论文部分内容阅读
目的表达、纯化结核杆菌H37Rv株重组PstS1(简称rPstS1)蛋白,为结核病血清学诊断价值研究及亚单位疫苗研制提供物质基础。方法重组质粒pET15b-PstS1转化大肠埃希菌表达菌株BL21(DE3)plysE,IPTG诱导rPstS1蛋白表达。经SDS-PAGE电泳和Western blot鉴定后,优化表达条件,用镍离子鳌合亲和层析柱纯化rPstS1蛋白,并用斑点金免疫渗滤法评价纯化蛋白的免疫反应性。结果成功构建了重组质粒pET15b-PstS1,相对分子质量为38×103的rPstS1蛋白约有30%以可溶性蛋白形式表达,经亲和层析后的rPstS1蛋白纯度为94.8%,有较好的免疫反应性。结论构建的重组质粒pET15b-PstS1能高效表达有免疫反应性的rPstS1蛋白,经亲和层析后可得到高纯度的纯化蛋白。
Objective To express and purify recombinant PstS1 (referred to as rPstS1) protein of Mycobacterium tuberculosis H37Rv strain for the purpose of providing a material basis for the research on serodiagnosis and subunit vaccine of tuberculosis. Methods The recombinant plasmid pET15b-PstS1 was transformed into Escherichia coli BL21 (DE3) plysE and IPTG induced the expression of rPstS1 protein. After identification by SDS-PAGE and Western blot, the expression of rPstS1 protein was purified by nickel ion chelate affinity chromatography. The immunoreactivity of purified protein was evaluated by dot immunogold filtration assay. Results The recombinant plasmid pET15b-PstS1 was successfully constructed. About 30% of the rPstS1 protein with a relative molecular mass of 38 × 103 was expressed as soluble protein. The purity of rPstS1 protein was 94.8% after affinity chromatography, which showed good immunoreactivity Sex. Conclusion The constructed recombinant plasmid pET15b-PstS1 can efficiently express immunoreactive rPstS1 protein, and the purified protein can be obtained by affinity chromatography.