结核分枝杆菌融合蛋白Ag85B-Hsp16.3的表达、纯化和鉴定  

Expression,purification and identification of the fusion protein Ag85B-Hsp16.3 of Mycobacterium tuberculosis

作　　者：杨巍[1,2] 师长宏[1] 赵佐庆[2] 赵勇[1] 江鹰[1] 

机构地区：[1]第四军医大学实验动物中心,西安710032 [2]第四军医大学唐都医院实验外科

出　　处：《中国人兽共患病学报》2010年第10期953-956,共4页Chinese Journal of Zoonoses

摘　　要：目的将结核分枝杆菌分泌蛋白Ag85B与Hsp16.3在大肠杆菌中融合表达并对其进行纯化和鉴定。方法采用PCR方法从质粒pProEX HTb-Hsp16.3扩增Hsp16.3基因,引入48bp的柔性linker结构以确保两蛋白的正确折叠。将目的片段克隆至pMD18-T载体中进行测序。将测序正确的基因片段双酶切后,替换质粒pProEX HTa-Ag85B-ESAT6中的ESAT6基因,从而获得重组质粒pProEX HTa-Ag85B-Hsp16.3,并转化入大肠杆菌DH5α。经IPTG诱导后进行融合表达,并分别用抗Ag85B多抗和抗Hsp16.3单抗以及活动期结核病人血清进行Western blot分析鉴定,采用镍柱亲合色谱法对融合蛋白进行纯化。结果 PCR扩增获得的目的基因与GenBank报道的一致。SDS-PAGE分析显示构建的融合蛋白在大肠杆菌中表达,且该融合蛋白能够分别与抗Ag85B多抗和抗Hsp16.3单抗以及结核病人血清反应。该融合蛋白主要以包涵体的形式存在,镍柱亲和层析可获得纯化的Ag85B-Hsp16.3融合蛋白。结论结核分枝杆菌融合蛋白Ag85B-Hsp16.3在大肠杆菌中高效表达,为进一步研究其生物学功能提供了基础。Hsp16.3 gene was firstly amplified by PCR from plasmid pProEX HTb-Hsp16.3 that was preserved in the laboratory,and a flexible chain with 48bp was linked between Ag85B and Hsp16.3 genes in order to ensure the correct folding of the fusion protein.The Hsp16.3 gene was further cloned into vector pMD18-T.By DNA sequence analysis and enzymatic digestion,Hsp16.3 gene took the place of ESAT6 gene in the recombinant plasmid pProEX HTa-Ag85B-ESAT6,which was also preserved in laboratory to produce the recombinant plasmid pProEX HTa-Ag85B-Hsp16.3.The fusion protein was induced by IPTG in E.coli DH5α and purified by Ni-NTA purification system under denaturing condition.Following analysis of SDS-PAGE and Western blotting,the protein was detected and purified efficiently.It mainly existed in inclusion body.It is evident that the prokaryotic expression vector of pProEX HTa-Ag85B-Hsp16.3 is successfully constructed and the fusion protein is expressed efficiently,which enables further study to work on the biological function of Ag85B-Hsp16.3.

关 键 词：AG85B HSP16.3 融合蛋白 原核表达 纯化 

分 类 号：R378.9[医药卫生—病原生物学]