人端粒酶逆转录酶基因RNAi表达载体的构建、鉴定

Construction of shRNA expression vector system targeting hTERT gene plasmid

目的构建端粒酶逆转录酶(hTERT)基因的RNA干扰(RNAi)表达载体。方法化学合成能编码针对hTERT基因的短发夹RNA(shRNA)序列的寡核苷酸,退火处理后克隆到pSilencer1.0-U6 shRNA表达载体的U6RNA聚合酶Ⅲ(PolⅢ)启动子的下游,构建重组RNAi质粒pSliencer-hTERT。结果经酶切电泳及测序分析证实,目的序列成功插入到预计位点。结论已成功构建pSliencer-hTERT载体,为进一步研究其对端粒酶活性的抑制作用打下基础。

Objective To construct the expressing vector of small hairpin RNA（shRNA） targeting human telomerase reverse transcriptase （hTERT） gene. Methods Two template DNA oligonucleotides for shRNA expression targeted hTERT gene were chemically synthesized. The annealed shRNA templates were processed to produce and identify the recombinant RNAi plasmid sPilencer - hTERT. Results It was demonstrated that shRNA template targeting hTERT gene had been inserted at the expected site and the insertion sequence was perfectly correct. Conclusion RNAi expression vector targeting hTERT gene has been constructed successfully and will be used as a useful method to develop specific hTERT- silencing therapeutics.