector plasmid and the helper plasmid that contains the rep-cap gene in the cell of adenovirus or herpes simplex infections.From the cell of culture supernatant and pathology, rAAV virus be can gather in the crops after 2-3 days, used adenovirus or hsv also contained simultaneously.Adenovirus and hsv all available heat are handled (55 ℃ 30 minutes to 2 hours) and deactivation, but do not influence the activity of AAV virus.
Though the method for this production rAAV virus is fairly simple, but still has many significant disadvantages.All need transfectional cell when preparing rAAV virus at first, at every turn.Because the restriction of transfection method self, transfection and cotransfection efficient are lower, are to produce one of lower reason of rAAV virus titer.And, also be difficult to extensive transducer cell at present with transfection method, so the needs of incompatibility mass production rAAV virus.Therefore, be necessary to study a kind of system and method that can be used for mass production rAAV virus.
The patent of invention of Yan Ziying etc. 1996 applications once " can be used to herpes simplex virus vector of packing recombinant adeno-associated virus and uses thereof " by name (Chinese patent application number 96 1 20549.0, publication number CN 1159480A).This patent has been introduced and a kind of AAV-2 rep-cap gene has been placed HSV1 amplicon vector plasmid, is built into pHSV-AAV (+/-).In this plasmid transfered cell, in the presence of the HSV-1 wild-type virus, can obtain a kind of wild-type HSV-1 and the hybrid virus that contains the pseudovirus of rep-cap gene, this hybrid virus has whole subsidiary functions that rAAV virus replication and packing are provided.(Conway JE et al such as nearest Conway, Recombinant adeno-associated virus type 2 replication and packaging isentirely supported by a herpes simplex virus type 1 amplicon expressmg rep and cap J.Virol.71 (11): 8780-8789,1997) also reported similar research.But, the shared ratio less (＜10%) of pseudovirus in this hybrid virus, the subsidiary function that can provide is limited; And the ratio of pseudovirus and wild-type virus is unfixing in virus goes down to posterity, and is unfavorable for mass-produced quality control.
(3) summary of the invention:
The object of the present invention is to provide and be used for easy and technological method and global function helper virus HSV-rc mass production rAAV virus.The objective of the invention is by the reorganization clay that contains the rep-cap gene and construction process, HSV-rc virus and construction process thereof being provided and producing with HSV-rc that the method for rAAV virus realizes.
The global function helper virus HSV-rc that the present invention proposes is the HSV-1 virus of reorganization, it is characterized in that having inserted in the HSV-1 genome rep-cap gene (4.3kb, direction does not limit) of a copy.Among the constructed 2 kinds of HSV-rc of the present invention, the rep-cap gene is inserted in respectively in the XbaI site of HSV-1UL2 gene (coding uracil dna glycosylase) and HSV-1UL44 gene (coding gC), and (Fig. 1 a) and HSV-rc/ Δ UL44 (Fig. 1 b) to be called HSV-rc/ Δ UL2.UL2 and UL44 gene product for HSV-1 in cultured cell in vitro propagation and go down to posterity optional.These 2 kinds reorganization HSV1 viruses all can be bred in HSV sensitive cells (as BHK-21) and stable going down to posterity.
The HSV-rc virus that the present invention produced is by a cover being contained complete genomic cosmid (the SetC clay of HSV1 virus, comprise cos6, cos14, cos28, cos48, cos56) (Conningham C, Davision AJ.A cosmid-based system forconstructing mutants of herpes simplex virus type 1.Virology, 1993, produce on the basis of 197:116-124) transforming.At first, with recombinant DNA technology AAV-2 rep-cap gene is inserted in the contained HSV-1 genome of one of them clay.Then, the reorganization clay that has inserted rep-cap is gone to use liposome method cotransfection HSV1 sensitive cells such as BHK-21 after the clay skeleton part through enzyme with corresponding all the other 4 clays earnestly, and homologous recombination takes place and produces recombinant virus in 5 HSV-1 fragments in cell.
The rAAV vector plasmid cells transfected that contains reporter gene GFP (green fluorescence protein gene) with this reorganization HSV1 virus infection, the cell pyrolysis liquid supernatant that obtains is used to infect the mammalian cell of cultivation, and (excitation wavelength 490nm) can see a large amount of green cells under fluorescent microscope.The rAAV virus that shows generation has infectivity, and can will express in the foreign gene transfered cell.
The present invention is built into the recombinant plasmid pEBUF5 that contains the GFP gene on the basis of pBDZ (+) plasmid (Chinese patent application number 97 1 16981.0).This plasmid is imported 293c18 cell (ATCC CRL10852, F9766, the EBNA1 gene that contains and express EBV virus in this cell), the HygromycinB resistant cell strain called after 293c18/EBUF5 of acquisition.With the stable cell strain 293c18/EBUF5 that carries rAAV vector plasmid pEBUF5 of HSV1-rc virus infection, can produce easily has infective rAAV/GFP virion in a large number, and this method can realize the batch process of rAAV virus.
The primeval life material that the present invention is used for construction of recombinant virus HSV-rc has: the SetC clay: by load sharing successively complete genomic 5 clays of HSV-1 virus form: cos6, cos14 cos28, cos48, cos56 (Conningham C, Davision AJ.A cosmid-based system for constructing mutants ofherpes simplexvirus type 1.Virology, 1993,197:116-124).For Davision AJ gives.The segmental end of each HSV-1 viral genome that loads in this cover clay repeats with the segmental end sequence of HSV-1 that is loaded in another clay, and this is the basis that homologous recombination takes place in cell 5 HSV-1 genomic fragments.pSub201：Samulski?et?al，A?recombinant?plasmid?from?which?an?infectious?adeno-associated?virusgenome?can?be?excited?in?vitro?and?its?use?to?study?viral?replication。The preparation method of HSV-rc recombinant virus:
Adopt and identical strategy and the method for preparation HSV1-lacZ100 recombinant virus (Wu Xiaobing etc., Chinese patent application number 98101753.3).
An XbaI single endonuclease digestion site is respectively arranged in the HSV-1 genomic fragment of the loading in cos6 and the cos56 clay, lay respectively in UL2 and the UL44 gene.With XbaI the rep-cap gene is cut out from pSub201, insert respectively in the XbaI site of cos6 and cos56, (Fig. 2 a) and cos56-rc Δ UL44 (Fig. 2 b) to be built into reorganization clay cos6-rc Δ UL2.Two kinds the reorganization clay all be deposited at the bacillus coli DH 5 alpha strain (MAX EFFICIANCY DH5 α, GIBCO#18258-012) in.The bacterial strain that contains these two reorganization clays is stored in China Microbial Culture Preservation Commission common micro-organisms center on September 24th, 1998, and registering on the books is numbered CGMCC No.0361+1,2.
With cos6-rc Δ UL2 and cos14, cos28, cos48, the cos56 combination is called SetH, and (Fig. 2 is a); With cos56-rc Δ UL44 and cos6, cos14, cos28, the cos48 combination is called SetI (Fig. 2 b).The moles such as 5 clays of pressing SetH and SetI mix, cut the cos skeleton with the PacI enzyme, with liposome cotransfection BHK-21 cell, 5 HSV-1 fragments in cell homologous recombination take place and produce the HSV-rc recombinant virus: cell begins to occur pathology after 5 days, treat to receive the nutrient solution supernatant after the complete pathology of cell, the centrifugal 5min of 2000r/min, the supernatant packing is stored in-20 ℃.With the reorganization HSV-rc called after HSV1-rc/ Δ UL2 (accompanying drawing 1a) of SetH clay generation and the reorganization HSV-rc name HSV1-rc/ Δ UL44 (accompanying drawing 1b) that the SetI clay is produced.The probability of the reorganization HSV-1 virus that contains target DNA fragment that produces with this method reaches 50～100%.Be easy to obtain single recombinant virus by plaque select.
Embodiment:
Following examples have been done detailed description to preparation and the purposes that is used for the global function helper virus of recombinant adeno-associated virus production of the present invention, but and do not mean that the restriction content of the present invention.
The preparation of embodiment 1 cosmid DNA
With reference to Molecular Cloning--A Laboratory Manual, 2nd ed. (Sambrook J.et al, 1986) extracts cosmid DNA with the method that alkaline lysis prepares plasmid DNA in a large number, uses the polyethylene glycol precipitation purifying.The preparation of embodiment 2 reorganization HSV-rc
Respectively with SetH (cos6-rc Δ UL2 and cos14, cos28, cos48, cos56) and SetI (cos56-rc Δ UL44 and cos6, cos14, cos28, mole such as 5 clays cos48) mixes, and cuts cos skeleton (needn't separate removal) with the PacI enzyme, with phenol, phenol/chloroform (1: 1) and each extracting of chloroform once, draw supernatant, with 2.5 times of first water-ethanol deposit D NA.The BHK-21 cell (about 2 * 10 that is paved with by product description cotransfection 80% with lipofactamine (GIBCO BRL) 20ul and 10ug DNA
6) cell, homologous recombination will take place and produce the HSV-rc recombinant virus in 5 HSV-1 fragments in cell.Use the 37 ℃ of cultivations of RPMI-1640 that contain 2%FBS behind the transfection 24h instead, change liquid every day once.Cell begins to occur pathology after 5 days, treat to receive the nutrient solution supernatant after the complete pathology of cell, and the centrifugal 5min of 2000r/min, the supernatant packing is stored in-20 ℃.With the reorganization HSV-rc called after HSV1-rc/ Δ UL2 that the SetH clay produces, the reorganization HSV-rc that the SetI clay is produced names HSV1-rc/ Δ UL44.The recombinant virus that obtains is carried out the plaque purifying twice, can obtain single HSV1-rc/ Δ UL2 and HSV1-rc/ Δ UL44.The foundation of embodiment 3 293c18/pEBUF5 cell strains
Be built into the recombinant plasmid pEBUF5 that contains the GFP gene on the basis of pBDZ (+) plasmid (Chinese patent application number 97 1 16981.0), its structure is seen accompanying drawing 3.This plasmid is imported the 293c18 cell with liposome method, and (ATCC CRL10852 F9766), selects to cultivate 10-15d, the resistant cell strain called after 293c18/EBUF5 of acquisition with Hygromycin B 200ug/ml.Embodiment 4 usefulness HSV-rc infect the pAAV-GFP cells transfected and prepare rAAV-GFP
The rAAV vector plasmid cells transfected that contains reporter gene GFP (green fluorescence protein gene) with this reorganization HSV1 virus infection, 4 cracking cells of cytopathy (36-72h) back multigelation are to discharge the rAAV-GFP in the cell, low-speed centrifugal is removed cell debris, get 56 ℃ of deactivation 30min of supernatant, be used to infect the mammalian cell of cultivation.Embodiment 6 usefulness HSV-rc infect the 293c18/pEBUF5 cell strain and prepare rAAV-GFP
With the stable cell strain 293c18/EBUF5 that carries rAAV vector plasmid pEBUF5 of the HSV1-rc virus infection of 0.5～5moi, complete pathology takes place in cell behind 24～48h, with cell and its nutrient solution multigelation together 4 times, the centrifugal 5min of 1000r/min promptly contains a large amount of rAAV-GFP virus in the supernatant.Can produce easily in this way has infective rAAV/GFP virion in a large number, and can realize the batch process of rAAV virus.Embodiment 7 rAAV virus transduction culturing cell
Get rAAV-GFP virus supernatant 1ml and add in the bhk cell of cultivating (80% is paved with), (excitation wavelength 490nm) observation under fluorescent microscope can be seen a large amount of green cells behind the 24-48h.The rAAV virus that shows generation has infectivity, and can will express in the foreign gene transfered cell.
Claims (11)
1. carry the recombinant human herpes simplex types 1 virus HSV1-rc of the rep-cap gene order of 2 type AAV viruses in the genome.
2. the described hsv HSV1-rc of claim 1, wherein the rep-cap gene is inserted in the Xbal site of UL2 gene of the coding uracil dna glycosylase of HSV-1.
3. the described hsv HSV1-rc of claim 1, wherein the rep-cap gene is inserted in the Xbal site of UL44 gene of the coding gC of HSV-1.
4. carry the method for recombinant human herpes simplex types 1 virus HSV1-rc of the rep-cap gene order of 2 type AAV viruses in the producer gene group, comprise by a cover is contained the complete genomic clay SetC of HSV-1 and carry out genetic manipulation, the rep-cap gene is inserted in the HSV-1 genomic fragment, with itself and all the other 4 clay cotransfection cells, obtain to contain the reorganization HSV1-rc virus of rep-cap then.
5. according to the method for claim 4, wherein the rep-cap gene is the XbaI single endonuclease digestion site that is inserted in the UL2 among the cos6.
6. according to the method for claim 4, wherein the rep-cap gene is the XbaI single endonuclease digestion site that is inserted in the UL44 among the cos56.
7. recombinant plasmid cos6-rc Δ UL2, this plasmid carries out genetic manipulation by a cover is contained the complete genomic clay SetC of HSV-1, and the XbaI single endonuclease digestion site that the rep-cap gene is inserted into the UL2 among the cos6 obtains.
8. recombinant plasmid cos56-rc Δ UL44, this plasmid carries out genetic manipulation by a cover is contained the complete genomic clay SetC of HSV-1, and the XbaI single endonuclease digestion site that the rep-cap gene is inserted into the UL44 among the cos56 obtains.
9. intestinal bacteria (Escherichia coli) the DH5 α strain CGMCC No.0361-1 that comprises the described recombinant plasmid cos6-rc of claim 7 Δ UL2.
10. intestinal bacteria (Escherichia coli) the DH5 α strain CGMCC No.0361-2 that comprises the described recombinant plasmid cos56-rc of claim 7 Δ UL44.
11., be used for the preparation and the production of rAAV virus according to the described recombinant human herpes simplex types 1 virus of claim 1 HSV1-rc.
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| ES2371546T3 (en) * | 2001-11-13 | 2012-01-05 | The Trustees Of The University Of Pennsylvania | A PROCEDURE FOR DETECTION AND / OR IDENTIFICATION OF SEQUENCES OF ADENO-ASSOCIATED VIRUSES (AAV) AND ISOLATION OF NEW SEQUENCES AS? IDENTIFIED |
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| AU2003272868A1 (en) * | 2003-10-15 | 2005-04-27 | Agtc Gene Technology Company Ltd. | Method for large-scale production, isolation, purification and the uses of multi-type recombinant adeno-associated virus vectors |
| CN102676580B (en) * | 2012-05-30 | 2013-06-19 | 扬州大学 | Preparation method of herpes simplex virus (HSV) gG1 transgenic peanuts with high immunogenicity |
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| CN1159480A (en) * | 1996-12-13 | 1997-09-17 | 中国预防医学科学院病毒学研究所 | Monobleb viras currier capable of being used in packaging recombination of adenovirus concomitant virus and use thereof |
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| CN1159480A (en) * | 1996-12-13 | 1997-09-17 | 中国预防医学科学院病毒学研究所 | Monobleb viras currier capable of being used in packaging recombination of adenovirus concomitant virus and use thereof |
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