携带EGFP基因的逆转录病毒载体的构建及初步应用

Construction and Preliminary Application of Retroviral Vector Carrying EGFP Gene

【目的】构建携带有增强型绿色荧光蛋白(EGFP)基因的逆转录病毒载体,观察GFP的表达情况以及对靶细胞生物特性的影响。【方法】以质粒pEGFP-C1为模板进行PCR反应,获得完整保留pEGFP-C1载体多克隆酶切位点(MCS)的EGFP基因片段,定向插入逆转录病毒载体pLXSN,筛选出正确的重组逆转录病毒载体pLXSN-EGFP,转染PA317包装细胞后获得具有感染能力的病毒颗粒,转染人多型性脑胶质瘤细胞SW038-C2,通过荧光显微镜和流式细胞仪(FACS)观察EGFP做为报告基因在体外的表达情况、以及对靶细胞生物特性的影响。【结果】成功克隆携带有EGFP基因的逆转录病毒载体,该载体经包装细胞包装后可有效转染人多型性脑胶质瘤细胞SW038-C2并稳定表达,对靶细胞的生长无抑制。高表达GFP的靶细胞形态变得细长,FACS检测约有98%的细胞表达GFP,体外培养6个月荧光强度没有明显的衰减,而低表达GFP的靶细胞形态无变化,FACS检测有30%细胞表达GFP。【结论】使用GFP作为报告基因,检测到的病毒载体转染率比实际偏低,所构建的pLXSN-EGFP载体可在体外稳定表达,为再插入治疗用基因进行后续研究奠定基础。

[Objective] To construct the retroviral vector carrying enhanced green fluorescent protein （EGFP） gene and to observe the expression of green fluorescent protein （GFP） in vitro and its effect on biological characteristics of target cells. [Methods] EGFP gene fragment, which was ligated into the multiple cloning site （MCS） with pEGFP-C1 vector preserved completely, was obtained through PCR reaction using pEGFP-C1 plasmid as a template and directionally inserted into MCS of retroviral vector pLXSN. The right recombinant retroviral vector pLXSN-EGFP was selected to transfect packaging cell PA317 to gain virus particles with infection ability. Human glioblastoma mutiforme cells SW038-C2 were transfected. The expression of EGFP gene as reporter gene in vitro and its effect on the biological characteristics of target cells were observed by fluorescence microscope and fluorescence activated cell sorting （FACS）. [Results] The recombinant retroviral vector with EGFP gene was cloned successfully. Packaged by packaging cell PA317, the recombinant vector could transfect human glioblastoma mutiforme cells SW038-C2 effectively, express stably, and did not show inhibitory effect on the growth of target cells. Target cells with high expression of GFP became long and thin. FACS showed about 98% of these cells expressed GFP, and the intensity of the fluorescence did not decrease after culture in vitro for 6 months. The shape of target cells expressing low level GFP did not change and 30% of them expressed GFP was observed in FACS. [Conclusions] The transfection rate of virus vector being detected is lower than actual results while GFP is used as reporter gene. The constructed vector pLXSN-EGFP can express stably in vitro, which are useful for future research for inserting another therapy gene in the vector.