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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 highly expressed Mycobacterium tuberculosis early target antigen 6kDa protein (ESAT-6). Methods The fragment of ESAT-6 gene was amplified by PCR and cloned into the prokaryotic expression vector pET-21b (+). The recombinant plasmid of ESAT-6 gene was identified by enzyme digestion analysis and sequence analysis. , Which was then transformed into E. coli BL21 (DE3). The positive strains were induced by IPTG and the target proteins were analyzed by SDS-PAGE and Western blotting. The recombinant protein was purified by Ni-NTAHis. BindResins. Results The ESAT-6 gene fragment was successfully amplified and the recombinant expression plasmid pET-ESAT-6 was constructed. The molecular weight of the expressed product was about 6 kD by SDS-PAGE. The purity of the purified recombinant ESAT-6 protein was high, Tuberculosis patients identified by the serum. Conclusion The recombinant protein of Mycobacterium tuberculosis ESAT-6 was successfully expressed and purified successfully using pET prokaryotic expression system. The recombinant protein has good immunogenicity.