pLXSN-EGFP逆转录病毒载体的构建及体内外表达的研究  

Construction and Preliminary Application of Retroviral Vector which Carries EGFP Gene

作　　者：赵宁[1] 郭中敏[1] 陈系古[1] 

机构地区：[1]中山大学实验动物中心,广州510080

出　　处：《热带医学杂志》2009年第3期241-244,F0003,共5页Journal of Tropical Medicine

基　　金：广东省重点攻关课题(No.522203045);广东省博士启动基金(No.984058)

摘　　要：目的构建携带有绿色荧光蛋白(GFP)基因的逆转录病毒载体,观察该报告基因在动物体内外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,获得增强型绿色荧光蛋白(EGFP)基因片段,定向插入逆转录病毒载体pLXSN获得重组逆转录病毒载体pLXSN-EGFP,转染PA317包装细胞后收集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观察EGFP作为报告基因在体内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并在体内外稳定表达,对靶细胞及其所成肿瘤的生长无抑制。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30.7%细胞表达GFP。结论该载体可在体内外成功表达,对靶细胞的生长无明显抑制,高表达GFP的靶细胞有一定形态上的变化;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。Objective To construct the recombinant retroviral vector which carries enhanced green fluorescent protein （GFP） gene, analyze the level of GFP expressing in vitro and in vivo and observe the biological character of cell expressed EGFP gene. Methods EGFP gene was amplified by PCR and directionally inserted into the multiple cloning site of retroviral vector pLXSN. Positive clone was identified by enzyme digesting and PCR. Transfected replication defective retroviral vector was packaged by PA317 cells into SW038-C2 cells. The expression of EGFP gene in tumor cells and the biological character changing of tumor cells was observed in vitro and in vivo by fluorescence microscope and FACS. Results Successfully constructed the recombinant retroviral vector pLXSN-EGFP and was packaged by PA317. The recombinant vector can transfect the glioblastoma mutiforme cells of human SW038-C2 and express normally. The growth of transfected cells and tumor nods were not affected. The cells expressing high level EGFP gene became long and thin. Assaying by FACS, about 98% of these cells expressed GFP. The intensity of the fluorescence was not reduced after 6 months cultured in vitro. The shape of cells expressing low level EGFP gene is normal and 30% of them expressed GFP. Conclusion The EGFP gene could express normally in vitro and in vivo and didn＇t influence the growth of tumor cells. But the morphology of tumor cells expressed GFP gene was changed. Using GFP as the report gene, transfection rate of the retroviral vector is lower than expected.

关 键 词：EGFP基因 逆转录病毒载体 pLXSN—EGFP 转染 SW038-C2细胞 

分 类 号：R730.5[医药卫生—肿瘤]