腺病毒包裝、擴增、純化、滴度測定及感染

1、.腺病毒包裝、擴增、純化、滴度測定及感染一、 包裝1. 包裝細胞詳情見AD-293 Cells.pdf，AdEasy Adenoviral Vector System.pdf（P31-P32）2. 細胞轉(zhuǎn)染方法一、詳情見Adeno-X Expression System 1 User Manual.pdf（P28-P29）C0508磷酸鈣法細胞轉(zhuǎn)染試劑盒.pdf3. 病毒收集詳情見AdEasy Adenoviral Vector System.pdf（P34）二、 擴增詳情見Adeno-X Expression System 1 User Manual.pdf（P29-P30）三、 純化(i)

2、 病毒上清（接一、包裝 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 a

3、s 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.(i

4、x) 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 i

5、n 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混勻；c. 加入1.142ml的PBS混勻；d. 4°C下，孵育1.5h，每20-30min顛倒混勻；e. 4°C下，7000g離心10min。f.小心移除上清，切勿觸動白色沉淀，如果白色沉淀出現(xiàn)松動，可以在7000 g下短暫離心；g. 加入原病毒上清1/200 to 1/100 體積的PBS重

6、懸沉淀病毒粒子；h. 立即對濃縮純化的病毒粒子進行滴度測定，并分裝后（50-100ul/管）凍存于-80°C超低溫冰箱中；上述所需試劑配制方法見Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors.pdf（P3）四、 滴度測定病毒滴度測定-針對有綠色熒光蛋白標記的病毒.doc病毒滴度測定-針對沒有綠色熒光蛋白標記的病毒.doc五、 感染詳情見Adeno-X Expression System 1 User Manual.pdf（P31）a. 感染前24小時，把細胞傳代至35 mm平皿中，加入完全培養(yǎng)基至終體積3ml，培養(yǎng)過夜，感染時，細胞密度為8090%；b. 把凍存于-80°C的病毒取出并在室溫下凍融；c. 用新的完全培養(yǎng)基更換培養(yǎng)液，并加入凍融的病毒粒子（MOI為50-100）和poly