结核分枝杆菌Ag85B与ESAT6融合蛋白的表达、纯化及抗原活性的初步研究

Expression,purification of the fusion protein Ag85B-ESAT6 of Mycobacterium tuberculosis and its antigenic activity

目的将结核分枝杆菌分泌蛋白Ag85B与ESAT6在大肠杆菌中融合表达,并对其进行纯化、鉴定和免疫学特性的初步研究。方法采用PCR方法从MTB毒株H37Rv基因组DNA中扩增Ag85B和ESAT6基因,并在两基因中引入48bp的柔性linker结构,以确保两蛋白的正确折叠。将各目的片段克隆至pMD18-T载体中进行测序。将测序正确的目的基因片段双酶切后亚克隆至原核表达载体pPro-EXHTa,得到重组质粒pPROEXHTa-Ag85B-ESAT6,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和抗ESAT6的mAb进行Western blot分析鉴定。通过镍柱对融合蛋白进行纯化。在PBS中通过透析恢复重组蛋白的天然结构,将复性融合蛋白与8例临床可疑结核病人血清进行ELISA测定。结果PCR法扩增获得的各目的基因片段与GenBank报道的一致。构建的融合蛋白原核表达载体在大肠杆菌中表达后,经SDS-PAGE分析显示重组质粒在原核系统中得到了表达。融合蛋白能够分别与抗Ag85B和抗ESAT6的mAb反应。该融合蛋白主要以包涵体的形式存在,镍柱亲和层析纯化后得到了高纯度的Ag85B-ESAT6融合蛋白,并能够与结核病人血清发生反应。结论结核分枝杆菌融合蛋白Ag85B-ESAT6在大肠杆菌中得到了高效表达,为进一步研究其免疫原性和保护性提供了基础。

Ag85B and ESAT6 genes were firstly amplified by PCR from H37Rv virulent strain of Mycobacterium tuberculosis （MTB）, and a flexible chain with 48bp was linked between Ag85B and ESAT6 genes, in order to ensure the correct folding of the fusion protein. The two genes were further cloned into vector pMD18-T respectively. By analysis with enzymatic di- gestion and DNA sequence, Ag85B and ESAT6 genes were subcloned into the prokaryotie expression vector pPROEXHTa, in turns, to produce the recombinant plasmid pPROEXHTa-Ag85B-ESAT6. The fusion protein was induced by IPTG in E. coli DH5a, 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, which was fully renaturated by dialysis. The renaturated protein could reaet with sera of 8 cases with tuberculosis by indirect ELISA. It is evident that the prokaryotic expression vector of pPROEXHTa-Ag85B-ESAT6 was successfully eonstructed and the fusion protein was expressed efficiently, which enables further study on the immunogenieity and protective effect of Ag85B-ESAT6.