卡氏肺孢子菌主要表面糖蛋白上游保守序列基因microRNA表达载体的构建和鉴定

Construction and identification of plasmid vector of microRNA on pneumocystis carinii major surface glycoprotein gene's upstream conserved sequence

目的:构建和鉴定卡氏肺孢子菌(Pneumocystis carinii,PC)主要表面糖蛋白(Major surface glycoprotein,MSG)表达调控基因上游保守序列(Upstream conserved sequence,UCS)的microRNA表达载体,并在体外转染到PC中观察其对PC的抑制作用。方法:设计并人工合成针对PC MSG-UCS基因的1对microRNA序列并定向克隆到表达载体pcDNA^(TM)6.2-GW/EmGFPmiR上,序列正确的载体用脂质体转染PC细胞,RT-PCR检测其对PC MSG-UCS基因的抑制作用,扫描电镜观察PC的超微结构变化。结果:凝胶电泳和测序鉴定得到的产物与预期的目的基因一致,其在mRNA水平能明显抑制PC的表达并能破坏PC的超微结构。结论:PC MSG-UCS基因的microRNA表达载体pPC-UCSm构建成功,并能在体外抑制PC的表达。

Objective： To constructive and identify plasmid vector of microRNA on pneumocystis carinii （PC） major surface glycoprotein （MSG） gen＇s upstream conserved sequence （UCS）, then we researched its effect after the recombinant plasmid was transfected into PC. Methods ： microRNA oligonucleotides targeting PC MSG-UCS gene were chemically synthesized and inserted into plasmid vector pcDNA？ 6.2-GW/EmGFPmiR after annealing, then the recombinant plasmid was transfected into PC by liposome in vitro. The PC MSG-UCS mRNA expression was detected by RT-PCR and the fine structure of PC was observed by electronmicroscope. Results ： Gel electrophoresis and DNA sequencing showed that the recombinant plasmid containing the correct and full microRNA oligonucleotide, and it could knock down the expression of mRNA in PC in vitro, it could also destroy PC. Conclusion： The microRNA plasmid, pPC-UCSm, was constructed successfully, and it could knock down the expression of PC in vitro.