结核分枝杆菌16-kDa α晶体蛋白的原核表达及纯化

Prokaryotic Expression and Purification of 16-kDa α-Crystallin of Mycobacterium tuberculosis

目的:利用基因工程技术原核表达并纯化结核分枝杆菌α晶体蛋白(Acr)。方法:以结核分枝杆菌H37Rv株基因组DNA为模板,通过PCR方法扩增Acr的编码基因,以pCold为载体构建重组质粒,再转化到表达宿主菌大肠杆菌BL21(DE3)中,用IPTG诱导表达,经SDS-PAGE和Western印迹分析和纯化该表达产物。结果:构建了具有正确基因序列的Acr重组表达质粒,重组Acr在大肠杆菌BL21(DE3)中经低温诱导得到可溶性表达;分别用6×His的单克隆抗体和16-kDa单克隆抗体对表达产物进行Western印迹分析,结果显示在相对分子质量约19 000处均有特异性条带,与预计大小吻合;纯化后蛋白纯度达90%,浓度达0.8 mg/mL。结论:表达了重组可溶性Acr,为深入研究该蛋白的生物学、免疫学活性奠定了基础。

Objective： To prokaryotic express and purify the recombinant α-crystallin（Acr） of Mycobacterium tuberculosis. Methods： The Act gene was amplificated by PCR with the genome of M.tuberculosis H37Rv as a template, and was inserted to vector pCold to construct the recombinant plasmid. The recombinant plasmid was transformed into E.coli BL21（DE3） and the recombinant Act was induced with IPTG. Results： SDS-PAGE and Western blot analysis showed that the expressed recombined protein had a molecular weight of 19 kD. The Acr was expressed as soluble protein under the condition of low temperature. After purifing by Ni-NTA agarose, the concentration and the purity of the protein were 90% and 0.8 mg/mL respectivly. Conclusion： The target gene was cloned into the host bacterium and expressed correctly, and these results would establish the basis for the further research in biology and immunology of Act.