CN1139656C - Quick high-effective separation and purification method of virus related to rocombinant adenovirus and its application - Google Patents

Abstract

The present invention provides a method for fast and effectively separating and purifying rAAV viruses through a PEG/NaCl system and chloroform by utilizing the characteristic of chloroform treatment resistance of an AAV virus particle. The method is suitable for the separation and the purification of rAAV viruses in a large scale and technologized production, and is particularly suitable for the purification of rAAV produced by taking HSV viruses as helper viruses. The present invention can also be used for the purification of rAAV produced by a non-helper virus packing system or a non-cell in-vitro packing system. RAAV viruses purified by the present invention can be used for gene transfer and gene therapy.

Description

The method and the purposes of a kind of separation rapidly and efficiently and purification of Recombinant adenovirus correlated virus

Technical field the invention belongs to biological technical field, is specifically related to the separation of recombinant adeno-associated virus, concentrated and purification process, especially is fit to mass preparation reorganization AAV virus to be used for transgenosis and gene therapy.

The background technology virus vector is widely used in transgenosis and gene therapy.Adeno-associated virus (Adeno-associated virus wherein, AAV) thus more and more receive publicity because of having the characteristics such as long-term stability expression that no pathogenicity, physico-chemical property are stable, immunogenicity is weak, adjustable point is incorporated into mediate foreign gene in the eukaryotic cell karyomit(e), have broad application prospects in transgenosis and field of gene.

People's 2 type adeno-associated viruses (AAV-2) are the present the most frequently used adeno-associated viruses of making virus vector.AAV virus is helper-dependent virus, and its infection duplication needs the auxiliary of adenovirus or hsv etc.

The particle diameter of AAV-2 virus is 20～24nm, is 20 body structures.Virus is made up of 20～25%DNA and 75%～80% protein.Buoyant density in cesium chloride solution is 1.41g/cm ³Genome is the single stranded DNA of 4.7kb.Respectively there is the tumor-necrosis factor glycoproteins (ITR) of one section 145bp at the genome two ends, are the distinctive cis-acting elements of AAV virus, by the duplicating of AAV virus, integrate, save, packing etc. is essential.Be the encoding gene of AAV between the ITRs, the left side be the rep gene, the 4 kinds of Rep albumen Rep78 that encode, Rep68, Rep52, Rep40; The right be the cap gene, the 3 kinds of coat protein VP1 that encode, VP2, VP3, molecular weight is followed successively by 87,72,62KDal.VP3, VP2, the proteic ratio of VP1 are about 10: 1: 1 in the AAV virion.

(Cloning of adeno-associated virus into pBR322:rescue of intact virusfrom the recombinant plasmid in human cells such as Samulski RJ, Proc.Natl.Acad.Sci.USA, 79:2077-2031,1982) with complete double-stranded AAV dna clone in the pBR322 plasmid, find that the AAV provirus genome be arranged in plasmid also has the ability that forms infectious virus.Therefore exogenous gene expression unit is placed between two ITR of AAV virus, the function of rep and cap gene and helper virus then provides by alternate manner is trans, can obtain to contain the reorganization AAV virus of foreign gene in cell.

(recombinant adeno-associated virus, rAAV) the particulate structure is identical with wild-type AAV for recombinant adeno-associated virus.Different is that the genome of reorganization AAV virion packing is the foreign DNA that the two ends of strand have AAV ITR sequence.Open ended foreign DNA length is in 5.0kb in the reorganization AAV virus.

Classical rAAV production method (Xiao Xiao, Richard Jude Samulski Current Protocols inHuman Genetics, Vectors for Gene Therapy, Unit 12.1,1996) be to infect as 5 type adenovirus (Ad5) with two plasmid co-transfection 293 cells and helper virus.One is reorganization AAV vector plasmid in two plasmids, and another is the helper plasmid that contains AAV rep-cap gene.Because this method operation is complicated, the factor that influences the rAAV generation is many, is difficult to obtain the rAAV of high titre, is not easy to expand the scale of production.Therefore many investigators are devoted to improve the production method of reorganization AAV virus (rAAV).These improvement comprise following a few class: 1) with the repcap gene transfer in cell strain, build up a kind of package cell line.The expression of repcap wherein is subjected to promotor control (the Kenji Tamayose of himself, Yukihiko Hirai, and Takashi Shimada, A new strategy for large-scalepreparation of high-titer recombinant adeno-associated virus vectors by using packaging celllines and sulfalonated cellulose column chromatography, Human Gene Therapy7:507-513,1996), or change other composing type or inducible promoters (Inoue N into, Russell DW.Packagingcells based on inducible gene amplification for the production of adeno-associated virusvectors.J.Virol 72:7024-7031,1998); 2) AAV ITRs and the DNA of exogenous gene expression unit are therebetween inserted in the adenoviral gene group, be built into a kind of mosaic type recombinant adenovirus (Guang-Ping Gao, Guang Qu et al.High-titer adeno-associated viral vectors from a rep/cap cell line and hybrid shuttle virusHuman Gene Therapy 9:2353-2362,1998; Liu XL, Clark KR, Johnson PR Production ofrecombinant adeno-associated virus vectors using a packaging cell line and a hybridrecombinant adenovirus.Gene Ther 1999 Feb; 6 (2): 293-9).3) AAV ITRs and the DNA of exogenous gene expression unit are therebetween placed the EBV virus vector of self-replicating, transduce to cell, build up a kind of cell strain (Yan Ziying that carries reorganization AAV vector plasmid with self-replicating ability, Yao Ermei etc., novel reorganization AAV carrier package system based on the EBV replicon, Science Bulletin, 42 (17): 1860-1863,1997).4) the repcap gene is inserted in the adenoviral gene group, be built into a kind of recombinant adenovirus with complete subsidiary function.Yet manyly experiment showed, that this thinking is difficult to realize.May be because rep albumen can't produce the feasible recombinant adenovirus that contains the repcap gene of the strongly inhibited effect of adenovirus.5) carry the repcap gene with HSV virus amplification, generation has HSV hybrid virus (the James E.Conway of complete subsidiary function, Sergei Zolotukhin et al.Recombinant adeno-associated virus type 2 replication and packaging id entirely supportedby a herpes simplex virus type 1 amplicon expressing rep and cap, J Vorol 71 (11): 8780-8789,1997; The Su Yuelong, Wu Xiaobing, Yang Tianzhong, tribute favour space, Hou Yunde, novel reorganization AAV carrier package system's Chinese science (C collects) 28 (5) that the sub-Ying of face makes up with the HSV-1 amplicon vector: 457-462,1998).6) rAAV virosome outer packaging (acellular packing) (J Virol 72 (4): 3241-3247,1998 for Zhou XH, Muzyczka N.In vitro packaging ofadeno-associated virus DNA).

We had once proposed the production strategy (Chinese patent application number: 98120033.8 and 99119039.4 of " an a strain vector cell/strain helper virus "; Wu Zhijian, Wu Xiaobing etc., generation with reorganization HSV of AAV carrier package function, Science Bulletin, 44 (5): 506-509), from the cell of pathology, can obtain a large amount of rAAV virus with the reorganization HSV-1 virus (HSV1-rc) of the having carried AAV virus rep-cap gene carrier cell strain of reorganization AAV carrier DNA of having infected stable integration.During with the strain of rHSV-rc virus infection carrier cell, its DNA massive duplication also finally produced progeny virus after HSV1-rc virus entered cell.The rep-cap gene that carries during the HSV1-rc viral dna replication is synchronization replication also, thereby produces the rep-cap gene of high copy.The Rep genes encoding produces 4 kinds of Rep albumen, and (Rep52 Rep40), makes reorganization AAV carrier DNA come out and massive duplication finally becomes strand and is wrapped into the AAV capsomere from the cellular genome rescue for Rep78, Rep68; 3 kinds of coat protein VP1 of cap genes encoding, VP2, VP3 is assembled into capsomere in nucleus.This method has solved having problems on a large scale of rAAV effectively.

We find in the research, and the BHK-21 cell with reorganization HSV-1 virus HSV1-rc infects untransfected AAV carrier DNA also can produce a large amount of AAV virions.Be this virion be empty particle.Illustrate that AAV is genomic dna to be wrapping in the capsomere after installing empty particle in advance when virus forms virion again.

How efficiently separation and a large amount of rAAV of purifying are another key issues of its application.Less about the report of reorganization AAV separation and purifying at present.Traditional purification process adopts the fraction precipitation of ammonium sulphate method that rAAV is separated with cell debris and makes it concentrated, takes turns the super centrifugal acquisition rAAV component of cesium chloride gradient with 2～3 then, removes cesium chloride and other salt with dialysis process at last.This method both wasted time and energy, and make the infectivity of rAAV that bigger loss is arranged again, and the rate of recovery was low.Therefore, many investigators are devoted to develop new purification process.Bao Dao column chromatography method (Kenji Tamayose in recent years, Yukihiko Hirai, and Takashi Shimada, A new strategy for large-scalepreparation of high-titer recombinant adeno-associated virus vectors by using packaging celllines and sulfalonated cellulose column chromatography, Human Gene Therapy7:507-513,1996; S Zolotukhin, Bj Byrne et al.Recombinant adeno-associated viruspurification using novel methods improves infectious titer and yield.Gene Therapy 6:973-985,1999; Dirk Grimm, Andrea kern et al.Novel tools for production and purificationof recombinant adenoassociated virus vectors, Human Gene Therapy 9:2745-2760,1998) purge process of rAAV is simplified greatly, and the rate of recovery improve a lot.But these methods also have the cost height, handle that bulk sample is limited in one's ability, experiment condition and the high shortcoming of plant and instrument requirement.

Usually, reorganization AAV viral purification process can be divided into following steps:

1) lysis (except the cell free system): classic methods is to adopt the method for multigelation 3～4 times.The cell and the nutrient solution that will contain rAAV virus are collected in together, and multigelation is 3～4 times in dry ice/ethanol bath and 37 ℃ of water-baths, and purpose is that smudge cells is to discharge rAAV.The shortcoming of this method is that rAAV virus discharges incomplete.Other method also has ultrasonic disruption cell method, Septochol method etc.

2) helper virus deactivation (except the non-auxiliary virus packaging system): utilize adenovirus and hsv to heat sensitive characteristics, generally use 56 ℃ of water-bath 30～2hr deactivation helper viruses.

3) reorganization AAV virus is separated with cell debris: generally remove cell debris with low-speed centrifugal.

4) reorganization AAV virus is separated with other component and concentrated: the method for normal employing is a cesium chloride density gradient ultracentrifugation.It separates according to the buoyant density that is sophisticated AAV virion is 1.40～1.42g/cm ³Recently, wait with the monoclonal antibody of anti-AAV virion and make affinity column, be used for separating rAAV and obtain good purifying and concentrated effect.

5) purity of the reorganization AAV of purifying and titre detect: the main detection method of purity has HPLC, SDS-PAGE electrophoresis, ultraviolet spectro-photometric analysis, electronic microscope photos etc.Titer determination comprises the mensuration of physics titre (particles/ml) and infection titer (TU/ml).

Summary of the invention the present invention proposes a kind of novel method of separation and purification rAAV virus, this method is different from traditional rAAV virus separation purification method, is characterized in that easy rate of recovery height, cost quick, that purification effect is good, rAAV is viral are low and is easy to be amplified to the suitability for industrialized production scale.This method mainly is made up of following steps:

1) chloroform smudge cells, deactivation HSV helper virus and make a large amount of cell protein sex change precipitation;

2) handle cell pyrolysis liquid with degraded nucleic acid with DNaseI and RNase;

3) add NaCl and impel rAAV to separate, centrifugal removal cell debris with cell debris;

4) precipitate rAAV with PEG/NaCl;

5) remove foreign protein and remaining PEG with the chloroform extracting;

6) dialysis desalination;

7) be further purified rAAV with density gradient centrifugation or affinity chromatography.

In concrete operations, the rAAV virus separation purification method that the present invention proposes will carry out following operation:

1) a large amount of generations of rAAV: with HSV1-rc is that helper virus infects the AAV carrier cell strain that contains goal gene, treat that cell occurs that complete CPE changes and when floating (about 48～72hr), harvested cell culture (cell and nutrient solution) is measured its volume as rough lysate;

2) helper virus deactivation and lysis: handle the dual purpose that stock liquid can reach deactivation helper virus HSV1-rc and lysing cell with chloroform, but to the not influence of AAV virus.Have infective hsv particle that the lipid bilayer adventitia is arranged and be embedded in wherein multiple viral glycoprotein, this layer tunicle is that the HSV virus infected cell is necessary.Chloroform can lipin dissolving, and makes a large amount of protein denaturations.Handling with chloroform can 100% deactivation HSV virus, simultaneously efficiently lysing cell film and nuclear membrane.The AAV virion has the chloroform resistance, handles its structure and not influence of infection activity with chloroform.

3) removal of cell debris and metaprotein: in cell pyrolysis liquid, add solid sodium-chlor to final concentration 1.0～1.2mol/L, stirring and dissolving.Centrifugal 10～the 15min of 11000g.In bottle in the supernatant immigration one clean Erlenmeyer flask, estimate its volume.Discard centrifugal precipitation and lower floor's chloroform.Adding sodium-chlor and can impel the AAV virion to separate with cell debris, also is that next step is essential with polyethylene glycol precipitation AAV virus institute.

4) in supernatant, add solid polyethylene glycol 8000 to final concentration 6～12%, stirring and dissolving.Place more than 1 hour to spending the night for 4 ℃.Centrifugal 10～the 15min of 12000g.In another clean flask of supernatant impouring, allow supernatant flow to end as far as possible.Precipitate with an amount of PBS ²⁺Dissolving adds residual DNA and RNA outside DnaseI and the RNase digestion AAV virion.Add isopyknic chloroform extracting, the centrifugal 5min of 12000g, careful sucking-off upper strata water moves in the sterile tube under aseptic technique.This liquid is the rAAV virus liquid that concentrates with purifying.

5) the rAAV virus purity that obtains of the rAAV purification process that proposes of the present invention can reach＞99%.From 2 * 10 ⁹The rAAV titre for preparing in the rough lysate of cells (5 110 * 288mm rolling bottles) can reach 10 ^14～15Particles/ml, infection titer can reach＞and 10 ^12～13TU/ml.The rate of recovery of rAAV＞90%.The rAAV that obtains can be used for experiment in vitro and experimentation on animals, can obtain the AAV product of clinical grade after being further purified.

This virus liquid is further purified and can adopts the double liquid phase extraction method.With PEG/ salt system or PEG/Dex system.Final with dialysis removal PEG and salt, use heat sterilization.

Be further purified and also can adopt column chromatography (comprising sieve chromatography, affinity chromatography) or cesium chloride ultracentrifugation, and methods such as dialysis and ultrafiltration.

It is the purifying of the rAAV of helper virus generation that the rAAV purification process that the present invention proposes is particularly suitable for hsv, also is suitable for the purifying with the rAAV of non-auxiliary virus production system and the acquisition of external packaging system.Description of drawings Fig. 1 has showed with 10%SDS-PAGE detected through gel electrophoresis rAAV-GFP virus capsid protein (coomassie brilliant blue R250 dyeing).In this figure, M is expressed as: swimming lane M is the proteinic electrophoresis result of molecular weight standard, and its size from up to down is followed successively by 97400,66200,42700,31000,14400Dal (Promega).1 is expressed as: swimming lane 1 is the electrophoresis result that the rAAV-GFP virus liquid 5ul of the method purifying that proposes with the present invention adds 5ul2 * loadingbuffer.2 are expressed as: swimming lane 1 is the electrophoresis result that adds 5ul 2 * loading buffer without the extractive rAAV-GFP virus of final step chloroform liquid 5ul.(preparation formula of 2 * loading buffer: 100mmol/LThis.Cl (pH6.8), 200mmol/L DTT, 4%SDS, 0.2% tetrabromophenol sulfonphthalein, 20% glycerine.)。Fig. 2 has showed the electronic microscope photos result (magnification is * 54800) of the rAAV-GFP virus of purifying.Fig. 3 has showed the electronic microscope photos result (magnification is * 38000) of the AAV ghost virus of purifying.

The following listed examples of embodiment is rapidly and efficiently separated and the method and the purposes of purification of Recombinant adenovirus correlated virus have been done detailed description of the present invention, but and does not mean that and limit content of the present invention.Embodiment 1

Produce rAAV-GFP virus with rolling bottle

Reporter gene GFP is inserted among the universal AAV carrier pSNAV-1, be built into reorganization AAV carrier pSNAV-1/GFP.PSNAV-1/GFP is imported in the BHK-21 cell (available from ATCC, cultivating with the RPMI1640 nutrient solution 37% that contains 10%FBS) with Lipofectamine (GIBCO BRL) transfection reagent, add G418 800 μ g/ml and select to cultivate 10～15d.Obtain cell mixing clone's carrier cell.With this carrier cell enlarged culturing to 4 35cm ²The square glass culturing bottle in, covering with (has 8 * 10 approximately ⁷Individual cell) trysinization is used in the back, and (in 110 * 288mm), 37 ℃ slowly run (1 rev/mins) are cultivated to be inoculated into 1 rolling bottle.The nutrient solution volume is the 200ml/ rolling bottle.Import the cell in this rolling bottle in 5 rolling bottles enlarged culturing with trysinization behind the 3d.Treat that cell covers with the back and (has 2 * 10 approximately ⁹Individual cell), nutrient solution is inclined to, add helper virus HSV-rc/ Δ UL2 and (in the XbaI site of the virus genomic UL2 gene of HSV1, insert AAV repcap gene, Chinese patent application numbers 98120033.8) 5～10ml (MOI=0.5～2), (1 rev/min) viral adsorption 1～2hr slowly runs.Add 37 ℃ of 200ml/ rolling bottle serum-free RPMI-1640s slowly run (1 rev/min) cultivate.Treat the complete pathology of cell, when coming off easily, cover the violent jolting of tight bottle cap, the cell on the bottle wall all is eluted in the nutrient solution.Collect the culture that merges 5 rolling bottles, estimate its volume, divide to be filled in the 500-ml Erlenmeyer flask 250ml/ bottle.Be used for next step purifying.Embodiment 2

Purification process purifying rAAV with the present invention's proposition

Meet embodiment 1.In each triangular flask, add chloroform 25ml (10: 1v/v), place the violent jolting 1～1.5hr of 37 ℃ of shaking tables.Take out and at room temperature leave standstill 10min.Add DNase and RNase to final concentration 1 μ g/ml.Mixing digests 30～60min under the room temperature gently.Add solid sodium chloride to final concentration 1mol/L, the jolting dissolving.4 ℃ of centrifugal 15min of 12000rpm.Take out the upper strata water, discard chloroform and precipitation.Add PEG8000 to final concentration 10% (w/v).The jolting dissolving.4 ℃ of placements are spent the night.4 ℃ of centrifugal 15min of 11000rpm/min.Supernatant is poured in the clean container.Centrifuge tube is tipped upside down on the thieving paper, allow supernatant flow to end as far as possible.Use 5ml PBS ²⁺Damping fluid with each centrifuge tube pipe at the bottom of and the precipitation on tube wall piping and druming elute merging, its branch is filled to (0.6ml/ pipe) in the 1.5-ml plastic centrifuge tube, add isopyknic chloroform extracting.4 ℃ of centrifugal 5min of 12000g, careful sucking-off upper strata water moves in the sterile tube under aseptic technique.This liquid is the rAAV-GFP virus liquid that concentrates with purifying.This virus liquid volume ratio original volume has concentrated 200 times.

Embodiment 3

The production and the purifying of AAV ghost virion

Cultivate the BHK-21 cell with rolling bottle.Adding helper virus HSV-rc/ Δ UL2 after cell covers with uses the method identical with embodiment 1 to obtain the sick cell culture.Extract the AAV virus of this culture with the rAAV purification process of the present invention's proposition.The viral liquid that obtains carries out electron microscopic observation (referring to Fig. 3 of Figure of description), visible a large amount of virions, and particle center density is higher, is indicated as empty particle.This presentation of results infects with helper virus HSV-rc/ Δ UL2 does not have the bhk cell of transfection AAV carrier DNA (not containing the ITR sequence) can produce AAV empty particle particle effectively.

Embodiment 4

RAAV virus titer and purity detecting

Meet embodiment 2.Detect the titre (particles/ml) of rAAV-GFP virus in the viral liquid of purifying with the GFP probe points hybridizing method of digoxigenin labeled (Boehringer Mannhein test kit).Get the viral liquid PBS of 10ul purifying ²⁺10 times of damping fluid dilutions.Add DNase and RNase to final concentration 1ug/ml37 ℃ of digestion 1hr.Place ice bath after the boiling water bath 5min.With putting film behind the dilution buffer 10 multiple proportions serial dilutions, the 1ul/ point.120 ℃ of roasting film 30min.68 ℃ of prehybridization 1hr.Adding 68 ℃ of hybridization of probe spends the night.Wash film, colour developing.As a result the 1st～4 clear and definite positive, positive a little less than the 5th.The sensitivity that the postulated point hybridizing method detects DNA is 10 ⁶Molecule calculates virus titer=10 ^4～5* 10 ⁶* 10 * 1000=10 ^14～15Particles/ml.

Purity with AAV virus in the viral liquid of SDS-PAGE electrophoretic method detection purifying.Record SDS-PAGE separation gel and spacer gel.Resolving gel concentration is 10%.Get the viral liquid of purifying and the viral liquid 20ul before the extracting of final step chloroform respectively, add 2 * loading buffer 20ul.Boiling water bath 3min.Every hole application of sample 10ul, 200V electrophoresis 1hr.Electrophoresis finishes the back with coomassie brilliant blue R250 staining fluid (dissolving 0.25g coomassie brilliant blue R250 in 45ml methyl alcohol 45ml water and 10ml Glacial acetic acid) dyeing, with corresponding destainer decolouring.The size of protein molecular weight Markers is followed successively by 97400,66200,42700,31000,14400Dal (Promega).Electrophoresis result is seen Fig. 1 of Figure of description.Electrophoresis result shows, the rAAV virus liquid electrophoresis of the purifying that the purification process that proposes with the present invention obtains shows as three bands (lane1), its size is consistent with the size of 3 kinds of AAV coat protein respectively, and (wild-type AAV-2 coat protein VP1 is 87kDal, VP2 is 72kDal, VP3 is 62KDal,), account for total protein more than 99%; The chloroform extracting can be removed foreign protein very effectively, does not but lose the AAV viral protein; Handling through lane1 being carried out computer scanning, obtain the strength ratio of three protein bands, is 10: 1: 1 just, matches with VP3, the VP2 of AAV-2 virion and the ratio of VP1.

Embodiment 5

The electronic microscope photos of rAAV virus

With the rAAV-GFP of purifying among the embodiment 2 virus liquid through under Electronic Speculum, observing after the negative staining, visible big or small uniformity, clear and legible solid virion.Particle diameter is about 20～24nm.Electronic Speculum result is referring to Fig. 2 of Figure of description.Embodiment 6

The mensuration of rAAV-GFP infection titer

With 37 ℃ of the RPMI1640 nutrient solutions that contains 10%FBS, 5%CO2 cultivates the HeLa cell.Inoculation HeLa cell on 24 orifice plates, 5 * 10 ⁵Cells/well.After the overnight incubation, the sucking-off nutrient solution; The viral liquid of getting the 10ul purifying is diluted to 1ml, and with 10 multiple proportions serial dilutions, every hole adds different dilution viral liquid 0.5ml, cultivates 1hr for 37 ℃.Every hole adds 5 type adenovirus (Ad5) 50ul (MOI=5), and nutrient solution 0.5ml.Observe the green fluorescence cell behind 37 ℃ of cultivation 36hr under inverted fluorescence microscope, the counting wherein green cell in certain hole is counted n (10＜n＜100).Calculate the rAAV-GFP virus titer: n * extension rate * 1000/5=n * 10 ⁹* 200=2n * 10 ¹¹TU/ml.The infection titer of estimation rAAV-GFP is 2 * 10 ^12～13Between the TU/ml.

Claims (18)

1. RAAV virus separate, the method for purifying, it is characterized in that, with the cell that contains rAAV virus and nutrient solution as rough lysate, through following steps separate, purifying:

   1) in rough lysate, adds chloroform, deactivation HSV helper virus, lysing cell, make a large amount of cell protein sex change precipitation, obtain cell pyrolysis liquid;

   2) in cell pyrolysis liquid, add solid sodium-chlor to final concentration 1.0～1.2mol/L, stirring and dissolving, centrifugal, stay supernatant;

   3) precipitate rAAV with PEG/NaCl, in step 2) add solid polyethylene glycol in the supernatant of described sodium chloride-containing, stirring and dissolving, leave standstill, centrifugal, abandon supernatant, stay precipitation;

   4) handle cell pyrolysis liquid with degraded nucleic acid, dissolving step 3 with DNaseI and RNase) described in precipitation, add the remaining nucleic acid beyond DnaseI and the RNase dissolving rAAV virion;

   5) remove foreign protein and remaining PEG with the chloroform extracting, add the chloroform extracting, centrifugal, draw the upper strata water;

   6) dialysis desalination;

   7) be further purified rAAV with density gradient centrifugation or affinity chromatography.
2. 2. rAAV virus according to claim 1 is separated, the method for purifying, it is characterized in that, the cell of the described rAAV of containing virus and nutrient solution can obtain in order to following method: with HSV1-rc infection carrier cell strain, when the complete CPE of appearance changed and be floating, harvested cell and nutrient solution were as rough lysate.
3. 3. rAAV virus according to claim 1 is separated, the method for purifying, it is characterized in that this method can be used in combination with the double liquid phase extraction technology, is used for separation, the purifying of rAAV virus.
4. 4. rAAV virus according to claim 1 is separated, the method for purifying, it is characterized in that this method can be used in combination with column chromatography technology, is used for separation, the purifying of rAAV virus.
5. 5. rAAV virus according to claim 1 is separated, the method for purifying, it is characterized in that this method can be used in combination with the dialysis technology, is used for separation, the purifying of rAAV virus.
6. 6. rAAV virus according to claim 1 is separated, the method for purifying, it is characterized in that this method can be used in combination with ultra-filtration technique, is used for separation, the purifying of rAAV virus.
7. 7. rAAV virus according to claim 1 is separated, the method for purifying, it is characterized in that described step 1) " adds chloroform deactivation HSV helper virus, lysing cell, makes a large amount of cell protein sex change precipitations " and can be used for separation, the purifying of rAAV virus separately in rough lysate.
8. 8. rAAV virus according to claim 1 is separated, the method for purifying, it is characterized in that, described step 2) " in cell pyrolysis liquid, add solid sodium-chlor to final concentration 1.0～1.2mol/L, stirring and dissolving, centrifugal, stay supernatant " can be used for separation, the purifying of rAAV virus separately.
9. 9. rAAV virus according to claim 1 is separated, the method for purifying, it is characterized in that, described step 3) " precipitate rAAV with PEG/NaCl; in step 2) add solid polyethylene glycol in the supernatant of described sodium chloride-containing; stirring and dissolving; leave standstill, centrifugal, abandon supernatant, stay precipitation " can be used for separation, the purifying of rAAV virus separately.
10. According to the described rAAV of claim virus separate, the method for purifying, it is characterized in that described step 5) " is removed foreign protein and remaining PEG with the chloroform extracting, added the chloroform extracting; centrifugal, draw the upper strata water " and can be used for separation, the purifying of rAAV virus separately.
11. 11. according to the described rAAV of claim virus separate, the method for purifying, it is characterized in that described step 1) and step 2) be used and can be used for the separating of rAAV virus, purifying.
12. 12. rAAV according to claim 1 virus is separated, the method for purifying, it is characterized in that described step 1) and step 2) and step 3) be used and can be used for the separating of rAAV virus, purifying.
13. 13. rAAV according to claim 1 virus is separated, the method for purifying, it is characterized in that described step 1) and step 2), step 3) and step 7) be used and can be used for the separating of rAAV virus, purifying.
14. 14. rAAV according to claim 1 virus is separated, the method for purifying, it is characterized in that described step 2) be used with step 3) and can be used for the separating of rAAV virus, purifying.
15. 15. rAAV according to claim 1 virus is separated, the method for purifying, it is characterized in that described step 2) be used with step 3) and step 7) and can be used for the separating of rAAV virus, purifying.
16. 16. rAAV according to claim 1 virus is separated, the method for purifying, it is characterized in that, described step 3) and step 7) are used and can be used for the separating of rAAV virus, purifying.
17. 17. rAAV according to claim 1 virus is separated, the method for purifying, it is characterized in that, described step 5) and step 7) are used and can be used for the separating of rAAV virus, purifying.
18. 18. rAAV virus according to claim 1 is separated, the method for purifying, it is characterized in that, this method can be used for not containing in the AAV virus a large amount of separation and the purifying preparation of the what is called " AAV empty particle " of gene.

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