Zhang Jiming, Yang Chun, He Yonglin, Mu Liuqing, Zhang Chunyan, Fan Yu, Xu Lei
Center for Molecular Medicine and Tumor Research, Department of Pathogen Biology, School of Basic Medicine, Chongqing Medical University, Chongqing 400016, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014 Jun;30(6):573-5, 580.
To construct a prokaryotic expression plasmid for zinc-dependent metalloprotease-1 (Zmp1) gene from Mycobacterium tuberculosis and express the plasmid in E.coli.
Zmp1 gene was amplified by PCR using the genome of Bacillus Calmette-Guerin vaccine (BCG) as a template and inserted into a multiple cloning site of prokaryotic expression vector pET32a(+). The constructed prokaryotic expression vector pET32a±Zmp1 was transformed into E.coli BL21(DE3), and the recombinant proteins were expressed via IPTG induction. Finally, the expression of Zmp1 protein was detected by SDS-PAGE and Western blotting.
Restriction analysis and sequencing proved that the recombinant plasmid pET-32a±Zmp1 was constructed correctly. The relative molecular mass of the expressed recombinant protein was about 94 000 which was the same with that of presumed fusion protein. Recombinant Zmp1 protein showed a specific binding to monoclonal antibody with His tag.
The prokaryotic expression vector for Zmp1 gene was successfully constructed and the Zmp1 fusion protein was effectively expressed in E. coli BL21(DE3).
构建结核分枝杆菌锌依赖性金属蛋白酶-1（Zmp1）基因的原核表达质粒，并在大肠杆菌中表达该质粒。
以卡介苗（BCG）基因组为模板，通过PCR扩增Zmp1基因，并将其插入原核表达载体pET32a(+)的多克隆位点。将构建好的原核表达载体pET32a±Zmp1转化至大肠杆菌BL21(DE3)中，通过IPTG诱导表达重组蛋白。最后，通过SDS-PAGE和Western印迹检测Zmp1蛋白的表达情况。
酶切分析和测序证明重组质粒pET-32a±Zmp1构建正确。表达的重组蛋白相对分子质量约为94 000，与推测的融合蛋白一致。重组Zmp1蛋白与His标签单克隆抗体表现出特异性结合。
成功构建了Zmp1基因的原核表达载体，Zmp1融合蛋白在大肠杆菌BL21(DE3)中得到有效表达。
