绿色荧光蛋白示踪的HSV1-tk/GCV系统重组慢病毒载体的构建及活性检测

Construction of Recombinant HSV1-tk/GCV Green Fluorescent Protein Monitoring Lentivirus System and Detection of Its Activity

【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 To construct recombinant herpes simplex virus type 1 thymidine kinase/ganciclovir（HSV1-tk/GCV） green fluorescent protein（GFP） monitoring lentivirus system,and to examine the activity of package cells transfected with the constructed lentiviurs vector and infected with human cutaneous melanoma cell line A375. Methods The recombinant lentiviurs vector p LV-tk-GFP expressing suicide gene HSV1-tk and GFP gene was constructed,and then was identified by restriction endonuclease digestion and DNA sequencing. After the recombinant plasmid and packaging plasmid were cotransfected to packaging cell line HEK293 T, the animated virus was obtained and was used to infecting A375 to screen cell line with stable expression by puromycin. A standard fluorescence microscope was used for florescent detection,and the tk-GFP gene activity was evaluated by GCV anti-tumor effect. Results The results of restriction endonuclease digestion and DNA sequencing showed that tk gene had been successfully inserted into the correct position of the recombined plasmid p LV-tk-GFP,showing no sequence mutation. After A375 cells were infected with the packaging virus,tk-GFP gene expression was present under fluorescence microscope,indicating that cell line A375/tk-GFP was obtained. GCV had obvious killing effect on cell line A375/tk-GFP,indicating that tk gene activity was normal.Conclusion The recombinant lentivirus vector p LV-tk-GFP has been successfully constructed,and the vector can produce animated virus and has the bioactivity of tk-GFP gene in the infected human cutaneous melanoma cell line A375.