双启动子shRNA表达载体对hTERT和bcl-2基因表达的抑制作用

Inhibitory Effect of Dual-Promoter shRNA Vector on hTERT and bcl-2 Gene

目的:研究双启动子shRNA表达载体对人端粒酶蛋白催化亚单位(hTERT)和B细胞淋巴瘤/白血病-2(bcl-2)基因表达的抑制作用。方法:分别从构建好的大肠杆菌DH5α菌体中提取含有双启动子shRNA的表达载体pdPRO-TB、单启动子对照pdPRO-T、pdPRO-B和阴性对照质粒pdPRO,通过脂质体介导瞬时转染Hela细胞,倒置显微镜下观察细胞形态。实时荧光定量RCR检测hTERT和bcl-2基因的表达水平。结果:4种菌体中均提取出5 000 bp左右的质粒,分别转染Hela细胞48 h后,可见转染pdPRO的Hela细胞形态无明显变化,转染pdPRO-T和pdPRO-B的Hela细胞有部分悬浮,转染pdPRO-TB的Hela细胞有大量悬浮。各载体质粒构建均成功,各目的基因相对管家基因的含量:T(hTERT)=39.8%,B(hTERT)=107.9%,TB(hTERT)=32.1%;T(bcl-2)=91.4%,B(bcl-2)=35.4%,TB(bcl-2)=31.4%。结论:双启动子shRNA表达载体构建成功,可同时抑制hTERT和bcl-2基因的表达。

Objective： To investigate the inhibitory effect of dual promoter shRNA-expression vector on human telomerase reverse transcriptase catalytic subunit （hTERT） and B-cell lymphoma/ leukemia-2 （bcl-2） gene. Methods： The dual-promoter shRNA expression vector pdPRO-TB, single promoter control pdPRO-T, pdPRO-B and the negative control plasmid pdPRO were extracted from E. coli DH5a bacteria, which were transiently transfected Hela cells. The inverted microscope was used to observe the cell morphology. The expression levels of hTERT and bcl-2 were detected by real time quantitative RCR. Results： Plasmids of about 5 000 bp were extracted from E. coli DH5a bacteria. There were no significant changes in transfected pdPRO Hela ceils 48 h after transfection. Hela ceils transfected pdPRO-T and pdPRO-B were partly inhibited. Hela ceils transfected pdPRO-TB were inhibited significantly. By PCR, expression vectors were constructed successfully. By qRT-PCR, the relative expression of genes were： T（hTERT）=39.8%, B（hTERT）=107.9%, TB（hTERT）=32.1%, T（bcl-2）= 91.4%, B（bcl-2）=35.4% and TB（bcl-2）=31.4%. Conclusion： The dual-promoter shRNA expression vectors were constructed successfully, which can inhibit the expression of two genes, hTERT and bcl-2.