腺病毒包装、扩增、纯化、滴度测定及感染

腺病毒包装、扩增、纯化、滴度测定及感染 一、 包装 1. 包装细胞 详情见AD-293 Cells.pdf，AdEasy? Adenoviral Vector System.pdf(P31-P32) 2. 细胞转染 方法一、 详情见Adeno-X Expression System 1 User Manual.pdf(P28-P29) C0508磷酸钙法细胞转染试剂盒.pdf 3. 病毒收集 详情见AdEasy? Adenoviral Vector System.pdf(P34) 二、 扩增 详情见Adeno-X Expression System 1 User Manual.pdf(P29-P30) 三、 纯化 (i) 病毒上清(接一、包装 3.病毒收集). (ii) Add 51 ml of a 50% PEG 6000 solution. (iii) Add 21.7 ml of a 4 M NaCl stock solution. (iv) Add 23.3 ml of PBS. This will result in a final volume of 300 ml. The final PEG 6000 concentration will be 8.5% and the final NaCl concentration will be B0.3 M. (v) Distribute the sample as 150-ml aliquots in two 250-ml polypropylene wide-mouthed bottles. (vi) Store the bottles at 4 1C for 1.5 h. Mix contents every 20–30 min. (vii) Centrifuge bottles at 7,000g for 10 min at 4 1C using a Beckman fixed-angel JLA-10.500 rotor. (viii) After centrifugation, a white pellet should be visible. (ix) Carefully decant the supernatant and add 1.2 ml of 50 mM Tris-HCl, pH 7.4, per bottle. Resuspend the pellets by vigorously pipetting liquid up and down. (x) Vortex the bottles vigorously for 20–30 s to further resuspend the pellets. (xi) Transfer the vector suspension into screw-cap microfuge tubes in aliquots of 100 ml. 。(xii) Snap-freeze the tubes in crushed dry ice and store at -80 C. a.按照10ml的病毒上清加入2.5ml的50%PEG6000混合; b. 加入1.064ml的4M的NaCl混匀; c. 加入1.142ml的PBS混匀; d. 4?C下，孵育1.5h，每20-30min颠倒混匀; e. 4?C下，7000g离心10min。 f.小心移除上清，切勿触动白色沉淀，如果白色沉淀出现松动，可以在7000 g下短暂离心; g. 加入原病毒上清1/200 to 1/100 体积的PBS重悬沉淀病毒粒子; h. 立即对浓缩纯化的病毒粒子进行滴度测定，并分装后(50-100ul/管)冻存于-80?C超低温冰箱中; 上述所需试剂配制方法见Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors.pdf(P3) 四、 滴度测定 病毒滴度测定-针对有绿色荧光蛋白标记的病毒.doc 病毒滴度测定-针对没有绿色荧光蛋白标记的病毒.doc 五、 感染 详情见Adeno-X Expression System 1 User Manual.pdf(P31) a. 感染前24小时，把细胞传代至35 mm平皿中，加入完全培养基至终体积3ml，培养过夜，感染时，细胞密度为80–90%; b. 把冻存于-80?C的病毒取出并在室温下冻融; c. 用新的完全培养基更换培养液，并加入冻融的病毒粒子(MOI为50-100)和polybrene(4 μg/ml)，培养液终体积为3ml; d. 感染8-24h后，用新3-4ml新完全培养基更换感染培养液; e. 感染48小时后收取细胞进行QPCR检测，感染72小时后收取细胞进行WB 检测;