rAAV-hBMP7的包装纯化及感染滴度的测定

Package and purify the rAAVhBMP_7 virus and infection titer measurement

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-hrG-FP-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-associ- ated 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 - hBMP7, 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 con- centrate. Obtained virus fluid were processed by chloroform precipitate treatment, PEG8000/NaC1 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 HT1080 cells were identified by the in situ beta galactosidase dye- ing. 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 X 1011v. 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 hBMP7 gene trans- fected eukaryotic cells and has high transfection efficiency and infection titer.