结核分枝杆菌热休克蛋白Hsp70在原核表达载体中的克隆表达与鉴定

目的对结核分枝杆菌的一种热休克蛋白Hsp70基因进行克隆,鉴定,并在原核系统中表达。方法以结核杆菌H37Rv菌株基因组DNA为模板,用PCR法对基因Hsp70进行扩增,产物与载体质粒pET-22b(+)构建表达Hsp70的重组质粒,将此重组质粒先转化到E.coliDH5α内提取质粒,酶切鉴定,再转化入表达宿主E.coliBL21(DE3)PlysS菌株内,对转化菌株以IPTG进行诱导后,破菌,离心,上清进行SDS-PAGE,通过电转移将胶中蛋白转到硝酸纤维素薄膜上后进行免疫印迹分析。结果电泳发现转化了重组质粒的菌株有表达蛋白,所表达的蛋白质相对分子质量为70 000,抗体检测有特异带大小为70kDa。结论成功进行了结核杆菌分泌热休克蛋白Hsp70基因的克隆表达。

Cloning, expression and identification of heat shock protein Hsp 70 of Mycobacterium tuberculosis in prokaryotic expression vector

The purpose of this study is to clone,express and identify the heat shock protein Hsp70 from Mycobacterium tuberculosis in order to provide the foundation for studies in the diagnosis of tuberculosis as well as for the development of tuberculosis vaccine,the gene encoding protein Hsp70 was amplified from the genomic DNA of M.tuberculosis H37Rv by using PCR technique.The amplified product was cloned initially into expression vector pET-22b(+),and then transformed to E.coli Dhαs train.Clones containing the vector were selected on LB plus ampicillin(100μg/ml) plate,and the plasmid DNA was extracted and digested with enzymes.Plasmids containing the right insert were sequenced to confirm its identity,and then re-transformed the recombinants to E.coli BL21(DE3) PlysS strain.Bacterial lysates from cultures induced with IPTG(0.6 mmol/L) were directly loaded onto SDS-PAGE,and the proteins on the SDS-PAGE gel were transferred to nitrocellulose membrane,detected with polyclonal antiserum against M.tuberculosis H37Rv.Through these procedures,a specific protein with a molecular mass of 70 kDa could be visualized on gel.This protein can be used for further studies in the detection of the effectiveness of immunity and the preparation of antigen and antibodies of Hsp70 in large scale