CN110184297A - A kind of AAV auxiliary package carrier, building screening technique and application for expressing shRNA - Google Patents
A kind of AAV auxiliary package carrier, building screening technique and application for expressing shRNA Download PDFInfo
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- CN110184297A CN110184297A CN201910443566.4A CN201910443566A CN110184297A CN 110184297 A CN110184297 A CN 110184297A CN 201910443566 A CN201910443566 A CN 201910443566A CN 110184297 A CN110184297 A CN 110184297A
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Abstract
The present invention relates to genetic engineering fields, and in particular to a kind of construction method, screening technique and the application of the AAV auxiliary package carrier and the carrier for expressing shRNA；The carrier is based on Rep2 and CapDJ and assists package carrier, and 2 × BsmBI of site of insertion clone shRNA between miR33-5 ' UTR and miR33-3 ' UTR under CMV promoter driving, shRNA expression small fragment connects the site 2xBsmBI into the skeleton carrier.The construction method of the carrier includes: preparation clone's skeleton；It prepares shRNA and expresses small fragment；ShRNA expression small fragment is connected on clone's skeleton carrier.The screening technique includes: to convert Stbl3 competent bacteria with the carrier, and plasmid extracts；By the plasmid co-transfection 293T cell of extraction；Titre compares screening.The present invention, which realizes, improves AAV production efficiency about 60% or more.
Description
Technical field
The present invention relates to genetic engineering fields, and in particular to a kind of AAV auxiliary package carrier and the carrier for expressing shRNA
Construction method, screening technique and application.
Background technique
Gene therapy at present is just gradually becoming reality from the dream of countless scientists in recent decades.The load of gene therapy
Body is always the key of whole gene treatment, but has to the requirement for meeting safety, validity, specificity.Gene viruses carry
Body plays crucial effect in gene therapy, and recombinant adeno-associated virus (rAAV) carrier is to be known as meeting above three at present
The gene therapy vector of a standard.But it seriously restricts AAV carrier due to the production yield Slow lifting of AAV for a long time and exists
Popularity in clinical application.
As first gene therapy medicament Glybera by the treatment fatty acid enzyme defect that European Union ratifies is listed, AAV makees
It is had been demonstrated for the feasibility of gene therapy vector, but the production efficiency of AAV is still the critical bottleneck of gene therapy.Usual situation
Under, one needs the genetic disease of whole body F-duction to need AAV about 1x1015The particle of AAV genome copy numbers (GC), and this
The other output of clinical grade needs 500,000 dollars.If can get with 50 15cm disks within the scope of current laboratory capabilities
1x1013The production efficiency of GC calculates, then needing to produce 100 times or 100 times production scales can just obtain medical treatment patient
Total amount.Therefore, how improving the yield of AAV and then reducing clinical expense becomes the bottle that those skilled in the art urgently break
Neck.
The main production process of AAV is three plasmid transfection 293T cellular processes at present.This method is simple and quick, but due to
293T is attached cell, and the extension difficulty in scale is larger.If suspension cell, the party attached cell can be domesticated for
Method can become the important candidate scheme of scale industrial production.Adherent 293T cell is domesticated for suspension cell to have in recent years
People is succeeded, but since production efficiency is still unable to meet demand, large-scale production can only be by solely increasing production scale.Cause
This, improves in method, innovates, obtain one of the hot spot that higher AAV yield is current gene therapy.
The 293 cells main host cell used as AAV production, and non-natural is suitble to the production of AAV.Letter in cell
Number access and the relevant key gene expression of each AAV and adjust that there is also very big optimization spaces, such as Stifani S. et al. to exist
2017 revealed, and the downward expression of certain genes can be greatly facilitated the efficiency of AAV packaging.Therefore, efficiently screening discovery
It is the important means for improving AAV yield that the downward expression of which kind of gene, which can promote the expression of AAV,.In selection shRNA table
In the mode reached, the mode of earliest period is the expression of shRNA to be driven with Pol III promoter U6 or H1, but began from 2006,
Having been reported that proves to drive the shRNA on expression miRNA skeleton more excellent using Pol II promoter, opens than conventional Pol III
Mover driving shRNA has higher efficiency and lower cytotoxicity.
Summary of the invention
In view of this, the present invention provides a kind of carrier for expressing shRNA and the construction method of the carrier, screening technique and answers
With the carrier is used as the expression of shRNA, can increase substantially AAV production efficiency；The construction method, Screening Platform exist
Each link realizes high-throughput processing, saves each carrier and individually clones and the step of sequence verification, thus obtaining Effective selection
As a result vast resources and manpower are saved on the basis of.
The invention is realized by the following technical scheme:
A kind of AAV auxiliary package carrier for expressing shRNA, structure is as shown in Figure 1, it is assisted in Rep2 and CapDJ
The site 2x of insertion clone shRNA between miR33-5 ' UTR and miR33-3 ' UTR in package carrier under CMV promoter driving
ShRNA expression small fragment is connected the site 2xBsmBI into skeleton carrier, and is terminated and turned with the pA terminator of synthesis by BsmBI
Record, wherein the sequence of miR33-5 ' UTR is as shown in SEQ ID NO:1, and the sequence of miR33-3 ' UTR is as shown in SEQ ID NO:2.
Further, the construction method of the carrier of expression shRNA described above, step include:
(1) clone's skeleton of preparation linearisation cohesive end；
(2) shRNA that synthesis Dual chain belt glues end expresses small fragment；
(3) the shRNA expression small fragment that step (2) double center chain band glues end is connected to clone in step (1) and uses skeleton
On.
Further, the construction method of the carrier of the expression shRNA, specific steps include:
(1) load is packed with the above-mentioned Rep/CapDJ auxiliary for containing CMV-miR33-5 ' UTR-2xBsmBI-3 ' UTR expression cassette
Body is named as mRRC as shRNA expression cloning skeleton carrier, obtains the clone of linearisation cohesive end using BsmBI digestion
Use carrier framework；
(2) the positive negative strand primer of the shRNA needed for synthesis, is made into 20 μM of concentration, by volume normal chain respectively: anti-chain:
10xT4DNA: sterilizing nuclease free H2The proportion of O=1:1:1:7 takes positive strand primer, negative strand primer that 10xT4DNA connection is added instead
Buffer is answered, and in sterilizing nuclease free H2It is mixed in O, 95 DEG C of denaturation after five minutes, are annealed with 5 DEG C of cooling rate per minute
To room temperature, the shRNA expression small fragment that Dual chain belt glues end is obtained；
(3) the shRNA expression small fragment that Dual chain belt obtained in step (2) glues end is connected to gained in step (1)
Linearisation skeleton carrier the site 2xBsmBI (in 200 μ l, 96 orifice plate): the Dual chain belt that annealing is generated glues end
After shRNA expresses 20 times of small fragment dilution, 1 μ l is taken, it is anti-to add 25ng mRRC carrier framework, 1 μ l 10xT4DNA ligase
Buffer, 0.5 μ l T4DNA ligase are answered, 10 μ l total volumes is added to water, is connected 16 hours at 16 DEG C；Afterwards plus 1.5 μ l
NEBuffer4 (10X) reaction buffer, 1.5 μ l 10mmol/l ATP, 0.5 μ l Exonuclease V (5units), 37 DEG C
The reaction 0.5 hour AAV to get expression shRNA assists package carrier.
Further, the screening technique of the AAV auxiliary package carrier of the expression shRNA, specific steps include:
(1) package carrier is assisted by the AAV of above-mentioned steps building connection expression shRNA；
(2) convert: (ThermoFisher is public for the conversion of 2~10 μ l of carrier described in step (1) Stbl3 competent bacteria
Department, article No. C737303) 20~100 μ l (96 orifice plate of 2ml), 42 DEG C put 2 minutes on ice, then plus 1.5ml after heat shock 45~60 seconds
TB culture medium 32 DEG C shaking table culture 20 hours；Culture bacterium solution 1.2ml is taken to make the extraction of endotoxin-free mini-scale plasmid；
(3) transfect: (carrier comes by the library plasmid mRRC that step (2) Small Amount is extracted and Ad helper plasmid pAd-deltaF6
Source: carrier portion, Univ Pennsylvania USA UPENN vector core PL-F-PVADF6) it can be driven in wide spectrum promoters CAG
Purpose carrier pITR-CAG..Gluc (the expression Gluc with both ends with AAV ITR sequence of dynamic lower expression Gluc luciferase
Luciferase is voluntarily constructed by the prior art) (293T is derived from cotransfection 293T cellCRL-3216TM), it uses
10% fetal calf serum of Media Components, 2mM L-glutamine (ATCC 30-2214), 1%Penicillin/
In the DMEM in high glucose of Streptomycin (hereinafter referred to as complete medium), at 37 DEG C, 5%CO2Under the conditions of culture it is 48~72 small
When, if mRRC is not inserted into the empty carrier of shRNA as control sample；
(4) titre compares screening: whole plate cell carries out Frozen-thawed cycled 3~4 times；Whole plate cell 4000g is centrifuged 15 minutes, is taken
Supernatant makees the infection that suspends: dividing 2E+5~1E+6 293T cell per well into 24 orifice plates, 5~100 μ l of supernatant is added, and add butyric acid
Sodium is to 2~50 μm of ol/l of final concentration；It is changed to 200 hole μ l/ of complete medium after 16 hours, supernatant is taken to detect Gluc enzyme after 24 hours
Activity intensity quantifies multiple compared with the control；Retain the clone that can improve 30% or more AAV yield, the rear strain saved is made
Plate streaking culture chooses clone bacterium and determining shRNA sequence is sequenced.
Further, application of the AAV auxiliary package carrier of the expression shRNA in production AAV.
A method of AAV being produced using the AAV auxiliary package carrier of expression shRNA, specific steps include:
(1) about 1E+5 293T cell is spread into 24 orifice plates, with 37 DEG C of complete medium, 5%CO2Under the conditions of culture it is 16 small
When；
(2) each with the carrier and Ad assistant carrier pAd-deltaF6 and pITR-CAG..Gluc carrier of the expression shRNA
0.2~2ug is added in 0.05~0.2ml DMEM, adds 15 μ l of PEI (1 μ g/ μ l), mixes at once, places 10 under room temperature
It is added in cell culture medium after minute；
(3) cell and supernatant are collected after cultivating 72 hours, under the conditions of 37 DEG C and in dry ice multigelation three times, 10000g
Supernatant is AAV crude extract after ten minutes for centrifugation；
(4) gained AAV coarse body fluid in (3) is made up of the method for Iodixanol gradient ultra centrifugation or column chromatographic elution
AAV。
Compared with prior art, the present invention has the following advantages:
1) present invention expresses any shRNA using the 5 ' UTR and 3 ' UTR of the expression skeleton miR33 of efficient shRNA.
Based on the positive negative strand primer of shRNA needed for skeleton synthesis, annealing forms the shRNA expression small fragment connected into skeleton.This
The process of invention simplified vector construction and transfection cell, realization is high-throughput to handle each link, saves independent gram of each carrier
The step of grand and sequence verification, realizes high-throughput method to screen, thus on the basis of obtaining Effective selection result
Save vast resources and manpower.
2) by increasing expression shRNA in the AAV production method of three plasmid transfections to increase the production efficiency of AAV.
The selection of shRNA is based on an own vector selection skeleton: mRRC.The present invention also innovatively establish one it is simple but effective
Carrier construction method constructs a large amount of shRNA expression vector using this platform and simple and easy method to realize and improve AAV yield
High flux screening.MRRC-shNPM1 clone is obtained, realizes and improves AAV production efficiency about 60% or more.
Detailed description of the invention
Fig. 1 is carrier structure schematic diagram of the present invention.
Fluorescein after Fig. 2 infects 293T cell 48 hours for acquisition shNPM1-AAV-Gluc after mRRC-shNPM1 cotransfection
Activity ratio relatively schemes:
The control Control- generated after the mRRC empty carrier cotransfection that wherein left side cylindricality is inserted into figure for no shRNA
AAV-Gluc, right hand column shNPM1-AAV-Gluc；The longitudinal axis represents the expression activitiy of luciferase after AAV infection, with first
A cylindricality is control.
Specific embodiment
The problem of solved in order to better illustrate the present invention, used technical solution and effect achieved, are now tied
It closes specific embodiment and related data is further described.It should be noted that the content of present invention is including but not limited to following implementation
Example and combinations thereof embodiment.This part is by taking the downward shRNA (name shNPM1) of NPM1 gene as an example, to the technology of the present invention side
Case is described further.
Embodiment one constructs shNPM1 (inhibiting NPM1 gene expression) carrier library on mRRC carrier and makees to promote AAV yield
Screening
1, the carrier of expression shNPM1 of the invention, it is restricted interior which introduces two Type IIS at cloning site
The characteristics of enzyme cutting site BsmBI, this fermentoid, is cleavage site and recognition site 2~20 bases apart, therefore draws at the same time
When entering two contrary sites of identification, the identification sequence of itself can be removed after dicing, it is viscous after generating accurate cutting
End is connected for Insert Fragment, not remaining to express sequence useless sequence.
2, the construction method of the carrier of present invention expression shNPM1, specific steps include:
(1) made with the Rep/CapDJ auxiliary package carrier for containing CMV-miR33-5 ' UTR-2xBsmBI-3 ' UTR expression cassette
For shRNA expression cloning skeleton carrier, it is named as mRRC, after BsmBI digestion, agarose electrophoresis, which recycles to obtain, linearizes sticky end
Clone's skeleton at end；
(2) from the positive negative strand primer of shNPM1 needed for the synthesis of primer company:
ShNPM1-F:
5’-gcagGCGCCAGTGAAGAAATCTATACTCGAGTATAGATTTCTTCACTGGCGC-3’
ShNPM1-R:5 '-gcctGCGCCAGTGAAGAAATCTATACTCGAGTATAGATTTCTTCACTGGCGC-3 '；
It is made into 20uM concentration, respectively takes 1 μ l that 1 μ l 10xT4 DNA connection reaction buffer, 7 μ l H are added2It is mixed in O, 95
DEG C denaturation after five minutes, with per minute 5℃Speed cooling be annealed to room temperature, form shNPM1 (the SEQ ID that Dual chain belt glues end
NO:3 small fragment) is expressed；
(3) shNPM1 expression small fragment is connected into the site 2xBsmBI (200 μ l, 96 orifice plates into above-mentioned skeleton carrier
In): after the shRNA expression small fragment that the Dual chain belt that annealing is generated glues end dilutes 20 times, take 1 μ l, then plus 25ng mRRC bone
Frame carrier, 1 μ l 10x T4DNA connection enzyme reaction buffer solution, 0.5 μ l T4DNA ligase, add to 10 μ l total volumes with water,
16 DEG C connect 16 hours；Afterwards plus 1.5 μ l NEBuffer4 (10X) reaction buffers, 1.5 μ l10mmol/lATP, 0.5 μ l
Exonuclease V (5units), 37 DEG C are reacted 0.5 hour.
This method operates vector construction with 96 orifice plate high-flux parallels.Due to apply Exonuclease V (RecBCD,
NEB Cat#M0345S), the not connected carriers of the overwhelming majority are removed, loop connecting successful product is left behind, prevent from linear not connecting
It is connected into function plasmid and enters the useless clone being inserted into after bacterium by the non-target fragment of recombination generation, greatly reduce final clone's back
Scape, therefore converted product can not choose monoclonal as bed board；Using the result for screening the AAV yield that gets a promotion as standard, the positive is obtained
Confirmation is sequenced again after the selection result, and it detects the sequence in expression cassette, saves clone's routine link of the overwhelming majority.In contrast,
Exist in routine techniques due to accounting for a high proportion of non-purpose clone, needs to choose multiple clones, through too small upgrading grain and sequencing,
Correct clone is verified, then transfects, infect, screen.The low background cloning process application of high throughput of the invention introduces removal line
Property DNA excision enzyme V, can remove the not connected carriers of the overwhelming majority, leave behind successful connection product, it is ensured that conversion enters competence
The loop connecting product of the substantially successful connection of bacterium,
In the prior art due to the presence of unrelated clone, need to choose multiple clones, through too small upgrading grain and sequencing, verifying
Correct clone out, then transfect, infect, screen.High-flux clone method of the invention applies exonuclease
Exonuclease V removes not connected carrier, substantially reduces clone's background, so as to without clone's verifying is chosen the case where
The lower screening operation for making downstream experiment.
3, the screening technique of the carrier of present invention expression shNPM1, specific steps include:
(1) above-mentioned carrier is constructed by above-mentioned steps；
(2) convert: 2~10 μ l of connection product carrier described in (3) converts Stbl3 competent bacteria
(ThermoFisher company, article No. C737303) 20~100 μ l (96 orifice plate of 2ml), 42℃Heat shock puts on ice 2 after 45~60 seconds
Minute, then plus 1.5ml TB culture medium 32 degree shaking table culture 20 hours；Culture bacterium solution 1.2ml is taken to make endotoxin-free mini-scale plasmid
It extracts.
(3) it transfects: the library plasmid mRRC and Ad helper plasmid pAd-deltaF6 that (2) Small Amount is extracted (carrier source:
Carrier portion, Univ Pennsylvania USA UPENN vector core PL-F-PVADF6) it can be under wide spectrum promoters CAG driving
Express carrier pITR-CAG.Gluc (expression Gluc luciferase, voluntarily construct) cotransfection with ITR mesh of Gluc luciferase
(293T is derived from 293T cellCRL-3216TM), if mRRC is not inserted into the empty carrier of shRNA as control sample,
It is cultivated 48~72 hours in 37 degree of 5%CO cell incubators；
(4) titre compares screening: whole plate cell carries out Frozen-thawed cycled 3~4 times；Whole plate cell 4000g is centrifuged 15 minutes, is taken
Supernatant makees the infection that suspends: dividing 2E+5~1E+6 293T cell per well into 24 orifice plates, 5~100 μ l of supernatant is added, and add butyric acid
Sodium is to 2~50 μm of ol/l of final concentration；Change complete medium after 16 hours into, 200 holes μ l/ take supernatant to detect after 24 hours
Gluc enzyme activity, quantifies multiple compared with the control；Determination can improve the clone of 30% or more AAV yield, afterwards with preservation
Strain scribing line chooses clone bacterium and determining shRNA sequence is sequenced.
The method that embodiment two produces AAV using the carrier of expression shNPM1
Experimental material: 293T cell, Ad helper plasmid pAd-deltaF6, AAV assist packaging plasmid mRRC-shNPM1,
The carrier pITR-CAG.Gluc, PEI (1ug/ μ l) of ITR mesh, DMEM high glucose medium.
1, divide 1E+5 293T cell into 24 orifice plates in advance, with 37 DEG C of complete medium, 5%CO2Under the conditions of cultivate 16
Hour；
2, with the shNPM1 carrier and Ad assistant carrier each 0.2~2ug of pAd-deltaF6 and pITR-CAG.Gluc carrier,
It is added in 0.5ml DMEM, adds 15 μ l of PEI (1ug/ μ l), mix at once, placed under room temperature and cell training is added after ten minutes
It supports in base；
3, cell and supernatant are collected after 72 hours, 37℃Three times, 10000g is centrifuged 10 points to multigelation in water-bath and dry ice
Supernatant is AAV crude extract after clock.
Comparative experiments: in addition to mRRC carrier replaces with the RC carrier of conventional AAV production, remaining reagent and step are complete
Equally.
Uciferase activity knot after being infected 293T cell 48 hours using present invention expression shNPM1 carrier and conventional carrier
Fruit is as shown in Fig. 2, interpretation of result: the present invention need not change in originally production AAV method, only replace original table
Carrier RC (X) up to AAV replication protein Rep2 and AAV-X serotype coat protein (CapX) is mRRC (X), that is, can reach promotion
The effect of AAV yield 60%.The no significant difference in terms of AAV mass.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
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Claims (8)
1. a kind of AAV for expressing shRNA assists package carrier, structure is as shown in Figure 1, it is wrapped in Rep2 and CapDJ auxiliary
Load the site 2x of insertion clone shRNA between miR33-5 ' UTR and miR33-3 ' UTR in body under CMV promoter driving
ShRNA expression small fragment is connected the site 2xBsmBI into skeleton carrier, and is terminated and turned with the pA terminator of synthesis by BsmBI
Record, wherein the sequence of miR33-5 ' UTR is as shown in SEQ ID NO:1, and the sequence of miR33-3 ' UTR is as shown in SEQ ID NO:2.
2. the construction method of the AAV auxiliary package carrier of expression shRNA described in claim 1, which is characterized in that its step packet
It includes:
(1) clone's skeleton of preparation linearisation cohesive end；
(2) shRNA that synthesis Dual chain belt glues end expresses small fragment；
(3) the shRNA expression small fragment that step (2) double center chain band glues end is connected in step (1) on clone's skeleton.
3. construction method according to claim 2, which is characterized in that the construction method of the carrier of the expression shRNA, tool
Body step includes:
(1) made with the above-mentioned Rep/CapDJ auxiliary package carrier for containing CMV-miR33-5 ' UTR-2xBsmBI-3 ' UTR expression cassette
For shRNA expression cloning skeleton carrier, it is named as mRRC, is carried using the clone that BsmBI digestion obtains linearisation cohesive end
Body skeleton；
(2) the positive negative strand primer of the shRNA needed for synthesis, is made into 20 μM of concentration, by volume normal chain respectively: anti-chain:
10xT4DNA: sterilizing nuclease free H2The proportion of O=1:1:1:7 takes positive strand primer, negative strand primer that 10xT4 DNA connection is added instead
Buffer is answered, and in sterilizing nuclease free H2It is mixed in O, 95 DEG C of denaturation after five minutes, are annealed with 5 DEG C of cooling rate per minute
To room temperature, the shRNA expression small fragment that Dual chain belt glues end is obtained；
(3) the shRNA expression small fragment that Dual chain belt obtained in step (2) glues end is connected to line obtained in step (1)
Property skeleton carrier the site 2xBsmBI: annealing generate Dual chain belt glue end shRNA expression small fragment dilute 20 times
Afterwards, 1 μ l is taken, 25ng mRRC carrier framework, 1 μ l10xT4DNA connection enzyme reaction buffer solution, 0.5 μ lT4 DNA connection are added
Enzyme adds to 10 μ l total volumes with water, connects 16 hours at 16 DEG C；Afterwards plus 1.5 μ lNEBuffer4 (10X) reaction buffers,
1.5 μ l10mmol/lATP, 0.5 μ lExonuclease V, 37 DEG C of reaction 0.5 hour AAV to get expression shRNA assist packet
Load body.
4. a kind of screening technique of the AAV auxiliary package carrier of expression shRNA described in claim 1, step include:
(1) prepare carrier described in claim 1, or construct connection table suddenly by claim 2-4 any one the method
AAV up to shRNA assists package carrier；
(2) convert: the carrier described in (1) converts Stbl3 competent bacteria, and culture takes culture bacterium solution to make endotoxin-free a small amount of
Plasmid extracts；
(3) it transfects: by the plasmid of (2) Small Amount extraction and Ad helper plasmid pAd-deltaF6, with the carrier pITR- of ITR mesh
CAG..Gluc cotransfection 293T cell, if being not inserted into the empty carrier of shRNA as control sample, culture；
(4) titre compares screening: whole plate cell carries out freeze thawing, and centrifugation takes supernatant to make the infection that suspends；Then Gluc enzymatic activity is detected
Intensity quantifies multiple compared with the control；Determination can improve the clone of AAV yield, save；Make plate with the clone's strain saved afterwards
Scribing line culture chooses clone bacterium and determining shRNA sequence is sequenced.
5. screening technique according to claim 4, which is characterized in that the specific steps of the screening technique include:
(1) prepare carrier described in claim 1, or construct connection table suddenly by claim 2-4 any one the method
AAV up to shRNA assists package carrier；
(2) it converts: the conversion Stbl3 competent bacteria of 2~10 μ l of carrier described in (1), 42℃Heat shock is put on ice after 45~60 seconds
2 minutes, then plus 1.5ml TB culture medium 32 degree shaking table culture 20 hours；Culture bacterium solution 1.2ml is taken to make a small amount of matter of endotoxin-free
Grain extracts；
(3) it transfects: by the plasmid of (2) Small Amount extraction and Ad helper plasmid pAd-deltaF6, with the carrier pITR- of ITR mesh
CAG..Gluc cotransfection 293T cell, if being not inserted into the empty skeleton carrier of shRNA as control sample, 37 degrees Celsius of 5%CO2
It is cultivated 48~72 hours in incubator；
(4) titre compares screening: whole plate cell carries out Frozen-thawed cycled 3~4 times；Whole plate cell 4000g is centrifuged 15 minutes, takes supernatant
Make the infection that suspends: dividing 2E+5~1E+6 293T cell per well into 24 orifice plates, 5~100 μ l of supernatant is added, and add sodium butyrate extremely
2~50 μm of ol/l of final concentration；Complete medium is changed to after 16 hours, 200 holes μ l/ take supernatant to detect Gluc enzyme activity after 24 hours
Property intensity, quantifies multiple compared with the control；Determination can improve the clone of AAV yield, save；Make plate with the strain saved afterwards to draw
Line culture chooses clone bacterium and determining shRNA sequence is sequenced.
6. screening technique according to claim 5, which is characterized in that the conversion operation of the step (2) is in 96 orifice plates
It completes.
7. a kind of method of the AAV auxiliary package carrier production AAV using expression shRNA, specific steps include:
(1) about 1 × 10 is spread5A 293T cell is into 24 orifice plates, with 37 DEG C of complete medium, 5%CO2Under the conditions of cultivate 16 hours；
(2) take expression shRNA carrier and Ad assistant carrier pAd-deltaF6 and pITR-CAG..Gluc carrier each 0.2~
2ug is added in 0.05~0.2mlDMEM, adds PEI15 μ l (1ug/ μ l), mixes at once, places under room temperature and adds after ten minutes
Enter in cell culture medium；
(3) cell and supernatant are collected after cultivating 72 hours, 37℃Under the conditions of and dry ice in multigelation three times, 10000g centrifugation
Supernatant is AAV crude extract after ten minutes；
(4) AAV is made in gained AAV coarse body fluid in (3).
8. expressing application of the AAV auxiliary package carrier of shRNA in production AAV described in claim 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105980551A (en) * | 2014-01-31 | 2016-09-28 | 英国卫生部 | High titer production of adeno-associated viral vectors |
| CN107384957A (en) * | 2017-07-07 | 2017-11-24 | 广州派真生物技术有限公司 | The AAV auxiliary package carriers of expression miRNA a kind of and construction method, screening technique and the application of the carrier |
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2019
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105980551A (en) * | 2014-01-31 | 2016-09-28 | 英国卫生部 | High titer production of adeno-associated viral vectors |
| CN107384957A (en) * | 2017-07-07 | 2017-11-24 | 广州派真生物技术有限公司 | The AAV auxiliary package carriers of expression miRNA a kind of and construction method, screening technique and the application of the carrier |
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