结核杆菌Mtb8.4抗原在大肠杆菌中表达的初步研究

目的构建结核杆菌低分子质量抗原Mtb8.4的原核表达质粒,并检测其在大肠杆菌中的表达。方法PCR扩增目的基因,克隆到质粒pETHisT7启动子的下游;酶切和PCR法鉴定重组子;阳性重组子转化大肠杆菌BL21(DE3)pLysS,经IPTG诱导后,SDSPAGE电泳分析。结果重组质粒pETHisMtb8.4成功构建,含阳性重组子的菌体蛋白经SDSPAGE电泳出现一新的蛋白带,与预计相符。结论初步证明构建的大肠杆菌重组体能够表达目的蛋白,为进一步对其免疫效应的研究奠定了基础。

Construction of prokaryotic recombinant plasmid encoding Mtb 8.4 gene of Myco-bacterium tuberculosis and its expression in E.coli

To constract the prokaryotic recombinant plasmid encoding Mtb 8.4 gene of Myco-bacterium tuberculosis and to detect the expression of proteins in E.coli, the gene encoding the protein Mtb 4.8 was amplified and cloned into the down-stream of the T7 promoter pET-His. The positive clones were screened by using the double digestion and polymerase chain reaction (PCR) and the recombinant plasmid was transformed into E.coli BL21 (DE3) as induced by IPTG, and analyzed by SDS-PAGE.It was found that the recombinant expression plasmid pET-His-His-Mtb 8.4 was constructed successfully in the present study, and the gel staining with coomassie blue G-250 showed that the induced E.coli carried the recombinant plasmid that could produced the Mtb 8.4 protein.It concludes that the constrcted recombinant of E.coli can express the target protein, thus providing the basis for the further studies on the production of antigens and antibodies of Mtb 8.4 protein in large scale and for the detection of their immune effectiveness.