GFP-Rv2626c融合蛋白表达载体的构建及表达纯化

目的构建含Rv2626c基因的原核表达载体,获得GFP-Rv2626c融合蛋白,为今后开发鉴别结核感染的血清学诊断试剂打下基础。方法以结核杆菌H37Rv基因组DNA为模板,经PCR法扩增得到Rv2626c基因DNA片段。将扩增片段克隆到pGM-T载体中测序,并进一步将Rv2626c亚克隆到pET28a-GFP中,构建原核重组表达载体pET28a-GFP-Rv2626c。将pET28a-GFP-Rv2626c转化E.coli BL21(DE3),用IPTG诱导目的基因表达,经SDS-PAGE和Western-blot分析和纯化该表达产物。结果经核苷酸序列测定和酶切鉴定,成功构建了重组质粒pET28a-GFP-Rv2626c。用IPTG诱导该质粒转化的E.coliBL21后,获得分子量约46kD的GFP-Rv2626c融合蛋白。经Ni-NTA Agarose试剂盒进行蛋白纯化,获得纯化的GFP-Rv2626c融合蛋白。结论成功制备出重组GFP-Rv2626c融合蛋白,为开发鉴别结核感染的血清学诊断试剂打下了基础。

Construction of the prokaryotic expression vector for gene encoding the GFP-Rv2626c fusion protein and its purification

To construct the prokaryotic expression vector for gene encoding the GFP-Rv2626c fusion protein in order to search a serological method to distinguish the state of tuberculous infection, the Rv2626c gene was amplified from Mycobacterium tuberculosis H37 Rv DNA by PCR, and the PCR-amplified product was then cloned into vector pGM-T and then sequenced. The prokaryotic expression vector pET28a-GFP-Rv2626c was constructed by using the purified Rv2626 gene that had been subcloned to the expression vector pET28a-GFP. Then, the resultant product pET28a-GFP-Rv2626c was transformed to E. coli BL21 with IPTG induction. As demonstrated by SDS-PAGE and Western blotting, an exogenous protein with molecular mass of 46 kDa approximately was obtained. This expressed protein could be purified with Ni-NTA agarose kit. Consequently, this GFP-Rv2626c fusion protein may be used as the antigen candidate for the serological identification of state in tuberculous infection.