结核杆菌DnaA蛋白在耻垢分枝杆菌中的同源表达

目的:在耻垢分枝杆菌中表达重组结核杆菌DnaA蛋白并对表达产物进行鉴定。方法:用PCR的方法扩增结核杆菌dnaA基因并克隆至表达载体pMF406中,构建重组大肠杆菌-分枝杆菌穿梭质粒pMF-dnaA。经双酶切及测序鉴定后,用电转化的方法将重组质粒转至耻垢分枝杆菌mc2155中。用0.02%乙酰胺诱导重组耻垢分枝杆菌,对表达产物进行SDS-PAGE和Western blotting检测和鉴定。结果:重组耻垢分枝杆菌构建成功,SDS-PAGE及Western blotting结果显示该重组耻垢杆菌可以实现结核杆菌DnaA蛋白的同源高效表达。结论:结核杆菌DnaA蛋白的同源表达为结核杆菌DNA复制机制的研究奠定了基础。

Homologous Expression of M.Tuberculosis DnaA Protein in M.smegmatis

Objective To express Mycobacterium tuberculosis DnaA protein in Mycobacterium smegmatis and identify the expression product. Methods The dnaA gene of M. tuberculosis was amplified by PCR and cloned into expression vector pMF406 to generate the shuttle vector of E.coil and Mycobacterium pMF-dnaA. It was comfirmed by restriction endonuclease digestion and sequence analysis. The recombinant plasmid was transformed into M. Smegmatis mc2155 by electroporation. The recombinant M. smegmatis was induced by 0.02% acetamide and the expression product was analyzed with SDS-PAGE and Western blotting. Results The recombinant M. smegmatis was successfully constructed and the M. tuberculosis DnaA protein was expressed in the recombinant M. Smegmatis at a relatively high level identified by SDS-PAGE and Western blotting. Conclusion The successful homologous expression of the M. tuberculosis DnaA protein will be very helpful for the further study on the mechanism of M. tuberculosis DNA replication .