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目的构建结核分枝杆菌PPE60的GST融合蛋白表达载体,并在大肠杆菌中诱导表达融合蛋白。方法以结核分枝杆菌标准株H37Rv基因组DNA为模板,经PCR技术扩增目的基因,将目的基因克隆到pGEX-4T-1载体中,转化大肠杆菌DH5α,筛选阳性克隆。进行质粒的酶切鉴定和序列分析;对重组的大肠杆菌进行诱导表达,SDS-PAGE分析菌体蛋白,凝胶密度扫描分析目的蛋白的表达情况,Western-blot鉴定融合蛋白。结果正确构建了pGEX-PPE60融合表达载体,载体能在大肠杆菌中表达分子量约为70 kDa的重组蛋白,占菌体总蛋白的50%。该融合蛋白能与GST抗体产生阳性反应。结论成功构建了GST-PPE60融合蛋白的表达载体,并能在大肠杆菌中高效表达,为PPE60蛋白的功能研究奠定了基础。
Objective To construct GST fusion protein expression vector of Mycobacterium tuberculosis PPE60 and express the fusion protein in E. coli. Methods The genomic DNA of Mycobacterium tuberculosis H37Rv was used as template to amplify the target gene by PCR. The target gene was cloned into pGEX-4T-1 vector and transformed into E. coli DH5α. The positive clones were screened. The plasmid was identified by restriction enzyme digestion and sequence analysis. The recombinant E.coli was induced to express, the bacterial protein was analyzed by SDS-PAGE, the expression of target protein was analyzed by gel density scanning, and the fusion protein was identified by Western-blot. Results The pGEX-PPE60 fusion expression vector was correctly constructed. The vector can express a recombinant protein with a molecular weight of about 70 kDa in E. coli, accounting for 50% of the total bacterial proteins. The fusion protein can react positively with GST antibody. Conclusion The expression vector of GST-PPE60 fusion protein was successfully constructed and expressed efficiently in E. coli, which laid the foundation for the study of the function of PPE60 protein.