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【目的】构建绿色荧光蛋白(GFP)示踪的单纯疱疹病毒1型胸苷激酶基因(HSV1-tk)的重组慢病毒载体,并检测该系统对人皮肤黑色素瘤细胞株A375的感染活性。【方法】构建含有HSV1-tk、GFP融合基因的重组慢病毒表达载体p LV-tk-GFP,并使用限制性内切酶消化进行鉴定及序列测定;用该重组质粒与辅助质粒共转染包装细胞HEK293T,获得重组活病毒并感染A375,以嘌呤霉素筛选稳定表达细胞株;使用荧光显微镜观察GFP基因的表达,更昔洛韦(GCV)杀伤实验验证tk-GFP基因的活性。【结果】重组质粒p LV-tk-GFP经限制性酶切鉴定和DNA序列测定分析显示,tk基因已连接到载体正确位置,序列无突变;包装病毒并感染细胞后,荧光显微镜下观察显示tk-GFP基因在A375中表达;筛选得到稳定表达细胞株A375/tk-GFP,GCV能显著杀伤A375/tk-GFP细胞,表明tk基因活性正常。【结论】成功构建了重组p LV-tk-GFP慢病毒载体,该载体能包装产生活病毒且在感染的人黑色素瘤细胞株A375中tk-GFP具有生物活性。
【Objective】 The recombinant lentiviral vector of herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) traced by green fluorescent protein (GFP) was constructed and the activity of the system against human melanoma cell line A375 was tested. 【Method】 The recombinant lentiviral vector p LV-tk-GFP containing HSV1-tk and GFP fusion gene was constructed and identified by restriction endonuclease digestion and sequencing. The recombinant plasmids were co-transfected with helper plasmids HEK293T cells were infected with recombinant live virus and infected with A375. The cells were stably transfected with puromycin. The expression of GFP gene was observed by fluorescence microscopy. The activity of tk-GFP gene was tested by GCV killing assay. 【Result】 The recombinant plasmid p LV-tk-GFP was verified by restriction enzyme digestion and DNA sequence analysis. The tk gene was ligated to the vector at the correct position and had no mutation in the vector. After packaging the virus and infecting the cells, the fluorescence microscopy showed that tk -GFP gene was expressed in A375 cells. Stably expressing cell line A375 / tk-GFP was screened, and GCV could kill A375 / tk-GFP cells significantly, indicating that tk gene activity was normal. 【Conclusion】 Recombinant p LV-tk-GFP lentiviral vector was successfully constructed and was able to package live virus and tk-GFP was bioactive in infected human melanoma cell line A375.