重组慢病毒载体pLKO-1-EGFP-puro的构建

Construction of a noval lentiviral vector pLKO-1-EGFP-puro

目的:构建具有绿色荧光蛋白(GFP)标记及嘌呤霉素(puro)抗性的重组慢病毒载体。方法:改建慢病毒载体质粒pLKO-1-puro的多克隆位点(MCS),以质粒pEGFP-C3为模板扩增出CMV-EGFP并与pLKO-1-puro连接,形成重组的慢病毒载体质粒pLKO-1-EGFP-puro,后者与包装质粒Δ8.2和包膜蛋白质粒VSV-G在293T细胞中进行包装,产生重组慢病毒颗粒,感染非洲绿猴肾细胞Vero细胞,观察GFP的表达。结果:成功改建了慢病毒载体质粒pLKO-1-puro并插入了CMV-EGFP,形成了新型慢病毒载体质粒pLKO-1-EGFP-puro。3质粒共转染293T细胞产生的重组慢病毒颗粒感染Vero细胞后高表达绿色荧光。结论:成功构建了高效的带GFP报告基因及嘌呤霉素抗性的慢病毒载体。

Aim：To construct lentiviral vector with green fluorescent protein（GFP） and puromycin（puro）-resistant gene.Methods：The multiple clone site（MCS） of lentiviral plasmid pLKO-1-puro was reconstructed to be more compatible,and CMV-EGFP was also amplified from plasmid pEGFP-C3 by PCR. After double digestion, the new pLKO-1-puro and CMV-EGFP sequences were ligated to form another lentiviral plasmid pLKO-1-EGFP-puro,which was cotransfected into 293T cells with packaging plasmid Δ8.2 and envelop plasmid VSV-G to produce recombined lentivirus particles. Finally, the GFP expression was assayed by lentivirus infected Vero cells.Results：The MCS of pLKO-1-puro was successfully reconstructed.The new pLKO-1-puro was also inserted correctly by CMV-EGFP to form recombined pLKO-1-EGFP-puro. Lentiviral particles produced by cotransfection of three plasmids into 293T cells had high GFP expression in Vero cells.Conclusion： Recombinant lentiviral vector with GFP gene and puro resistance has been successfully constructed.