结核分枝杆菌分泌蛋白MPT64的克隆表达和纯化

目的构建结核分枝杆菌分泌蛋白MPT64原核表达载体并进行表达和纯化。方法以结核分枝杆菌标准株H37Rv基因组DNA为模板扩增出MPT64基因,产物经纯化回收后与载体pMD-T连接转化,酶切鉴定,克隆到pET21a原核表达载体,测序鉴定插入序列完全正确者转化大肠杆菌BL21,诱导表达MPT64融合蛋白,利用亲和层析纯化表达产物,SDS-PAGE电泳进行鉴定。结果成功构建MPT64表达载体,SDS-PAGE电泳鉴定成功表达MPT64蛋白。并以此蛋白进行结核抗体的检测,其特异性和敏感性分别为96%和43%。结论成功表达结核分枝杆菌MPT64蛋白,并进行特异性和敏感性的检测,证明重组的MPT64蛋白有希望成为结核病血清学诊断的组合抗原的候选者之一。

Cloning, expression and purification of MPT64 from Mycobacterial tuberculosis

To construct an expressing vector MPT64 of Mycobacterium tuberculosis and to identify and purificate the protein in the E coll. Methods The gene encoding protein MPT64 was amplified from Mycobacterium tuberculosis H37RV chromosomal DNA by using PCR, then cloned into pMD-T vector after being identified, then cloned pET21a expressing vector, transformed into E coli BL21. Bacterial lyastes prepared from IPTG induced cultures were loading SDS-PAGE. Results A MPT64 expressing vector was contructed, and the fusion protein was obtained. Conclusions The study based on successful expressed MPT64 protein laid the groundwork for its application.