抑制端粒酶活性的pSUPER RNAi系统的构建

Construction of pSUPER RNAi system of inhibiting telomerase activity

目的 构建抑制端粒酶活性的 si RNA表达载体。方法 化学合成 2段编码短发夹 RNA序列的、靶向端粒酶 h TERT基因的寡核苷酸 ,各 6 4个碱基 ,退火 ,克隆到经 Bgl 、Hind 双酶切同时 CIP处理后的 p SUPER载体的 pol H1启动子的下游 ,重组构建 RNAi质粒 ,同时设立非特异对照。结果 重组构建的 p SUP- h TE载体经双酶切电泳分析及插入基因片段序列分析 ,结果表明 6 4个碱基成功插入到预计位点 ,并且序列完全一致。结论 载体的成功构建 ,为进一步研究其对端粒酶活性的抑制作用打下基础。同时使我们发展了在体内合成 si RNA的方法 ,这项技术能广泛的用在分析基因功能、肿瘤治疗等 ,更加便捷地把

Objective To construct expressing vector of siRNA in order to inhibit telomerase activity. Methods 64 base pair oligos for hairpin RNA expression which targeted telomerase hTERT gene were chemically synthesized and annealed. pSUPER vector was linearized with BglⅡ and HindⅢ treated with CIP. Finally, anneled oligos were inserted into the downstream of treated pSUPER's polⅢ H1 promoter to construct RNAi plasmid (pSUP hTE). Oligos with a scrambled sequence were used as a negative control. Results Recombinant pSUP hTE vector was identified by digestion with EcoRⅠand Hind Ⅲ and confirmed by sequencing analysis with T3 primer. The results demonstrated that 64 bp had been inserted the expected site. Furthermore, the insertion sequence was exactly correct. Conclusion pSUP hTE RNAi system has been constructed successfully. This will facilitate the study of telomerase activity inhibition. Based on the results, we can develop the RNAi synthesis in vivo. This technique not only facilitates a wide range of gene functional analysis and cell culture but also offers therapeutic means for treatment of tumors.