摘要
目的克隆结核分枝杆菌Ag85B基因,在大肠埃希菌中进行表达,获得纯化的重组Ag85B蛋白。方法以结核分枝杆菌H37Rv基因组DNA为模板,应用PCR方法扩增Ag85B基因,以质粒PET23a(+)为表达载体构建Ag85B重组质粒,转化大肠埃希菌BL21(DE3),以IPTG诱导表达,通过SDS-PAGE电泳和Western blot技术分析鉴定Ag85B蛋白表达,采用Novagen公司生产的HisBind蛋白纯化试剂盒纯化Ag85B蛋白。结果构建了具有正确基因序列的Ag85B重组表达质粒,经SDS-PAGE分析,诱导表达的重组蛋白分子质量单位约为33ku,Western blot鉴定能被抗His单抗识别,该重组Ag85B蛋白在大肠埃希菌BL21(DE3)中以包涵体形式存在。结论目的基因克隆入宿主菌中并表达成功,纯化的重组Ag85B蛋白为进一步的研究与应用奠定了基础。
Objective To clone and express the gene coding for Mycobacterium tuberculosis antigen Ag85B, and obtain the purified recombinant protein Ag85B. Methods The Ag8513 gene was amplified from the genomic DNA by polymerase chain reaction （PCR） in vitro, and cloned into vector PET23a （＋） to construct the recombinant plasmid. The recombinant plasmid was transformed into expressive vector Escherichia coli BL21 （DE3）, and induced with IPTG; the presence of recombinant protein in the expression vector was analyzed by SDS-PAGE and Western blot. The recombinant Ag85B protein was purified with His Bind Purification Kit. Results The recombinant plasmid Ag85B was correctly constructed, and the recombinant Ag85B protein was expressed as inclusion body in E. coli BL21 （DE3）. Conclusion The target gene has been cloned into host bacterium. The purified recombinant protein Ag85B paved the way for further study.
出处
《中国病原生物学杂志》
CSCD
2009年第2期85-87,94,共4页
Journal of Pathogen Biology
