腺病毒包装、扩增、纯化、滴度测定及感染.doc

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.pdf3. 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 thefinal 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 2030 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 byvigorously pipetting liquid up and down.(x) Vortex the bottles vigorously for 2030 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. 4C下，孵育1.5h，每20-30min颠倒混匀；e. 4C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-80C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8090%；b. 把冻存于-80C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poly