重组结核分枝杆菌38kDa蛋白的表达、纯化和复性及免疫特性的研究

目的构建结核分枝杆菌38kDa的重组质粒,在大肠杆菌中表达、纯化和复性重组蛋白,并评价其在结核病血清学诊断方面的价值。方法用PCR方法从结核分枝杆菌H37Rv基因组中扩增出psts1基因片段,连接到表达载体PET30a中,在大肠杆菌中表达;应用组氨酸标签(His-Tag)纯化重组蛋白;在PBS中通过半透膜恢复重组蛋白的天然结构。结果构建了含38kDa重组质粒的大肠杆菌工程菌,并以包涵体形式表达重组蛋白38kDa,其占细胞总蛋白的55%;重组蛋白变性后经镍柱纯化,其纯度达90%以上;通过半透膜完全恢复重组蛋白的天然结构,经蛋白印迹证实重组蛋白能与结核杆菌多克隆抗体结合。结论具有天然结构和高纯度的重组融合蛋白有可能成为有效的结核病血清学诊断的候选抗原。

Research immunological characteristics and expression, purification and renaturation of recombinant protein 38kDa about Mycobacterium tuberculosis

To construct the recombinant plasmid of protein 38kDa of M. tuberculosis and express, purificate and renaturate fusion protein 38kDa and to evaluate its potential value for TB serodiagnosis. The gene coding 38kDa protein was amplified by polymerase chain reaction (PCR) from genome of Mycobacterium tuberculosis H37Rv ,then cloned into expression vecter PET30a and expressed fusion protein 38kDa in E.coli BL21(DE3). It was purificated by his-tag of the fusion protein 38kDa and renaturated by semipermeable membrane. The recombinant 38kDa protein existed in inclusion bodies of E.coli.and amounted to 55% of totle cell protein. The inclusion bodies were fully renaturated by semipermeable membrane and specific immunogenicity was confirmed by Western blot analysis. The recombinant protein of purification and renaturation may be a potential candidate to be a serodiagnostic reagent.