作　　者： ; ; ; ; ; ;

机构地区： 中国农业科学院研究生院

出　　处： 《中国兽医科技》 2005年第12期946-949,共4页

摘　　要： 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。 The DNA fragments of mpb64 and Ag85B gene were amplified by PCR from genome DNA of Mycobacterium boris 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. boris.

关 键 词： 牛分枝杆菌 融合基因 原核表达

领　　域： [农业科学] [农业科学] [农业科学] [生物学]