吸水链霉菌TetR基因的克隆与表达

Cloning and Expression of the Gene Encoding Tetracycline Resistance Repressor in Streptomycesis hygroscopius

根据吸水链霉菌"应城变种10-22"的一个文库质粒pHZ1392的测序结果设计了1对引物,聚合酶链反应扩增编码TetR的663bp基因,利用T载体和蓝白斑筛选构建了克隆质粒T TetR,经序列测定确认扩增序列正确后克隆至pET24a,构建了表达质粒pET24a TetR,再经序列测定确认未发生移码后转化表达宿主菌E coliDL21(DE3),IPTG诱导后的SDS-PAGE分析显示蛋白表达获得成功,并进一步利用Ni-NTA树脂纯化了TetR蛋白。

According to the annotation results of DNA sequence of pHZ1392, a library cosmid of streptomyces hygroscopicus 'yingchengenesis 10-22', a pair of primers were synthesized and used to amplify the gene coding for tetracycline resistance repressor with polymerase chain reaction, the purified DNA fragment was inserted into T vector to construct the cloning plasmid T-TetR. After being sequenced and ascertained correctly, the gene was then inserted into pET24a to construct the expression plasmid pET24a-TetR, the expression host strain E.coli DL21(DE3) was transformed by pET24a-TetR and induced with isopropyl-beta-D-thiogalactopyranoside (IPTG ). The analysis result of SDS-PAGE showed that the TetR was successfully expressed and purified with Ni-NTA resin.