摘要
目的获得高效表达的结核分枝杆菌早期分泌抗原靶6kDa蛋白(ESAT-6)。方法利用PCR方法扩增出ESAT-6基因片段,将酶切处理后的基因片段定向克隆到原核表达载体pET-21b(+),酶切分析和序列测定鉴定出ESAT-6基因的阳性重组子,将其转化入E.coliBL21(DE3),阳性菌株经IPTG诱导,SDS-PAGE和免疫印迹分析靶蛋白的表达;重组蛋白经Ni-NTAHis.BindResins纯化。结果成功扩增出ESAT-6基因片段,构建了重组表达质粒pET-ESAT-6,SDS-PAGE显示表达产物分子量约为6kD,经亲和纯化后的ESAT-6重组蛋白纯度高,且能被结核病人血清所识别。结论利用pET原核表达系统成功的对结核分枝杆菌ESAT-6重组蛋白进行高效表达和纯化,重组蛋白具有良好的免疫活性。
Objective To obtain the high expressed early secretory antigentic target-6 （ESAT-6） protein of mycobacterium tuber- culosis. Methods ESAT-6 gene was amplified by PCR from mycobacterium tuberculosis and the digestion fragment was cloned into pET- 21b（ ＋ ）. After being verified with restriction endonuclease digestion and DNA sequencing, the positive recombinant vectors were transformed into E. coli BI21 （ DE3 ） which was then induced with IPTG and examined the expression of recombinant by SDS-PAGE and westernblotting. The fusion protein was purified by affinity chromatograph. Results ESAT-6 gene was amplified and the pET-ESAT-6 prukaryotlc recombinant plasmid was obtained. The recombinant protein was highly expressed by the induction of IPTG and can be recognized by serum of patient with Tuberculosis. Conclusion pET prokaryotic expression system can be used to construct the ESAT-6 recombinant and highly expressed the ESAT-6 protein. Its antigen specificity was confirmed as it could be recognized by antigen-specific antibody（ anti-ESAT-6 ）.
出处
《临床肺科杂志》
2010年第6期823-825,共3页
Journal of Clinical Pulmonary Medicine
基金
深圳市科技计划项目(200701008)
关键词
结核分枝杆菌
早期分泌抗原靶6
kDa蛋白
原核表达
mycobacterium tuberculosis
early secretory antigentic target-6
prokaryotic expression
