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目的:通过eGFP基因慢病毒颗粒的包装和感染,明确重组慢病毒对外源基因在细胞中的转导能力。方法:把慢病毒质粒Lv105-eGFP转染到293 Ta细胞中并包装为慢病毒颗粒,经电镜检测病毒颗粒和实时定量PCR(qPCR)测定病毒滴度,之后感染H1299细胞,通过荧光显微镜观察比较eGFP在靶细胞中的表达效率。结果:eGFP在包装细胞中转染和表达效率在48h时达90%以上,到72h时细胞病变效应(CPE)逐渐显著;电镜检测可看到典型的慢病毒颗粒形态特征;病毒原液的滴度是4.4×107VG/mL、浓缩液的是2.68×1010VG/mL;1μL病毒原液感染H1299细胞时,eGFP表达水平可达10%、增加到100μL感染时表达水平达到99%以上,证明eGFP基因得到有效转导并可在细胞中高水平表达绿色荧光蛋白。结论:成功包装了eGFP慢病毒颗粒并可在靶细胞中获得高效表达。
OBJECTIVE: To clarify the transduction ability of recombinant lentivirus to foreign genes in cells by packaging and infecting lentiviral particles of eGFP gene. Methods: The lentiviral plasmid Lv105-eGFP was transfected into 293Ta cells and packaged as lentivirus particles. The virus particles were detected by electron microscopy and the virus titer was determined by real-time quantitative PCR (qPCR), then infected with H1299 cells and observed by fluorescence microscopy The efficiency of eGFP expression in target cells. Results: The efficiency of transfection and expression of eGFP in packaging cells reached more than 90% at 48h, and the cytopathic effect (CPE) gradually became obvious at 72h. The morphology of typical lentivirus particles could be observed by electron microscopy. The titer Is 4.4 × 107VG / mL, concentrated solution is 2.68 × 1010VG / mL; 1μL virus stock infected H1299 cells, eGFP expression levels up to 10%, increased to 100μL infection, the expression level of 99% or more, that eGFP gene is valid Transduced and expressed high levels of green fluorescent protein in the cell. Conclusion: The eGFP lentivirus particles were successfully packaged and highly expressed in target cells.