重组腺相关病毒2/8的聚乙烯亚胺制备法

Preparation of Recombinant Adeno-Associated Virus 2/8 by Polyethylenimine

重组腺相关病毒2/8(recombinant adeno-associated virus 2/8,rAAV2/8)是基因治疗中的一种非复制型病毒,包装困难、耗时、费力。本研究中,一种采用非病毒载体聚乙烯亚胺(polyethylenimine,PEI)来高效包装重组腺相关病毒的方法被成功建立。在成功构建并鉴定包装rAAV2/8所需的pAAV-neo-eGFP、p5E18-VD286和pSH3-D 3个质粒后,用优化过转染条件的PEI转染方法共转染这3个质粒到HEK293细胞,获得了高效稳定的转染效率,并成功包装出携带有绿色荧光蛋白eGFP的rAAV2/8。rAAV2/8的细胞感染实验表明包装出的病毒具感染活性,证明了用PEI共转染三质粒高效包装rAAV2/8具可行性。

Recombinant adeno associated virus 2/8（rAAV2/8）is a nonreproductive gene therapy vector which is difficult, time-and labor-consuming to package. In present work, a novel method exploring the non-viral vector Polyethylenimine （PEI） to produce recombinant adeno-associated virus efficiently has been successfully established. It successfully constructs and identifies the three plasmids pAAV-neo-eGFP, p5E18 VD286 and pSH3-D which are required for rAAV2/8 preparation, and then co-transfects the three plasmids into HEK293 cells with optimized PEI method, successfully bringing out eGFP carried rAAV2/8 preparation with stable and high transfection efficiency. Cell transduction experiment of rAAV2/8 shows that the virus are functional, and simultaneously proves the feasibility of using PEI to produce rAAV2/8 efficiently.