人Bcl—2基因短发夹样RNA真核表达载体的构建和鉴定

目的构建人Bcl-2基因序列特异性的短发夹样RNA（Short Hairpin RNA，shRNA）质粒载体。方法根据Bcl-2基因序列及shRNA设计原则，化学合成4段编码短发夹RNA的寡核苷酸序列，将其定向克隆到带有卡那霉素抗性和增强绿色荧光蛋白的真核表达载体pGPH1／GFP／Neo中H1启动子的下游，重组构建RNAi质粒，同时设立阴性对照，并对重组质粒进行酶切分析和DNA序列测定。结果限制性内切酶PstⅠ和BamH Ⅰ酶切显示设计合成的shRNA编码序列被成功插入pGPH1／GFP／Neo质粒中，测序结果证实插入片断与设计序列完全一致。结论针对人Bcl-2的shRNA真核表达载体成功构建及鉴定为进一步研究Bcl-2功能奠定了良好的基础。

Construction and Identification of Human Bcl-2 shRNA Eukaryotic Expressing Vectors

Objective To construct eukaryotic vectors expressing short hairpin RNA(shRNA) sections targeting human Bcl-2 gene.Methods Four pairs of specific Bcl-2 shRNA oligoes and a non-coding sequence shNC were designed and synthesized,according to Bcl-2 cDNA sequence and the principle of shRNA designing.The complement form was obtained by annealing and cloned into plasmid vector pGPH1/GFP/Neo,then the recombinant plasmid was transformed into strain DH5a.Finally the vectors were identified by restriction enzyme analy...