论文部分内容阅读
目的构建及鉴定含有GST标签的结核分枝杆菌esat-6基因的原核表达载体,并在大肠埃希菌中高效表达融合蛋白。方法 PCR扩增结核分枝杆菌esat-6基因,并构建到pGEX4T-1上,重组质粒经测序鉴定后进行诱导表达,采用聚丙烯酰胺凝胶电泳和免疫印迹分析重组蛋白。结果扩增出了结核分枝杆菌esat-6基因,构建了具有正确基因序列的质粒载体pGEX4T-1-ESAT-6,转化大肠埃希菌BL21后经诱导产生了高水平的表达产物,蛋白纯化后可被结核病患者血清特异性识别。结论构建了质粒载体pGEX4T-1-ESAT-6,并经诱导后高效表达了ESAT-6融合蛋白,为进一步研究结核菌的免疫机制奠定了基础。
Objective To construct and identify the prokaryotic expression vector of GST-tagged Mycobacterium tuberculosis esat-6 gene and express the fusion protein efficiently in Escherichia coli. Methods The gene of Mycobacterium tuberculosis esat-6 was amplified by PCR and cloned into pGEX4T-1. The recombinant plasmids were identified by sequencing and induced to express. The recombinant protein was analyzed by polyacrylamide gel electrophoresis and immunoblotting. Results The esat-6 gene of Mycobacterium tuberculosis was amplified. The plasmid vector pGEX4T-1-ESAT-6 with the correct gene sequence was constructed and transformed into Escherichia coli BL21 to induce a high level of expression product. The protein was purified It can be specifically recognized by the serum of tuberculosis patients. Conclusion The plasmid vector pGEX4T-1-ESAT-6 was constructed and highly expressed ESAT-6 fusion protein after induction, which laid the foundation for further study on the immunological mechanism of Mycobacterium tuberculosis.