卡氏肺孢子胸腺嘧啶核苷酸合酶基因siRNA表达载体的构建和鉴定

Construction and identification of plasmid vector of short interfering RNA on pneumocystis carinii thymidylate synthase gene

目的:构建和鉴定大鼠卡氏肺孢子(Pneumocystis carinii,PC)胸腺嘧啶核苷酸合酶(Thymidylate synthase,TS)基因的siRNA(Short interfering RNA)表达载体。方法:人工合成针对PCTS基因的1对shRNA(Short hairpin RNA)序列并定向克隆到空载体pTZU6+1上,构建siRNA表达重组质粒pPC-TS,并采用双酶切产物凝胶电泳和基因测序法鉴定。结果:酶切电泳和基因测序鉴定得到的产物与预期的目的基因一致。结论:成功构建卡氏肺孢子胸腺嘧啶核苷酸合酶基因siRNA表达载体。

Objective：To construct and identify a plasmid vector of short interfering RNA （siRNA） on pneumocystis carinii （PC） thymidylate synthase. Methods： Short hairpin RNA oligonucleotides of thymidylate synthase were chemically synthesized and inserted into plasmid vector pTZU6＋ 1 after annealing. The recombinant plasmid, pPC-TS, transformed into E. coil. TOP10 and amplified, was digested by restriction endonucleases Hind Ⅲ and EcoRⅠ and identified by gel electrophoresis and DNA sequencing. Results： Gel electrophoresis and DNA sequencing showed that the recombinant plasmid containing the correct and full shRNA oligonucleotide. Conclusion ： The siRNA plasmid, pPC-TS, was constructed successfully.