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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,采用聚合酶链反应扩增目的基因片段;通过pET28a构建表达载体pET28a-pncA,序列测定证实正确后转化大肠埃希菌DH10b,经IPTG诱导表达His-吡嗪酰胺酶融合蛋白,采用SDS-PAGE和Western blot分析重组蛋白。结果扩增出结核分枝杆菌pncA基因并构建了具有正确基因序列的质粒载体pET28a-pncA,转化大肠埃希菌BL21后经诱导产生了分子质量单位约20ku的表达产物,并得到纯化的带His标签的目的蛋白。结论构建了结核分枝杆菌pncA基因原核表达质粒,并诱导表达了His-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。
Objective To construct the prokaryotic expression plasmid of pncA gene of Mycobacterium tuberculosis and obtain the expressed protein of Mycobacterium tuberculosis pyrazinamidase. Methods Mycobacterium tuberculosis genomic DNA was prepared and the target gene fragment was amplified by polymerase chain reaction (PCR). The expression vector pET28a-pncA was constructed by pET28a. After confirmed by sequencing, Escherichia coli DH10b was transformed into E.coli DH10b and expressed by IPTG. Enzyme-fused protein, recombinant protein was analyzed by SDS-PAGE and Western blot. Results The pncA gene of Mycobacterium tuberculosis was amplified and a plasmid vector pET28a-pncA with the correct gene sequence was constructed. After transforming into Escherichia coli BL21, the expression product of about 20ku molecular weight unit was induced and the purified product with His The target protein of the tag. Conclusion The prokaryotic expression plasmid pNcA of Mycobacterium tuberculosis was constructed and the His-pyrazinamidase fusion protein was induced and expressed, which lays the foundation for further study of pyrazinamide resistance.