CN101173300A - Method for separating and purifying recombined glandulae correlation viral vectors - Google Patents
Method for separating and purifying recombined glandulae correlation viral vectors Download PDFInfo
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- CN101173300A CN101173300A CNA2006100325121A CN200610032512A CN101173300A CN 101173300 A CN101173300 A CN 101173300A CN A2006100325121 A CNA2006100325121 A CN A2006100325121A CN 200610032512 A CN200610032512 A CN 200610032512A CN 101173300 A CN101173300 A CN 101173300A
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- chloroform
- viral vectors
- associated virus
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- recombined glandulae
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Abstract
The invention relates to an isolation and purification method of a recombinant adeno-associated virus vector, which isolates, concentrates and purifies the adeno-associated virus vector through six steps of ''chloroform treatment-PEG/NaClprecipitation-chlorooform extracts-dialysis-rotary evaporation-concentration''. The method has the advantages of getting the recombinant adeno-associated virus vector with higher purity and titer, simplicity, low cost, not affecting the activity of the virus particles, and having no toxic effects on human health.
Description
Affiliated technical field
The present invention relates to a kind of method that is used to separate with purifying recombined glandulae correlation viral vectors.
Background technology
Traditional separation and the method for purifying recombined glandulae correlation viral vectors are to adopt the fraction precipitation of ammonium sulphate method that recombinant adeno-associated virus is separated with cell debris and make it concentrated, use cesium chloride gradient ultracentrifugation 48 hours then, obtain the recombinant adeno-associated virus component.This method rate of recovery that wastes time and energy is low, and the infectivity of recombinant adeno-associated virus also has bigger loss simultaneously, and cesium chloride has potential toxicity to human body.Because heparin is the analogue of recombinant adeno-associated virus natural receptor, the purification process of report heparin affinity chromatography is arranged in recent years.The potential problems that adopt this purification process are that a lot of cell proteins are also with the heparin combination, therefore needing to solve the people such as problem .Zolotukhin that how to remove foreign protein has taked first with iodine Di alcohol density gradient centrifugation, obtain the recombinant adeno-associated virus of " half purifying ", carry out purifying with heparin affinity chromatography again.Although these methods improve a lot the rate of recovery of recombinant adeno-associated virus, the cost height is also arranged, plant and instrument requires high shortcoming, is difficult for promoting.Therefore, still lack at present a kind of efficient, easy, recombined glandulae correlation viral vectors purification process cheaply.
Summary of the invention
The objective of the invention is provides a kind of easy in order to overcome the shortcoming of prior art, efficient, cheaply the method for separation and purifying recombined glandulae correlation viral vectors.Chloroform is handled lysing cell, adds the chloroform of 1/10 cell suspension volume, and violent jolting is 1 hour in 37 ℃ of shaking tables.1mol/L NaCl/10%PEG concentrates adeno-associated virus.Residual PEG and protein are removed in the chloroform extracting, and jolting dissolving back ice bath was placed 1 hour.Residual inorganic salt in the viral liquid are removed in dialysis, and as in the 2LPBS solution, 4 ℃ are spent the night with viral liquid.Viral liquid with 4 ℃ of evaporations of Rotary Evaporators 2 hours, is removed residual minim chloroform.Evaporating column concentrating virus liquid, 3000 rev/mins are centrifugal, and simmer down to 500 μ L improve viral titre.
The present invention has following advantage: not only can obtain the recombined glandulae correlation viral vectors of higher degree and titre, and method is easy, cost is lower, and virion active unaffected is to the human body nontoxicity.
Embodiment
(1) packing cell of collection recombined glandulae correlation viral vectors is suspended in the PBS solution.The chloroform that adds 1/10 volume places the violent jolting of 37 ℃ of shaking tables 1 hour.Add solid sodium chloride to final concentration 1mol/L, the jolting dissolving, 4 ℃, 12000 rev/mins are centrifugal 15 minutes.Take out the upper strata water, discard chloroform and precipitation.
(2) add PEG8000 to final concentration 10% (w/v), jolting dissolving back ice bath was placed 1 hour, and 11000 rev/mins centrifugal 15 minutes.Supernatant is discarded, with 5mL PBS damping fluid with each centrifuge tube pipe at the bottom of and the piping and druming of precipitation on the tube wall elute merging, its branch is filled to (0.6mL/ pipe) in the 1.5mL plastic centrifuge tube, adding DNA enzyme and RNA enzyme digest 30min to final concentration 1 μ g/mL under the room temperature.
(3) add isopyknic chloroform extracting, 4 ℃, 12000 rev/mins centrifugal 5 minutes.
(4) carefully sucking-off upper strata water is to the dialysis band, and in 2LPBS solution, 4 ℃ are spent the night.
(5) move to again in the 1.5mL plastic centrifuge tube, place on the Rotary Evaporators, 4 ℃ of rotary evaporations 2 hours,
(6) then viral liquid is packed in the Millpore evaporating column, 3000 rev/mins centrifugal, and simmer down to 500 μ L are the viral liquid of purifying.
The adeno-associated virus titre that obtains with this method can reach 1.0 * 10
12Virion/ml detects the visible VP1 that significantly represents virus coat, VP2, three kinds of protein bands of VP3 with polyacrylamide gel electrophoresis.
Claims (6)
1. one kind is rapidly and efficiently separated and the method for purifying recombined glandulae correlation viral vectors.Its process is:
1. chloroform is handled lysing cell, adds the chloroform of 1/10 cell suspension volume, and violent jolting is 1 hour in 37 ℃ of shaking tables.
2. with 1mol/L NaCl/10%PEG adeno-associated virus is concentrated.
3. residual PEG and protein are removed in the chloroform extracting, and jolting dissolving back ice bath was placed 1 hour.
4. residual inorganic salt in the viral liquid are removed in dialysis, and as in the 2LPBS solution, 4 ℃ are spent the night with viral liquid.
5. viral liquid was evaporated 2 hours for 4 ℃ with Rotary Evaporators, remove residual minim chloroform.
6. evaporating column concentrating virus liquid, 3000 rev/mins are centrifugal, and simmer down to 500 μ L improve the titre of virus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100325121A CN101173300A (en) | 2006-11-02 | 2006-11-02 | Method for separating and purifying recombined glandulae correlation viral vectors |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100325121A CN101173300A (en) | 2006-11-02 | 2006-11-02 | Method for separating and purifying recombined glandulae correlation viral vectors |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101173300A true CN101173300A (en) | 2008-05-07 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
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| CNA2006100325121A Pending CN101173300A (en) | 2006-11-02 | 2006-11-02 | Method for separating and purifying recombined glandulae correlation viral vectors |
Country Status (1)
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| CN (1) | CN101173300A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016006658A1 (en) * | 2014-07-10 | 2016-01-14 | タカラバイオ株式会社 | Production method for non-enveloped virus particles |
| CN107630037A (en) * | 2017-10-19 | 2018-01-26 | 和元生物技术（上海）股份有限公司 | A kind of purifying process for obtaining high-purity gland relevant viral vector |
-
2006
- 2006-11-02 CN CNA2006100325121A patent/CN101173300A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016006658A1 (en) * | 2014-07-10 | 2016-01-14 | タカラバイオ株式会社 | Production method for non-enveloped virus particles |
| US10023846B2 (en) | 2014-07-10 | 2018-07-17 | Takara Bio Inc. | Production method for non-enveloped virus particles |
| CN107630037A (en) * | 2017-10-19 | 2018-01-26 | 和元生物技术（上海）股份有限公司 | A kind of purifying process for obtaining high-purity gland relevant viral vector |
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| Date | Code | Title | Description |
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| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20080507 |