重组逆转录病毒载体的包装

Packaging of Recombinant Retroviral Vectors in Vitro

将 3.7kb的 Lac Z基因片段克隆到含标志基因 Neor的逆转录病毒载体 p LXSN与 p LNCX的多克隆位点 ,构建了含有 Lac Z基因的重组逆转录病毒载体 p LLSN和 p LNCL。在测定了 G4 1 8对包装细胞系PA31 7的最小致死剂量后 (最小致死剂量为 0 .3g/ L) ,通过脂质体 Lipofectin与磷酸钙 2种介质 ,分别将 2种重组逆转录病毒载体导入包装细胞系 PA31 7进行包装 ,并以 0 .3g/ L的 G4 1 8进行筛选 ,得到多个 G4 1 8抗性克隆。经扩大培养、传代 ,分别得到包装 p LLSN与 p LNCL的 2株阳性细胞。电镜下可见阳性细胞的培养上清中具有逆转录病毒形态特征的成熟病毒粒子。本研究为运用逆转录病毒载体系统 。

Recombinant retroviral vectors, pLLSN and pLNCL carrying LacZ have been constructed.The two vectors were transfected into PA317 for packaging by Lipofectin and calcium phosphat precipitation.After G418 selection at a concentration of 0.3 g/L for 2 weeks ,G418-resistant PA317 colonies were obtained,and then they were amplified.The supernatants of the cell cultures were harvested, concentracted and oberserved under transmission electron microscope, the mature virus particles possessed the typically morphological feature of retrovirus have been found.