人端粒酶逆转录酶基因shRNA慢病毒表达载体的构建及其病毒包装鉴定

Construction and identification of human telomerase reverse transcriptase-targeted small hairpin RNA-expressing plasmid and its stable transfected cervical cancer line Caski

目的:构建携带人端粒酶逆转录酶基因(hTERT)慢病毒表达载体LV3-shRNA-hTERT及其病毒包装鉴定并稳定转染宫颈癌Caski细胞系。方法:使用Designer3.0(Genepharma)软件设计靶向hTERT的小发卡RNA(shRNA)序列,将hTERT-shRNA基因片段插入慢病毒载体pGLV3/H1/GFP+Puro中,构建慢病毒表达质粒LV3-shRNA-hTERT。通过酶切、DNA测序验证hTERT片段的准确性,将LV3-shRNA-hTERT与包装质粒共转染293T细胞,浓缩上清并测定病毒滴度获得重组慢病毒,将该重组慢病毒感染Caski细胞,通过观测绿色荧光蛋白(GFP)的表达判断转染成功与否以及估计转染效率,RTPCR、Western blot分别测定转染后不同时间点的hTERTmRNA水平和hTERT蛋白表达情况,TRAP-ELISA法测定细胞端粒酶活性。结果:LV3-shRNA-hTERT携带正确hTERT基因,重组病毒经PCR证实hTERT-shRNA基因插入。利用重组慢病毒介导将重组质粒LV3-shRNA-hTERT高效转导入宫颈癌Caski细胞并稳定表达,荧光显微镜观察转染后24 h Caski细胞即开始发出绿色荧光,72 h后有大量荧光表达,而在稳定转染空质粒的Caski细胞中不表达。检测转染后的Caski细胞,发现有3条链能明显抑制hTERT mRNA和蛋白水平的表达,并显著降低了细胞端粒酶活性,其中以72H-TERT-1515抑制作用最强,对hTERTmRNA和蛋白的抑制率分别达75.0%和70.3%,端粒酶活性下降74.6%。结论:成功构建携带hTERT-shRNA慢病毒表达载体LV3-shRNA-hTERT,并稳定转染Caski细胞系,实验设计的shRNA能有效抑制宫颈癌Caski细胞hTERT mRNA水平和hTERT蛋白表达,并显著降低细胞端粒酶活性。为端粒酶在宫颈癌发病机制的研究、宫颈癌基因治疗的体外干扰治疗奠定了工作基础。

Objective： To construct the human telomerase reverse transcriptase -targeted small hairpin RNA -expressing plasmid （hTERT- targeted shRNA -expressing plasmid） system, and to observe its effect on hTERT expression and telomerase activity in cervical cancer cells Caski. Methods： Sequence of hTERT - targeted shRNA was designed by Designer3.0 （Genepharma） based on the mRNA se- quence of hTERT which was obtained from the Genbank. They were reeombined with the plasmid pGLV3/H1/GFP ＋ Puro, and then those plasmids were identified by gene sequencing to make sure they were correctly connected. Then those plasmids were transfeeted into cervical cancer cells Caski and fluorescence expression was observed. The expression of hTERT mRNA and protein was identified by RT - PCR and Western blot respectively. The telomerase activity was detected by TRAP - ELISA. Results： The hTERT - targeted shRNA - expressing plas- raids were successfully constructed, which was proved by gene sequencing. Then the pGLV3/H1/GFP ＋ Puro reeombined plasmids modified by fluoreseein was synthesized and transfected into Caski cells, the transfection efficiency of shRNA was evaluated by calculating the ratio of fluorescent cells to total ceils. Observed by fluorescence microscopy, 24 h after transfection, Caski ceils began to emit green fluorescence, 72 h after transfection, a large number of fluorescent expression, while there was no expression in Caski cells that stably transfeeted with empty vector . Caski cells after transfection were detected and the results showed that there were three chains could significantly inhibit the expression of hTERT mRNA and protein levels and significantly reduced telomerase activity, 72H -TERT- 1515 was the strongest , inhibi- tion rate of hTERTmRNA and protein was 75 . 0% and 70 . 3% , respectively, telomerase activity decreased 74. 6%. Conclusion： The hTERT- targeted shRNA -expressing plasmids have been successfully constructed, and the designed small inteffereneing RNA can effec- tively block the expression of hTERT and then inhibit the telomerase activity. It provides a basis for further study that the role of telomerase in pathogenesis and interference in vitro of gene therapy treatment of cervical cancer