结核分枝杆菌吡嗪酰胺酶-GST融合蛋白表达载体构建及在大肠杆菌中的表达

目的构建结核分枝杆菌pncA基因的原核表达质粒,获得结核分枝杆菌吡嗪酰胺酶的表达蛋白。方法制备结核分枝杆菌基因组DNA,采用聚合酶链反应技术扩增目的基因片段;通过pGEX4T-1构建表达载体pGEX4T-pncA,经序列测定证实正确后转化大肠杆菌DH10b,再经IPTG诱导表达谷胱甘肽S转移酶(GST)-吡嗪酰胺酶融合蛋白;用聚丙烯酰胺凝胶电泳和免疫印迹分析重组蛋白。结果扩增出了结核分枝杆菌pncA基因,构建了具有正确基因序列的质粒载体pGEX4T-pncA,转化大肠杆菌BL21后经诱导产生了高水平的表达产物。结论构建了质粒载体,并诱导表达了GST-吡嗪酰胺酶融合蛋白,为进一步研究吡嗪酰胺耐药性奠定了基础。

Recombination of Mycobacterium tuberculosis pyrazinamidase-GST fusion protein and its expressions in E.coil

Objective To construct glutathione S-transferase （GST）-tag prokaryotic expression plasmid of the pncA gene of Mycobacterium tuberculosis, and to express the fusion proteins efficiently in Escherichia coli BL21. Methods The pncA gene was amplified by PCR with specific primers from genomic DNA of M.tuberculosis H37Rv strain, and was cloned into pGEX4T-1 expression vector. E.coli DH10b strain was transformed with the recombinant vector that con- formed by sequencing and induced to express recombinant proteins. The relative moleculer size of the proteins was analyzed by SDS-PAGE and immunoblot. Results The length of PCR products pncA was 561 bp and identical with what the GenBank reported. The recombinant expressive vector pGEX4T-1-pncA was constructed. The E.coli BL21 strains with recombinant vector showed high level of pyrazinamidase fusion protein expressions after IPTG induction. Conclusions The pncA gene prokaryotic expression plasmid was constructed, and pyrazinamidase fusion protein was efficiently expressed after induction. The expression of recombinant pyrazinamidase protein lays a basis for further study pyrazinamide-resistance of M.tuberculosis.