目的:包装纯化具有感染性的腺相关病毒rAAV-hBMP7,检测其包装效率及感染滴度.方法:应用AAV Helper-Free System、磷酸钙三质粒共沉淀法,将病毒质粒pAAVIRES-hrGFP-hBMP7、辅助质粒pAAV-Helper和包装质粒pAAV-RC转染AAV-293细胞,进行病毒包装,荧光计数法测定病毒包装效率;反复冻融收获病毒液,按氯仿处理、PEG/NaCl沉淀的方法浓缩纯化;纯化的病毒液感染AAV-HT1080细胞观测荧光表达,原位β-半乳糖苷酶染色测定病毒感染效率和滴度.结果:转染AAV-293细胞后6h即有绿色荧光表达,包装效率为94.16±0.13%.纯化的rAAV-hBMP7在HT1080细胞中的感染效率为78%,计算病毒物理滴度为2.34×1011v·g/ml.结论:磷酸钙三质粒共沉淀法可实现病毒在AAV-293细胞中的高效包装,纯化的rAAV病毒载体能介导hBMP7基因转染真核细胞并具有较高的转染效率和感染滴度.%Objective : To package and purify the infectious virus which are the recombined adeno-associated viral vector of human bone morphogenetic protein-7(hBMP-7) and testing the packaging efficiency and infection titer . Methods : Application of AAV Helper-Free System and calcium phosphate three plasmid coprecipitation meth -od ,the virus plasmid pAAVIRES - hrGFP - I1BMP7 , auxiliary plasmid pAAV - Helper and packing plasmid pAAV -RC were transfected into AAV-293 cells to virus packaging .Through the repeated freezing and thawing get virus concentrate .Obtained virus fluid were processed by chloroform precipitate treatment , PEG8000/NaCl precipitation and chloroform extraction for purification . The purified virus fluid infected HT-1080 cell and observed the fluorescence expression . Virus titer and infection efficiency to HT 1080 cells were identified by the in situ beta galactosidase dyeing . Results : After transfected AAV-293 cells , green fluorescent protein was expressed in 6 hours and the packing efficiency is 94.16±0.13% ; the transfected efficiency to AAV-HT1080 cells was 78% .Measuring virus titer is a-bout 2 .34×1011 v .g/ml. Conclusion; Calcium phosphate three plasmid coprecipitation method can realize the virus in AAV-293 cells of the efficient packing . The purified rAAVhBMP7 virus carrier can mediated I1BMP7 gene transfected eukaryotic cells and has high transfection efficiency and infection titer .
展开▼
