pGenesil-1-Atg7-shRNA真核表达载体的构建

Construction of eukaryotic expression vector of pGenesil-1-Atg7-shRNA

目的利用RNA干扰技术,构建pGenesil-1质粒载体介导靶向自噬基因Atg7的shRNA真核表达载体。方法根据GenBank中Atg7基因的cDNA序列(NM-001136031),设计有小发卡结构的三条寡核苷酸序列,连接到空载体pGenesil-1中,转化到JM109菌株,选取阳性克隆,进行酶切鉴定和DNA测序分析。结果经双酶切结果显示合成的shRNA序列成功连接到pGenesil-1质粒载体中,DNA测序结果证实干扰序列和目的序列相同,重组载体pGenesil-1-Atg7-shRNA构建成功。结论成功构建干扰人Atg7基因的重组表达载体,为研究Atg7蛋白的生物学功能提供了研究基础。

Objective To construct the eukaryotic expression vector expressing short hairpin RNA （shRNA） section targeting human Atg7 gene by using RNA interference.Methods According to the sequence of human Atg7 cDNA （GenBank：NM-001136031）,three oligonucleotides of shRNA were designed and cloned into pGenesil-1 plasmid which had then been transformed into JM109 E.coli.The positive results were chosen to be cloned and the positive clones were digested with restriction endonuclease for identification and DNA sequencing.Results Restriction endonuclease analysis indicated that three oligonucleotides of shRNA had been connected into pGenesil-1 plasmid successfully.DNA sequencing confirmed the correct construction of the eukaryotic expression vector pGenesil-1-Atg7-shRNA.Conclusions The eukaryotic expression vector expressing shRNA targeting human Atg7 is successfully constructed.It provides a favorable foundation for further study on the function of Atg7.