牛分枝杆菌mpb64和Ag85B融合基因在大肠埃希氏菌中的原核表达

以牛分枝杆菌Vallee株基因组DNA为模板,应用PCR扩增获得mpb64和Ag85B两个目的基因片段,采用重叠延伸剪接技术(SOE)剪接mpb64和Ag85B,得到融合基因mpb64-Ag85B;将融合基因片段先克隆于pMD 18-T载体,再亚克隆到表达载体pET32a( )中,得到重组质粒pET64-85。该重组质粒经核苷酸序列测定,显示其中的外源片段与期望的序列一致;其BL21(DE3)转化菌经IPTG诱导表达带有6个组氨酸标签的融合蛋白;用Ni2 螯合层析方法纯化融合蛋白,Western-blotting分析结果显示,该融合蛋白能与抗牛分枝杆菌阳性血清发生反应。

Prokaryotic expression of fusion gene mpb64-Ag85B of Mycobacterium bovis in Escherichia coli

The DNA fragments of mpb64 and Ag85B gene were amplified by PCR from genome DNA of Mycobacterium bovis Vallee strain.The fusion gene mpb64-Ag85B was obtained by splicing the mpb64 gene with the Ag85B gene,using overlapping extension technique.The fusion gene was inserted initially(into) pMD 18-T vector and was then sub-cloned(into) the expression vector pET32a( ),and the recombinant plasmid pET64-85 was obtained.Sequence analysis showed that the fusion gene was inserted correctly as expected.The recombinant protein was obtained in the pET64-85 transformed E.coli BL21 cells induced by IPTG and was purified by affinity chromatography.Western-blotting analysis showed that the protein could be recognized by sera from bovines infected with M.bovis.