呼吸道合胞病毒F2蛋白与结核分枝杆菌早期分泌抗原靶6蛋白的融合表达及纯化

目的在大肠杆菌中融合表达呼吸道合胞病毒(Respiratory syncytial virus,RSV)F2蛋白与结核分枝杆菌(Mycobacterium tuberculosis,MTB)早期分泌抗原靶6(Early secretory antigenic target-6,ESAT-6)蛋白,并进行纯化。方法分别从MTB标准菌株H37Rv及含F蛋白全长基因的pMD18-T-f质粒中扩增esat-6-f2基因,插入原核表达载体pET-30a中,构建重组表达质粒pET-30a-esat-6-f2,转化大肠杆菌BL21(DE3),IPTG诱导表达,表达的融合蛋白经SDS-PAGE和Western blot鉴定后,经镍离子亲和层析柱纯化。结果重组表达质粒经双酶切及测序鉴定,证明构建正确;重组融合蛋白ESAT-6-F2相对分子质量约为21 000,主要以包涵体形式表达,可与小鼠抗His单克隆抗体特异性结合;纯化的融合蛋白纯度可达90%以上。结论成功在大肠杆菌中表达并纯化了重组融合蛋白ESAT-6-F2,为进一步研究其免疫原性和免疫保护性奠定了基础。

Fusion expression and purification of respiratory syncytial virus F2 protein and Mycobacterium tuberculosis early secretory antigenic target-6 protein

Objective To express a fusion protein ESAT-6-F2 comprising respiratory syncytial virus F2 protein and Mycobacterium tuberculosis(MTB)early secretory antigenic target-6(ESAT-6)protein in E.coli,and purify the expressed product.Methods The esat-6-f2 gene was amplified from the genome of MTB H37Rv and plasmid pMD18T-f containing the full-length gene of F protein respectively and inserted into prokaryotic expression vector pET30a.The constructed recombinant plasmid pET-30a-esat-6-f2 was transformed into E.coli BL21(DE3) for expression under induction of IPTG.The expressed fusion protein ESAT-6-F2 was identified by SDS-PAGE and Western blot,and purified by Ni-NTA affinity chromatography.Results Restriction analysis and sequencing proved that recombinant plasmid pET-30a-esat-6-f2 was constructed correctly.Recombinant fusion protein ESAT-6-F2,with a relative molecular mass of about 21 000,mainly existed in a form of inclusion body,which showed specific binding to mouse anti-His monoclonal antibody and reached a purity of more than 90% after purification.Conclusion Recombinant fusion protein ESAT-6-F2 was expressed successfully in E.coli and purified,which laid a foundation of further study on its immunogenicity and protective effect.