hTERTshRNA表达质粒的构建及鉴定.docVIP

hTERT shRNA表达质粒的构建及鉴定 胡凡果1，史玉荣2，牛瑞芳2，刘彤1，只向成3 （1 天津医科大学总医院普通外科 300000；2天津医科大学附属肿瘤医院中心实验室300000；3天津医科大学附属肿瘤医院乳腺三科 300000） 通讯作者：只向成 doctorx888@ [摘要] 目的 构建针对人端粒酶催化亚基hTERT的shRNA表达质粒并加以鉴定。 方法 以具有G418抗性的pBAsi-hU6-Neo（BamHI/HindⅢ）质粒构建针对hTERT的shRNA表达载体，应用基因测序加以验证。经脂质体转染shRNA质粒入乳腺癌T47D细胞，抗生素G418筛选转染成功的细胞株。采用半定量RT-PCR及western blot法检测细胞中hTERT在mRNA和蛋白质水平上的表达；TRAP-ELISA法检测细胞的端粒酶活性变化。结果 成功构建了5种针对hTERT的shRNA质粒，经测序验证无误。将质粒转染入T47D细胞后经G418筛选获得了稳定转染的细胞株，hTERT基因在mRNA和蛋白水平的表达明显降低，端粒酶活性显著下降。 结论 质粒pBAsi-hU6-Neo（BamHI/HindⅢ）可用于构建hTERT shRNA表达载体,可转染到乳腺癌T47D细胞，并能显著降低hTERT基因mRNA和蛋白的表达，进而抑制细胞端粒酶活性。 [主题词] 端粒酶；人端粒酶逆转录酶；RNA干扰；乳腺癌 Construction and identification of hTERT-targeted shRNA-expressing plasmid HU Fan-guo, SHI Yu-rong, NIU Rui-fang, LIU Tong, ZHI Xiang-cheng Abstract [Objective] To construct and identify a hTERT-targeted shRNA-expressing plasmid vector. [Method] The plasmid pBAsi-hU6-Neo（BamHI/HindⅢ）which immune to antibiotic G418 was used to construct shRNA-expressing vector and identified by gene sequencing. Plasmid was transfected into breast cancer cell T47D with liposome and those cells expressing shRNA were selected by G418. RT-PCR and western blot were used to detect the expression of hTERT on mRNA and protein level. The telomerase activity was examined by TRAP-ELISA. [Results] 5 kinds of hTERT-targeted shRNA-expressing plasmid were successfully constructed, which was proved by gene sequencing, and they were introduced into breast cancer cell T47D. The expression of hTERT decreased both on mRNA and protein level, and telomerase activity was inhibited at the same time. [Conclusion] The plasmid pBAsi-hU6-Neo（BamHI/HindⅢ） can be used to construct shRNA-expressing vector and transfected into breast cancer cell T47D. The shRNA-targeted hTERT-expressing vector can inhibit the expression of hTERT on both mRNA and protein level and then reduce the telomerase activity. [key words] telomerase; hTERT; RNAi; breast cancer 端粒酶的活化与恶性肿瘤的发生、发展密切相关，通过激活端粒酶，端粒DNA得以延长从而使细胞获得永生化。端粒酶逆转录酶（telomer