hTERT基因启动子驱动eGFP的慢病毒载体构建及表达的初步研究

Construction and expression of lentivirus vector containing eGFP drived by hTERT promoter

目的构建人端粒酶逆转录酶(hTERT)基因启动子驱动eGFP报告基因的慢病毒载体,观察eGFP在肿瘤细胞中的表达情况。方法用hTERT基因启动子取代慢病毒载体eGFP上游的CMV启动子;将重组慢病毒载体pLenti-phTERT-eGFP用脂质体介导法转入端粒酶阳性的293FT、HepGⅡ、SGC7901细胞及端粒酶阴性的U2OS细胞中进行功能鉴定,以慢病毒载体pLenti-pCMV-eGFP作为对照,倒置荧光显微镜观察绿色荧光蛋白的表达情况。结果在端粒酶阳性的3株细胞中均可以观测到绿色荧光蛋白表达;在端粒酶阴性的U2OS细胞中无绿色荧光蛋白表达。结论hTERT基因启动子在端粒酶表达阳性的细胞中具有较强转录活性;所构建的慢病毒载体为进一步研究、利用hTERT基因启动子提供了较好的实验工具。

Objective To construct lentivirus vector containing eGFP reporter gene drived by hTERT promoter and to investigate the expression level of this recombined vector in different tumor cells. Methods First to replace the cytomegalovirus promoter with hTERT promoter in upstream of eGFP on lentivirus vector; second,with lipotectamine,to transfect recomhined lentivirus vector pLenti-phTERT eGFP into telomerase positive cells including 293FT, HepGⅡ,SGC7901 and also the telomerase negative cells U2OS,then to compare with lentivirus vector pLenti-pCMV-eGFP and investigate the expression status of our recombined vector under fluorescence microscope. Results Green fluorescence could be detected in telomerase positive cells （293FT, HepGⅡ, SGC7901） not the telomerase negative cells （U2OS）. Conclusion hTERT promoter has relative strong transcription activity in telomerase positive cells and our recombined lentivirus vector will be a good model for further study and utilization on hTERT promoter.