CN108330147A - A kind of foundation of recombined glandulae correlation viral vectors production technology - Google Patents
A kind of foundation of recombined glandulae correlation viral vectors production technology Download PDFInfo
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Abstract
The invention belongs to biotechnologies, and in particular to a kind of foundation of recombined glandulae correlation viral vectors production technology.Present invention firstly provides a kind of recombined glandulae correlation viral vectors preparation methods, include the following steps：（1）Plasmid transfection containing recombined glandulae correlation viral vectors genome sequence is entered to cultivate cell；（2）Incubation step under suitable conditions（1）Gained cell collects cell culture supernatant, and the cell culture supernatant refers to the cell containing recombined glandulae correlation viral vectors and the mixture of culture supernatant；（3）By step（2）The cell culture supernatant of gained is purified and is concentrated to get recombined glandulae correlation viral vectors.The preparation method applies generally to most of rAAV serotypes and mutant, and the recombined glandulae correlation viral vectors for producing acquisition have high titre and high-purity, and immunogenicity is low, safe and efficient advantage.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of foundation of recombined glandulae correlation viral vectors production technology.
Background technology
Adeno-associated virus (Adeno-associated virus, AAV) is a kind of parvovirus of replication defect type, is answered
System proliferation needs the assistance of the helper viruses such as adenovirus or herpesviral, and when lacking helper virus, AAV can only be had
The duplication of limit just enters latence after infection cell, is integrated into chromosome or exists in the form of episomal.Therefore,
AAV belongs to Parvoviridae dependovirus.
The core of AAV, which is one, has complementary single stranded DNA, and capsid protein is in 20 face body cubic symmetries, virus
The diameter of grain between 20~26nm, be human infection known viruse in minimum one of virus.Two ends of AAV genomes
For the inverted terminal repeat (inverted terminal repeat sequences, ITRs) of 145bp, ITR is DNA
Replication initiation and the AAV genomes of packaging recombination become the cis-acting elements needed for infective virion, have transcription
The activity of promoter is encoding viral area between sequence, containing there are two open reading frames (ORF).Wherein, left side ORF is compiled
Code four Rep albumen (Rep78, Rep68, Rep52, Rep40), these non-structural proteins for the integration of AAV viruses, duplication,
Rescue and packaging are critical；(molecular weight is followed successively by tri- viral capsid proteins of ORF coding VP1, VP2 and VP3 on right side
87KDa, 72KDa and 62KDa) and packaging activator protein AAP.The ratio of VP1, VP2 and VP3 in ripe AAV virions
About 1:1:10.According to the difference of capsid, AAV can be divided into 12 kinds of serotypes (AAV1~AAV12) at present and more than 100 kinds makes a variation
Body.Wherein, AAV2 is to obtain the serotype of infection clones earliest, and up to the present study to obtain a kind of most clear serum
Type.
AAV is as a kind of unique gene therapy vector by European Union's FDA certifications, because it extensively (can with host range
Infect dividing cell and non-dividing cell), safety good (no pathogenicity), low immunogenicity, in vivo expression external source steady in a long-term
The advantages that gene, good diffusion and physical property are stablized, be widely used in gene therapy basic research and
In clinical test.Moreover, more and more clinical tests demonstrate the validity of AAV carriers and good safety, it is allowed into
And it is considered as a kind of most promising gene transfer vehicle for the hot spot of gene therapy research.
It is exactly helper plasmid and vector plasmid there are one prodigious misgivings in the genes delivery system based on viral vectors
Recombination occurs and recovers wild-type virus.Classical production recombinant adeno-associated virus (Recombinant adeno-
Associated virus, rAAV) experimental program be need one vector plasmid of cotransfection and one express virus replication and
In the packaging plasmid of structural gene to the culture cell of adenovirus infection.This undoubtedly increases the possibility that wild type AAV is generated,
And this method is complicated for operation, influence factor is more, it is difficult to efficiently separate purifying rAAV, be also unsuitable for mass producing.Traditional
The technique of purifying rAAV is to use two-wheeled cesium chloride density gradient centrifugation, however this method takes time and effort, prepared AAV
Contain more protein and DNA impurity in carrier, and inside and outside transduction efficiency is relatively low.In addition, although AAV carriers itself have
The characteristics of low immunogenicity, but have multiple studies have shown that the injection of higher AAV carriers dosage can also cause the place significantly increased
Main immune response.Although existing purification technique cannot completely eliminate the immunogenicity of AAV carriers, prepare with high titre, height
The AAV products that purity and immunogenicity minimize are necessary for the research of field of gene.
Invention content
In order to overcome the problems of in the prior art, the purpose of the present invention is to provide a kind of recombinant adeno-associated virus
The foundation of carrier production technology.
To achieve the goals above and other related purposes, the present invention adopt the following technical scheme that：
The first aspect of the present invention provides a kind of recombined glandulae correlation viral vectors preparation method, includes the following steps：
(1) plasmid transfection containing recombined glandulae correlation viral vectors genome sequence is entered to cultivate cell；
(2) cell obtained by incubation step (1) under suitable conditions, collects cell culture supernatant, the cell culture
Supernatant refers to the cell containing recombined glandulae correlation viral vectors and the mixture of culture supernatant；
(3) it by the cell culture supernatant obtained by step (2), is purified and is concentrated to get recombinant adeno-associated virus load
Body.
Preferably, in the step (1), recombined glandulae correlation viral vectors genome sequence is located at three different loads
On constitution grain, three vector plasmids are respectively pHelper, prAAV-RC, prAAV-GFP.
The pHelper provides the ad gene products needed for the infective recombined glandulae correlation viral vectors of production tool
(such as：E2A, E4 and VA rna gene)；PrAAV-RC includes that replication protein rep coded sequences and viral capsid proteins cap are compiled
Code sequence；PrAAV-GFP provides eukaryotic promoter and mammal is thin when exogenous array is cloned into multiple cloning sites (MCS)
Other elements needed for born of the same parents' high level gene expression and instruct virus replication and the AAV inverted terminal repeats of packaging
(ITRs), remaining ad gene products is then provided by the host cell for stablizing expression adenovirus E 1 gene.By three plasmids
PHelper, prAAV-RC, prAAV-GFP cotransfection enter host cell, you can the recombinant adeno-associated virus for generating non-replicating carries
Body.PHelper, prAAV-RC, prAAV-GFP are commercially available plasmid, are purchased from Shanghai JiKai Gene Chemical Technology Co., Ltd.
Preferably, in the step (1), the culture cell (that is, host cell) is selected from HEK293T.
In the step (2), those skilled in the art can select suitable condition of culture pair according to the type of culture cell
Cell is cultivated, and collects cell culture supernatant on suitable opportunity, utmostly to reduce primary pollutant【Including core
Acid pollution object (referring mainly to DNA pollution object) and protein pollutant etc.】, and ensure viral crude extract (also known as cells and supernatant
Liquid) in active virion level.It, can be for example, can choose whether to be packed with serum-free according to actual conditions
It is produced in the case of being with or without the inductions such as chloroquine or sodium butyrate, and that cell culture supernatant can be after transfection is multiple
Time point is collected, such as can disposable harvests or respectively harvest is one inferior respectively at 48h and 72h of 48h/72h after transfection.No
The quality of the recombinant adeno-associated virus crude extract and final product that are obtained with production batch can be by measuring purity, titre and safety
Property is controlled.
In certain embodiments of the present invention, the actual conditions of culture are：Using containing 8%~20%, (volume basis is dense
Degree) FBS DMEM medium cultures in 37 DEG C of constant temperature incubations, before transfection, cell culture fluid change into low serum content DMEM training
Support base【DMEM culture mediums containing 2%~10% (concentration expressed in percentage by volume) FBS】, cell culture fluid changed into contain after transfecting 5~7h
The DMEM culture mediums of 2%~10% (concentration expressed in percentage by volume) FBS, and cell is placed in 37 DEG C, 5%CO2With the ring of saturated humidity
Continue to cultivate in border, and collects cell culture supernatant.Preferred cell culture supernatant collection scheme is, in transfection
After 72h, cell culture supernatant is collected.The cell culture supernatant can carry out subsequent recombined glandulae correlation viral vectors immediately
Purifying and concentration step, also of short duration can be put in 4 DEG C of refrigerators for future use.
Preferably, in the step (3), by the cell and culture supernatant in the cell culture supernatant obtained by step (2)
Liquid is detached, and PEG is then added into the culture supernatant after separation, and centrifugation recombined glandulae correlation viral vectors are resuspended
It obtains PEG and precipitates re-suspension liquid；Cell after separation is cracked by multigelation method, and cell pyrolysis liquid passes through millipore filter
(belonging to membrane filter) filters, and obtains filtered fluid；The filtered fluid is merged with PEG precipitation re-suspension liquids, obtains viral crude extract.
Preferably, the PEG is PEG8000.
Preferably, final concentration of 8%~12% (w/vs) of the PEG in supernatant.
Preferably, when being concentrated using PEG, processing method is 4 DEG C of 2~12h of placement.
Preferably, the aperture of the millipore filter is 0.22 or 0.45 μm.In an embodiment of the present invention, the micropore
The aperture of filter is 0.22 μm.
Preferably, the millipore filter is PES filters.
Further, the viral crude extract by all can nuclease handled, centrifuge and clarify, then pass through chloroform
It handles and clarifies, recycle virus liquid.
Preferably, the addition of the all-round enzyme is 5~300U/ml.
Further, the virus liquid of recycling is purified by Iodixanol density gradient centrifugation again, after recovery purifying
Virus liquid.
It includes four iodine gram sand that Iodixanol density gradient centrifugation purification step is listed in some embodiments of the present invention
Alcohol density gradient solution (9ml 15%, 6ml 25%, 5ml 40% be quality concentration expressed in percentage by volume with 5ml 60%, %).
Still further, the virus liquid after purification of recycling is concentrated by ultrafiltration using column is concentrated by ultrafiltration, ultrafiltration is obtained
Virus liquid after concentration.
Preferably, it is described be concentrated by ultrafiltration column molecular cut off (Molecular Weight CutOff, MWCO) be 30~
150KDa。
The depth concentration and purifying of recombined glandulae correlation viral vectors may be implemented in the ultrafiltration step.
In an embodiment of the present invention, molecular cut off (the Molecular Weight that column is concentrated by ultrafiltration
CutOff, MWCO) it is 100KDa.
Still further, the virus liquid after ultrafiltration concentration is filtered using millipore filter (belonging to membrane filter), into
The collection of row final product.
Preferably, the millipore filter is PVDF filters.
Preferably, the aperture of the millipore filter is 0.22 or 0.45 μm.In an embodiment of the present invention, the micropore
The aperture of filter is 0.22 μm.
The second aspect of the present invention provides aforementioned recombined glandulae correlation viral vectors preparation method and is carried in recombinant adeno-associated virus
The purposes of body preparation field.
Production technology of the present invention can prepare it is highly concentrated and purifying recombined glandulae correlation viral vectors product with
In gene transfer and gene therapy.
Compared with prior art, the present invention has the advantages that：
The present invention is using non-auxiliary virus packaging system (AAV Helper-Free System) as the life of rAAV carriers
Produce preparation system, using complete AAV virions can tolerate organic solvent characteristic and single Iodixanol density gradient from
Heart method isolates and purifies rAAV, and a kind of production technology of scalable recombined glandulae correlation viral vectors is established in exploration.The production work
Skill applies generally to most of rAAV serotypes and mutant.The present invention also provides the rAAV carrier granulars to be used for animal body
The application of interior experiment and three-dimensional fixed-point injection experiment.Due to high titre and high-purity, rAAV carriers provided by the invention
Grain immunogenicity is low, safe and efficient, can realize that foreign gene is stable and lasting expression.
Description of the drawings
Figure 1A：Helper plasmid pHelper and prAAV-RC collection of illustrative plates.
Figure 1B：Shuttle plasmid prAAV-GFP collection of illustrative plates.
Fig. 2：The rAAV carriers production diagram of the present invention, uses helper plasmid pHelper, prAAV-RC and shuttle plasmid
PrAAV-GFP cotransfections HEK293T produces cell, so that it generates the recombination rAAV carrier granulars of non-replicating.
Fig. 3：Show the flow chart of each step of rAAV viral vectors production technologies of the present invention.
Fig. 4：RAAV carrier granular products are carried out 10%SDS-PAGE gel electrophoresises, electrophoresis knot by purity detecting result figure
Shu Houyong eStainTML1 protein staining instrument dyes gel, and gel imaging system is used in combination to take pictures, wherein M swimming lanes represent
Protein molecular weight standard, swimming lane 1-4 respectively represent using technique productions provided by the invention AAV8, AAV9, AAV2 and
The purity detectings of tetra- kinds of serotype vectors grain products of AAV5 as a result, the visible three characteristic protein bands of electrophoresis result,
Size of the size respectively with three kinds of capsid proteins VP1, VP2 and VP3 of AAV is consistent, remaining miscellaneous band unobvious；Meanwhile it is right
Gel imaging result figure carry out quantitative analysis, testing result show VP1, VP2 and VP3 total amount account for about the 99% of total protein concentration with
On, show that the purity of the rAAV carrier granular products with technique productions provided by the invention is all very high.
Fig. 5：Experimental result picture in animal body, will be through technique productions provided by the invention using tail vein injection method
AAV5, AAV8, AAV9 serotype vectors particle (rAAV-Luciferase) are respectively with 1 × 1011The dosage of vg/ only injects 6 week old
C57BL/6 mouse, injection after three weeks, carry out living imaging with IVIS Lumina System, and it is (glimmering to analyze luciferase
Light element enzyme) expression, by experimental result in animal body it is found that with AAV5, AAV8 of technique productions provided by the invention and
Tri- kinds of serotypes of AAV9 all have preferable In vivo infection and infection has specificity.
Fig. 6：Three-dimensional fixed-point injection experimental result picture will use technique productions provided by the invention using three-dimensional fixed-point injection method
AAV9 light genetic carrier (rAAV9-mCherry) injection Cre mouse hippocampus, volume injected be 1.5 μ l, inject 14 days
Afterwards, brain tissue slice is taken to observe expression signal；Fig. 6 B are bat of the grey square frame institute's collar region under more high-amplification-factor in Fig. 6 A
Take the photograph image.
Specific implementation mode
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention.The test method of actual conditions is not specified in the following example,
Usually according to normal condition, or according to the condition proposed by each manufacturer.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical as the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment,
Outside material, the record according to those skilled in the art to the grasp of the prior art and the present invention can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related field.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.,
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.), Humana Press, Totowa, 1999 etc..
The preparation of 1 rAAV carrier granulars of embodiment
As shown in figure 3, a kind of preparation method of rAAV carrier granulars in this implementation, includes the following steps：
(1) culture of HEK293T cells：
The production of rAAV carrier granulars carries out in HEK293T.By HEK293T cells with every ware 1.4 × 106It is a thin
The amount of born of the same parents is seeded in the 10cm culture dishes containing 15% (concentration expressed in percentage by volume) FBS DMEM culture mediums, is placed in 37 DEG C, and 5%
CO2It is cultivated in the environment of saturated humidity.Inoculation implements transfection after 2 days.
(2) transfection of HEK293T cells：
Before transfection, cell culture fluid is changed into DMEM culture mediums that 5ml contains 5% (concentration expressed in percentage by volume) FBS.Use standard
PEI transfection methods, by instantaneous triple transfections by two assistant carrier plasmid pHelper (Figure 1A), prAAV-RC (Figure 1A)
With a shuttling expression plasmid vector prAAV-GFP (Figure 1B) according to every ware 7:7:7 mass are than cotransfection into HEK293T cells.Transfection
After mixture prepares, it is added dropwise to according to the amount of 1ml/ wares in cell culture medium.After transfecting 6h, cell culture fluid is changed
Contain the DMEM culture mediums of 8% (concentration expressed in percentage by volume) FBS at 12ml, and cell is placed in 37 DEG C, 5%CO2With saturated humidity
Continue to cultivate in environment.
(3) collection of cell culture supernatant：
After HEK293T cell transfectings 72h, the mixture of the cell containing rAAV carrier granulars and culture supernatant is collected,
For subsequent purifying and concentration step.
(4) purifying of rAAV carrier granulars：
First the cell of collection and the mixture of culture supernatant are centrifuged into 5min at 4 DEG C under the conditions of 3000g, with separation
Cell and supernatant.Then, into the supernatant after separation be added 40% (w/v) PEG8000/NaCl (PEG/NaCl be pre-configured,
The quality concentration expressed in percentage by volume of wherein PEG8000 is 40% (w/v), a concentration of 2.5M of NaCl) it is final concentration of to PEG8000
10% (w/v), mixing are placed on 4 DEG C of placement 2h；Then, 4 DEG C, 10000g centrifuges 30min, to precipitate rAAV carrier granulars.Together
Cell (carrying out four cycles altogether) after the multigelation method cracking separation of liquid nitrogen/37 DEG C Shi Caiyong.Cell pyrolysis liquid passes through diameter
For 0.22 μm of PES membrane filtrations, this filtered fluid is merged with the PEG of supernatant precipitation re-suspension liquids.The virus for merging gained slightly carries
Liquid passes through all-round nuclease (addition 50U/ml, also that is, the all-round enzymes of 50U, 37 DEG C of water-baths are added in per 1ml virus crude extracts successively
1h) and chloroform handles (chloroform:Viral sample=1:1, room temperature acutely shakes 30min) further purified, and recycle pure
Virus liquid after changing and concentrating.
The virus liquid of recycling can be purified by Iodixanol density gradient centrifugation again.Iodixanol in the present invention is close
It includes four Iodixanol density gradient solution (40% Hes of 9ml 15%, 6ml 25%, 5ml to spend gradient centrifugation purification step
5ml 60%, % are quality concentration expressed in percentage by volume), wherein contain 0.5% (w/v) in 15%, 25% and 60% gradient solution
It is phenol red.By Iodixanol linear gradient solution at 18 DEG C, 2h is centrifuged under the conditions of 63000rpm, and using conventional 5ml injections
Device collects 40% layer component.The rAAV viral samples recycled through this step further can be concentrated and detached by ultrafiltration
Purifying.Ultra-filtration process is completed in the ultrafiltration concentration column of MWCO=100KDa.Ultrafiltration step of the present invention includes often to surpass
It filters 1ml, that is, the program (carrying out three cycles altogether) of 150mM NaCl 20mM Tris-HCl solution ultrafiltration repeatedly is added.Finally
It is sterile filtered using 0.22 μm of PVDF filter membrane, carries out the collection of final product, the rAAV for so far being purified and being concentrated
Carrier granular product.
In order to reduce the generation of reproducible wild type rAAV to the greatest extent, in the present invention respectively gram by the genome sequence of rAAV
Above grand to three different vector plasmids.First vector plasmid is helper plasmid pHelper, provides production tool infection
Needed for the rAAV virions of property ad gene products (such as：E2A, E4 and VA rna gene).Second vector plasmid
It is helper plasmid prAAV-RC, contains rep the and cap genes of coding replication protein and viral capsid proteins.Third carrier
Plasmid is shuttle plasmid prAAV-GFP, provides eukaryotic promoter and is fed when exogenous array is cloned into multiple cloning sites (MCS)
Other elements needed for newborn zooblast high level gene expression, carrier contain the AAV for instructing virus replication and packaging simultaneously
Inverted terminal repeat (ITRs).Such three plasmid co-transfections HEK293T is produced into cell to generate non-replicating
RAAV carrier granulars, diagram are as shown in Figure 2.
The preparation of 2 rAAV carrier granulars of embodiment
As shown in figure 3, a kind of preparation method of rAAV carrier granulars in this implementation, includes the following steps：
(5) culture of HEK293T cells：
The production of rAAV carrier granulars carries out in HEK293T.By HEK293T cells with every ware 1.4 × 106It is a thin
The amount of born of the same parents is seeded in the 10cm culture dishes containing 8% (concentration expressed in percentage by volume) FBS DMEM culture mediums, is placed in 37 DEG C, 5%CO2
It is cultivated in the environment of saturated humidity.Inoculation implements transfection after 2 days.
(6) transfection of HEK293T cells：
Before transfection, cell culture fluid is changed into DMEM culture mediums that 5ml contains 2% (concentration expressed in percentage by volume) FBS.Use standard
PEI transfection methods, by instantaneous triple transfections by two assistant carrier plasmid pHelper (Figure 1A), prAAV-RC (Figure 1A)
With a shuttling expression plasmid vector prAAV-GFP (Figure 1B) according to every ware 7:7:7 mass are than cotransfection into HEK293T cells.Transfection
After mixture prepares, it is added dropwise to according to the amount of 1ml/ wares in cell culture medium.After transfecting 5h, cell culture fluid is changed
Contain the DMEM culture mediums of 10% (concentration expressed in percentage by volume) FBS at 12ml, and cell is placed in 37 DEG C, 5%CO2With saturated humidity
Continue to cultivate in environment.
(7) collection of cell culture supernatant：
After HEK293T cell transfectings 72h, the mixture of the cell containing rAAV carrier granulars and culture supernatant is collected,
For subsequent purifying and concentration step.
(8) purifying of rAAV carrier granulars：
First the cell of collection and the mixture of culture supernatant are centrifuged into 5min at 4 DEG C under the conditions of 3000g, with separation
Cell and supernatant.Then, into the supernatant after separation be added 40% (w/v) PEG8000/NaCl (PEG/NaCl be pre-configured,
The quality concentration expressed in percentage by volume of wherein PEG8000 is 40% (w/v), a concentration of 2.5M of NaCl) it is final concentration of to PEG8000
8% (w/v), mixing are placed on 4 DEG C of placement 12h；Then, 4 DEG C, 10000g centrifuges 30min, to precipitate rAAV carrier granulars.Together
Cell (carrying out four cycles altogether) after the multigelation method cracking separation of liquid nitrogen/37 DEG C Shi Caiyong.Cell pyrolysis liquid passes through diameter
For 0.45 μm of PES membrane filtrations, this filtered fluid is merged with the PEG of supernatant precipitation re-suspension liquids.The virus for merging gained slightly carries
Liquid passes through all-round nuclease (addition 300U/ml, also that is, 300U 37 DEG C of water-baths of all-round enzyme are added in per 1ml virus crude extracts successively
1h) and chloroform handles (chloroform:Viral sample=1:1, room temperature acutely shakes 30min) further purified, and recycle pure
Virus liquid after changing and concentrating.
The virus liquid of recycling can be purified by Iodixanol density gradient centrifugation again.Iodixanol in the present invention is close
It includes four Iodixanol density gradient solution (40% Hes of 9ml 15%, 6ml 25%, 5ml to spend gradient centrifugation purification step
5ml 60%, % are quality concentration expressed in percentage by volume), wherein contain 0.5% (w/v) in 15%, 25% and 60% gradient solution
It is phenol red.By Iodixanol linear gradient solution at 18 DEG C, 2h is centrifuged under the conditions of 63000rpm, and using conventional 5ml injections
Device collects 40% layer component.The rAAV viral samples recycled through this step further can be concentrated and detached by ultrafiltration
Purifying.Ultra-filtration process is completed in the ultrafiltration concentration column of MWCO=150KDa.Ultrafiltration step of the present invention includes often to surpass
It filters 1ml, that is, the program (carrying out three cycles altogether) of 150mM NaCl 20mM Tris-HCl solution ultrafiltration repeatedly is added.Finally
It is sterile filtered using 0.22 μm of PVDF filter membrane, carries out the collection of final product, the rAAV for so far being purified and being concentrated
Carrier granular product.
In order to reduce the generation of reproducible wild type rAAV to the greatest extent, in the present invention respectively gram by the genome sequence of rAAV
Above grand to three different vector plasmids.First vector plasmid is helper plasmid pHelper, provides production tool infection
Needed for the rAAV virions of property ad gene products (such as：E2A, E4 and VA rna gene).Second vector plasmid
It is helper plasmid prAAV-RC, contains rep the and cap genes of coding replication protein and viral capsid proteins.Third carrier
Plasmid is shuttle plasmid prAAV-GFP, provides eukaryotic promoter and is fed when exogenous array is cloned into multiple cloning sites (MCS)
Other elements needed for newborn zooblast high level gene expression, carrier contain the AAV for instructing virus replication and packaging simultaneously
Inverted terminal repeat (ITRs).Such three plasmid co-transfections HEK293T is produced into cell to generate non-replicating
RAAV carrier granulars, diagram are as shown in Figure 2.
The preparation of 3 rAAV carrier granulars of embodiment
As shown in figure 3, a kind of preparation method of rAAV carrier granulars in this implementation, includes the following steps：
(9) culture of HEK293T cells：
The production of rAAV carrier granulars carries out in HEK293T.By HEK293T cells with every ware 1.4 × 106It is a thin
The amount of born of the same parents is seeded in the 10cm culture dishes containing 20% (concentration expressed in percentage by volume) FBS DMEM culture mediums, is placed in 37 DEG C, and 5%
CO2It is cultivated in the environment of saturated humidity.Inoculation implements transfection after 2 days.
(10) transfection of HEK293T cells：
Before transfection, cell culture fluid is changed into DMEM culture mediums that 5ml contains 10% (concentration expressed in percentage by volume) FBS.Use mark
Accurate PEI transfection methods (are schemed two assistant carrier plasmid pHelper (Figure 1A), prAAV-RC by instantaneous triple transfections
1A) and a shuttling expression plasmid vector prAAV-GFP (Figure 1B) is according to every ware 7:7:7 mass are than cotransfection into HEK293T cells.Turn
After dye mixture prepares, it is added dropwise to according to the amount of 1ml/ wares in cell culture medium.After transfecting 7h, by cell culture fluid
It changes 12ml into and contains the DMEM culture mediums of 2% (concentration expressed in percentage by volume) FBS, and cell is placed in 37 DEG C, 5%CO2And saturated humidity
Environment in continue to cultivate.
(11) collection of cell culture supernatant：
After HEK293T cell transfectings 72h, the mixture of the cell containing rAAV carrier granulars and culture supernatant is collected,
For subsequent purifying and concentration step.
(12) purifying of rAAV carrier granulars：
First the cell of collection and the mixture of culture supernatant are centrifuged into 5min at 4 DEG C under the conditions of 3000g, with separation
Cell and supernatant.Then, into the supernatant after separation be added 40% (w/v) PEG8000/NaCl (PEG/NaCl be pre-configured,
The quality concentration expressed in percentage by volume of wherein PEG8000 is 40% (w/v), a concentration of 2.5M of NaCl) it is final concentration of to PEG8000
12% (w/v), mixing are placed on 4 DEG C of placement 8h；Then, 4 DEG C, 10000g centrifuges 30min, to precipitate rAAV carrier granulars.Together
Cell (carrying out four cycles altogether) after the multigelation method cracking separation of liquid nitrogen/37 DEG C Shi Caiyong.Cell pyrolysis liquid passes through diameter
For 0.22 μm of PES membrane filtrations, this filtered fluid is merged with the PEG of supernatant precipitation re-suspension liquids.The virus for merging gained slightly carries
Liquid passes through all-round nuclease (addition 5U/ml, also that is, the all-round enzymes of 5U, 37 DEG C of water-bath 1h are added in per 1ml virus crude extracts) successively
(chloroform is handled with chloroform:Viral sample=1:1, room temperature acutely shakes 30min) further purified, and recovery purifying and
Virus liquid after concentration.
The virus liquid of recycling can be purified by Iodixanol density gradient centrifugation again.Iodixanol in the present invention is close
It includes four Iodixanol density gradient solution (40% Hes of 9ml 15%, 6ml 25%, 5ml to spend gradient centrifugation purification step
5ml 60%, % are quality concentration expressed in percentage by volume), wherein contain 0.5% (w/v) in 15%, 25% and 60% gradient solution
It is phenol red.By Iodixanol linear gradient solution at 18 DEG C, 2h is centrifuged under the conditions of 63000rpm, and using conventional 5ml injections
Device collects 40% layer component.The rAAV viral samples recycled through this step further can be concentrated and detached by ultrafiltration
Purifying.Ultra-filtration process is completed in the ultrafiltration concentration column of MWCO=30KDa.Ultrafiltration step of the present invention includes per ultrafiltration
To 1ml, that is, the program (carrying out three cycles altogether) of 150mM NaCl 20mM Tris-HCl solution ultrafiltration repeatedly is added.Finally adopt
It is sterile filtered with 0.22 μm of PVDF filter membrane, carries out the collection of final product, the rAAV for so far being purified and being concentrated is carried
Body grain products.
In order to reduce the generation of reproducible wild type rAAV to the greatest extent, in the present invention respectively gram by the genome sequence of rAAV
Above grand to three different vector plasmids.First vector plasmid is helper plasmid pHelper, provides production tool infection
Needed for the rAAV virions of property ad gene products (such as：E2A, E4 and VA rna gene).Second vector plasmid
It is helper plasmid prAAV-RC, contains rep the and cap genes of coding replication protein and viral capsid proteins.Third carrier
Plasmid is shuttle plasmid prAAV-GFP, provides eukaryotic promoter and is fed when exogenous array is cloned into multiple cloning sites (MCS)
Other elements needed for newborn zooblast high level gene expression, carrier contain the AAV for instructing virus replication and packaging simultaneously
Inverted terminal repeat (ITRs).Such three plasmid co-transfections HEK293T is produced into cell to generate non-replicating
RAAV carrier granulars, diagram are as shown in Figure 2.
The quality control of 4 rAAV carrier granulars of embodiment
Embodiment 1, embodiment 2, embodiment 3 basis on, by following detection to embodiment 1, embodiment 2,
RAAV carrier granulars prepared by embodiment 3 carry out quality control.These detections include titre detection and purity detecting.
1 titre of detection detects：The present invention determines the titre of rAAV carrier granular products using QPCR absolute quantitation methods.It should
Method can be in the continuously fluorescent value variation of detection sample all the time of amplification, and can be obtained in wide quantification area good
Standard curve, accurate quantitative analysis, detection are carried out to target gene, and it is reproducible.QPCR detection primers used in the present invention are EGFP
Primer, upstream primer sequence are：5 '-TGCTTCAGCCGCTACCC-3 ' (SEQ ID NO.1), downstream primer sequence is：5’-
AGTTCACCTTGATGCCGTTC-3’(SEQ ID NO.2).First, by 20 μ l rAAV carrier granulars products, 1 μ l totipotent nucleus
Sour enzyme is in 37 DEG C of water-bath 30min；It 4 DEG C, 12000rpm, after centrifuging 10min, takes and resets and add 90 μ l dilution buffers on 10 μ l
(1.5MTris-HCl pH8.0,0.1M EDTA, 1M NaCl), in 37 DEG C of water-bath 30min；1 μ l eggs are added after natural cooling
White enzyme K, 65 DEG C of water-bath 1h；Finally, it is placed in 95 DEG C of water-baths and is incubated 20min, with inactivated proteases K.To be obtained through above-mentioned processing
Viral sample be template prepare QPCR reaction systems (20 μ l).QPCR detection reagents used in the present invention are 2 × SYBR Green
Mix.QPCR response procedures are：95℃10min；95 DEG C of 15s, 55 DEG C of 20s, 72 DEG C of 20s, totally 40 recycle；95 DEG C of 1min, 55 DEG C
30s, 95 DEG C of 30s.Finally, the rAAV genome copy numbers (vg/ml) in viral sample to be measured are calculated according to standard curve.Knot
Fruit, virus titer prepared by embodiment 1 can reach 1E+12vg/ml~2E+13vg/ml.Virus titer prepared by embodiment 2 can
Reach 1E+12vg/ml~2E+13vg/ml.Virus titer prepared by embodiment 3 can reach 1E+12vg/ml~2E+13vg/ml.
2 purity detecting of detection：eStainTML1 protein staining instrument is used for obtaining through production technology of the present invention
RAAV carrier granular products carry out purity detecting.eStainTML1 fast electric decoration methods incorporate traditional fixation, dyeing and take off
Color three-step reaction can carry out coomassie brilliant blue staining to the albumen on PAGE gel in a short time, and there is high dyeing to imitate
Rate and high sensitivity.First, rAAV carrier granular products are subjected to 10%SDS-PAGE gel electrophoresises, are used after electrophoresis
eStainTML1 protein staining instrument dyes gel, and PAGE gel is taken pictures and made using gel imaging system
Quantitative analysis, to determine the percentage composition of three kinds of capsid proteins in rAAV carrier prepared products.
RAAV carrier granular testing results prepared by embodiment 1 are shown in that Fig. 4, M swimming lanes represent protein molecular weight standard, swimming lane
1-4 respectively represents tetra- kinds of serotype vectors particle productions of AAV8, AAV9, AAV2 and AAV5 using technique productions provided by the invention
The purity detectings of product as a result, the visible three characteristic protein bands of electrophoresis result, size respectively with three kinds of capsid eggs of AAV
The size of white VP1, VP2 and VP3 are consistent, remaining miscellaneous band unobvious；Meanwhile gel imaging result figure is quantitatively divided
Analysis, testing result show that the total amount of VP1, VP2 and VP3 account for about 99% of total protein concentration or more, show with work provided by the invention
The purity of the rAAV carrier granular products of skill production is all very high.Similarly, embodiment 2 prepare rAAV carrier granulars in VP1,
The total amount of VP2 and VP3 accounts for about 99% of total protein concentration or more.VP1, VP2 and VP3 in rAAV carrier granulars prepared by embodiment 3
Total amount account for about 99% of total protein concentration or more.
It is noted with animal experiment in vivo and three-dimensional fixed point is done through the qualified rAAV carrier granular products of above-mentioned each detection detection
Experiment is penetrated, specifically, using tail vein injection method by AAV5, AAV8, AAV9 of the technique productions provided through the embodiment of the present invention 1
Serotype vectors particle (rAAV-Luciferase) is respectively with 1 × 1011The dosage of vg/ only injects the C57BL/6 mouse of 6 week old.
Injection after three weeks, carries out living imaging, and analyze the expression of luciferase (luciferase) with IVIS Lumina System
It is horizontal.By experimental result in animal body as shown in Figure 5 it is found that the AAV5 of the technique productions provided with the embodiment of the present invention 1,
Tri- kinds of serotypes of AAV8 and AAV9 all have preferable In vivo infection and infection has specificity.Using three-dimensional fixed-point injection method
The AAV9 light genetic carrier (rAAV9-mCherry) of the technique productions provided with the embodiment of the present invention 1 is injected to the sea of Cre mouse
Ma Ti, volume injected is 1.5 μ l, after injecting 14 days, takes brain tissue slice to observe expression signal, Fig. 6 B are grey square frames in Fig. 6 A
Shooting image of institute's collar region under more high-amplification-factor.Similarly, the technique productions provided with the embodiment of the present invention 2 and 3
Tri- kinds of serotypes of AAV5, AAV8 and AAV9 also all have preferable In vivo infection and infection has specificity.
In conclusion the techniques such as the processing of present invention combination chloroform, Iodixanol and ultrafiltration are from HEK293T cell cultures
It gradually extracts, purify and concentrate recombined glandulae correlation viral vectors.The present invention provides obtained based on above-mentioned purification process
RAAV carrier granular products, virus titer can reach 1E+12vg/ml~2E+13vg/ml, purity ＞ 99%.
The rAAV carrier granulars that production technology provided by the invention obtains can be used in animal experiment in vivo, immunogenicity
It is low, it can be achieved that foreign gene is stable and lasting expression.In fact, each link in production process, including cell quality, turn
Experiment parameter, the acquisition time of cell culture and the serum content etc. of cell maintenance medium are contaminated, the production can be influenced
The Toxin producing C of cell and subsequent concentration and purification step.Therefore, these experiment parameters are strictly controlled to finally obtaining
The quality of rAAV preparations is most important.RAAV carrier granulars product produced by the invention has high titre, high-purity and high stable
Property the characteristics of, transduction and animal experiment in vivo etc. all have advantageous use in vitro.Production technology of the present invention can be made
It is standby go out highly concentrated and purifying recombined glandulae correlation viral vectors product for gene transfer and gene therapy.
The above, only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation,
It should be pointed out that for those skilled in the art, under the premise of not departing from the method for the present invention, can also make
Several improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art,
Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more
Dynamic, modification and the equivalent variations developed, are the equivalent embodiment of the present invention；Meanwhile all substantial technologicals pair according to the present invention
The variation, modification and evolution of any equivalent variations made by above-described embodiment, still fall within the range of technical scheme of the present invention
It is interior.
SEQUENCE LISTING
<110>Shanghai JiKai Gene Chemical Technology Co., Ltd
<120>A kind of foundation of recombined glandulae correlation viral vectors production technology
<130> 170859
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 17
<212> DNA
<213> Artificial
<220>
<223>Sense primer
<400> 1
tgcttcagcc gctaccc 17
<210> 2
<211> 20
<212> DNA
<213> Artificial
<220>
<223>Downstream primer
<400> 2
agttcacctt gatgccgttc 20
Claims (11)
1. a kind of recombined glandulae correlation viral vectors preparation method, includes the following steps：
(1) plasmid transfection containing recombined glandulae correlation viral vectors genome sequence is entered to cultivate cell；
(2) cell obtained by incubation step (1) under suitable conditions, collects cell culture supernatant, the cells and supernatant
Liquid refers to the cell containing recombined glandulae correlation viral vectors and the mixture of culture supernatant；
(3) it by the cell culture supernatant obtained by step (2), is purified and is concentrated to get recombined glandulae correlation viral vectors.
2. production technology according to claim 1, which is characterized in that in the step (1), recombined glandulae correlation viral vectors
Genome sequence is located on three different vector plasmids, three vector plasmids be respectively pHelper, prAAV-RC,
prAAV-GFP。
3. production technology according to claim 1, which is characterized in that in the step (1), the culture cell is selected from
HEK293T。
4. preparation method according to claim 1, which is characterized in that, will be thin obtained by step (2) in the step (3)
Cell and culture supernatant in born of the same parents' culture supernatant detach, and PEG then is added into the culture supernatant after separation, from
The heart precipitates recombined glandulae correlation viral vectors, and PEG precipitation re-suspension liquids are resuspended to obtain；Cell after separation is split by multigelation method
Solution, cell pyrolysis liquid are filtered by millipore filter, obtain filtered fluid；The filtered fluid is merged with PEG precipitation re-suspension liquids, is obtained
Viral crude extract.
5. preparation method according to claim 4, which is characterized in that the PEG is PEG8000.
6. preparation method according to claim 4, which is characterized in that the millipore filter is PES filters.
7. preparation method according to claim 4, which is characterized in that the virus crude extract is carried out by all-round nuclease
Processing, centrifuges and clarifies, then handle and clarify by chloroform, recycles virus liquid.
8. preparation method according to claim 7, which is characterized in that the virus liquid of recycling passes through Iodixanol density level bands again
Degree centrifugation is purified, the virus liquid after recovery purifying.
9. preparation method according to claim 8, which is characterized in that the virus liquid after purification of recycling is dense using ultrafiltration
Contracting column is concentrated by ultrafiltration, the virus liquid after being concentrated by ultrafiltration.
10. preparation method according to claim 9, which is characterized in that the virus liquid after ultrafiltration concentration is used micropore mistake
Filter filters, and carries out the collection of final product.
11. if any one of claim 1~10 recombined glandulae correlation viral vectors preparation method is in recombined glandulae correlation viral vectors system
The purposes in standby field.
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| CN111557927A (en) * | 2020-04-26 | 2020-08-21 | 天津大学 | Synthesis method of nanoparticles for treating immune hemolytic anemia |
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| CN114317464A (en) * | 2021-12-27 | 2022-04-12 | 武汉汇研生物科技股份有限公司 | Separation and purification method of adeno-associated virus rAAV9 |
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