结核分枝杆菌inhA基因的克隆、表达及高纯度inhA蛋白的制备

目的：对结核分枝杆菌H37Rv的inhA基因进行克隆、表达并纯化表达产物。方法：构建含目的基因的表达质粒pET-24b/inhA，经酶切鉴定和测序检验后转化大肠杆菌BL21（DE3），然后以异丙基硫代-B-D-半乳糖苷（IPTG）对其诱导，获得稳定的高表达菌后对其产物以Nit-NTA Superflow亲和柱进行纯化。结果：酶切鉴定和测序显示所构建的重组质粒已成功地克隆了inhA基因；转化pET-24b/inhA的大肠杆菌可大量表达一相对分子质量约为28500的蛋白，产量最高可达细菌蛋白总量的30%左右；经Nit-NTA Superflow亲和柱纯化后可得到纯度99%以上的蛋白。结论：inhA基因克隆、表达获得成功，并得到了高纯度的inhA蛋白，为进一步研究结核分枝杆菌的耐药机制打下了基础。

Cloning,expression and purification of inhA from mycobacterium

Objective To clone and express inhA gene from mycobacterium tuberculosis , and purify the inhA protein. Methods Recombinant plasmid pET 24b/inhA was constructed and transferred into Escherichia coli . After restriction enzyme analysis and sequencing, the host bacteria were induced by IPTG and the product was identified by SDS PAGE. Furthermore, the overexpressed inhA protein was purified by Nit NTA Superflow system. Results The inhA gene was overexpressed in E. coli, the production was corresponding to 30 percent of total cell protein. Using Nit NTA Superflow,we can get more than 99% purified protein. Conclusions The cloning, expression and purification of inhA gene are successful.