结核分枝杆菌阿拉伯糖基转移酶AftA的原核表达及鉴定

Expression and identification of Mycobacterium tuberculosis protein arabinofuranosyltransferase (AftA)

目的构建高效表达菌株,以大量表达结核分枝杆菌AftA蛋白,为后续的抗结核药物的筛选奠定基础。方法采用PCR法从结核分枝杆菌H37Rv基因组DNA中扩增出AftA的基因片段,将其插入原核表达质粒PQE30,构建重组质粒PQE30-aftA。将PQE30-aftA转化大肠杆菌,用IPTG诱导目的基因表达。Western-blotting免疫印迹法鉴定后纯化该表达产物。结果经核苷酸序列测定和酶切鉴定结果表明,成功构建了重组质粒PQE30-aftA。在大肠杆菌M15中用IPTG诱导表达后,获得了AftA蛋白。蛋白经SDS-PAGE分析约为69.5kD。免疫印迹分析证实目的蛋白与阳性结核病患者抗血清产生特异性反应。结论成功制备出重组的AftA蛋白,为新型抗结核药物的筛选奠定了物质基础。

To clone and express the arabinofuranosyltransferase （AftA） protein to provide active protein as a target for new drug development,the aftA gene was amplified by PCR from genome of Mycobacterium tuberculosis H37Rv strain and inserted into prokaryotic expression vector pQE30 to obtain recombinant plasmid pQE30-aftA. The recombinant vector pQE30- aftA was transformed into E. coli after digestion by restriction endonuclease and sequence analysis. The E, coli M15 transformed with pQE30-αftA was induced with IPTG, and the expression product was analyzed by SDS-PAGE and Western-blotting. Then the expressed protein was purified by Ni-NTA purification system. It was found that the recombinant protein had a relative molecular mass of 69.5kDa. The Western-blotting analysis showed the expressed protein could react with sera from tuberculosis patients specifitally. It is apperent that the recombinant protein AftA was successfully prepared and this estabished some material foundations for the study to screen new anti-tuberculous drugs,