摘要
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.
出处
《生物技术通讯》
CAS
2012年第4期515-518,共4页
Letters in Biotechnology
关键词
结核分枝杆菌
α晶体蛋白
原核表达
纯化
Mycobacterium tuberculosis
α-crystallin
prokaryotic expression
purification