结核分枝杆菌pdhA基因的原核表达及其免疫原性分析

Prokaryotic expression of pdhA gene from Mycobacterium tuberculosis and immunogenicity analysis of the expressed product

以结核分枝杆菌H37Rv菌株的基因组DNA为模板,采用PCR方法扩增出1 100bp的pdhA(pyruvate dehydrogenase E1component alpha subunit)基因,经限制性酶切后,与pET-28a载体连接,转化到大肠杆菌BL21中,重组菌经1mmol/L IPTG诱导表达了pdhA蛋白,并用Ni-His-resin纯化了目的蛋白,经Western-blot分析鉴定了目的蛋白的免疫原性。结果显示,重组质粒的双酶切鉴定和DNA测序均正确;SDS-PAGE和Western-blot分析结果显示,在44ku处获得一目的条带,且具有很好的免疫原性。本研究成功获得了高纯度的重组蛋白pdhA,为深入研究其功能奠定了基础。

The pdhA（pyruvate dehydrogenase E1 component alpha subunit） gene was amplified from the genome of Mycobacterium tuberculosis H37Rv strain by PCR,cloned into pET-28a vector,and then transformed into Escherichia coli BL21.The recombinant plasmid was confirmed by restriction digestion and DNA sequencing.The pdhA protein was expressed in resultant strains induced with 1 mmol/L IPTG.The recombinant protein was purified with Ni-His-resin and the immunogenicity of it was identified by Western-blot.The recombinant protein with good immunogenicity was confirmed to be about 44 ku in size by SDS-PAGE.The results showed that the recombinant expression vector pET28a-pdhA was successfully constructed and the expressed recombinant protein with high purity provided the foundations for basis of further studies on the function of pdhA protein.