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rAAV vectorの精製方法
以下のReviewにおけるOptimized purificationの精製方法は、Ultracentrifugationを用いずにAAVを精製している文献である。
Transfection of HEK293, and post culture
20 Flasks of T-150, 3 Plasmid PEI
Centrifugation / cell pellet
3,000 xg 10min
(A) Lysis by Freeze-thaw, and (B) Nuclease, (C) sodium deoxycholate
(A) 50mM Tris-HCl, 0.15M NaCl, 2mM NgCl2, pH8, Dry ice-ethanol ↔︎ 37℃ water
(B) Benzonase: 50u/mL, RNase: 10U/mL, 37℃, 30min
(C) 0.5% sodium deoxycholate, 37℃, 30min
cfg (2,500 g x 10min)/sup
Pooling with cell culture supernatant
PEG 8000, NaCl Treatment / ppt
1. 8% PEG 8000, 0.5M NaCl by 40% PEG 8000, 2.5M NaCld, incubation 1h, RT.
2. centrifuge 2,000g x 30min
3. Re-suspend with HEPES buffer
Chloroform Extraction / cfg-sup /Evaporate
疎水性タンパクとPEGを除去
1. Add equal volume of chloroform
2. Vortex going , 2min , RT.
3. cfg (370g x 5min) (おそらく水層にAAV)
4.. Evaporate 30min , RT
PEG 8000, AmSO4 Treatment / AAV in sup
1. 10% PEG 8000, 13.2% AmSO4 pH8.0 adjusted by stocked solution
2., Incubation at RT.
(Impurity salt out in ppt), HEPES buffer pH has to be kept at pH8.0, AAV stable is Alkaline that acid.
Dialysis using 50kDa
diligent: PBS or MEMEM media with 0.001% puluronic F68 for preventing the aggregation.
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