【摘要】 目的:构建HLA-A*1101重链胞外域羧基端融合生物素化酶B irA底物肽(BSP)的融合蛋白(HLA-A11-BSP)原核表达载体,并在大肠杆菌中表达该融合蛋白。方法:以RT-PCR法扩增并克隆HLA-A*1101重链基因的cDNA,以PCR方法构建HLA-A11-BSP的表达载体,在大肠杆菌BL21(DE3)中诱导表达,并以免疫印迹法进行鉴定。结果:从HLA-A2阴性的供者外周血单个核细胞中克隆到HLA-A*1101重链基因的cDNA,以此cDNA为模板,将编码HLA-A*1101重链胞外域1~276序列与编码BSP的序列融合,构建HLA-A11-BSP融合蛋白表达载体,重组质粒经测序验证。融合蛋白在BL21(DE3)中诱导后获得高效表达,约占菌体总蛋白的20%;其相对分子质量约为35 000,与理论值一致。免疫印迹分析显示表达产物主要存在于包涵体中,上清液中几乎无任何产物存在。结论:成功构建表达HLA-A11-BSP融合蛋白的原核表达载体,该融合蛋白在大肠杆菌中以包涵体形式获得高水平表达。更多还原

【Abstract】 Aim: To construct the prokaryotic expression vector for HLA-A*1101 heavy chain ectodomain fused with a BirA substrate peptide(BSP) at its carboxyl terminus and express the fusion protein in Escherichia coli(E.coli).Methods: The cDNA of the heavy chain gene of HLAA*1101 was cloned by RT-PCR from HLA-A2-donors and verified by DNA sequencing. An expression vector,which encoded the ectodomain of HLA-A*1101 fused with BSP at the carboxyl terminus,was constructed by PCR with the cloned cDNA as a template.The recombinant protein was expressed in E.coli and identified with Western blotting.Results: The cDNA of HLA-A*1101 heavy-chain gene was cloned from peripheral blood mononuclear cells of HLA-A2-donors.The expression vector for the fusion protein of the ectodomain(residues 1-276) of HLA-A*1101 fused with BSP at the carboxyl terminus was constructed and the recombinant plasmid was verified by DNA sequencing.The fusion protein was over expressed in E.coli BL21(DE3),which accounted for 20% of total bacterial proteins.The relative molecular weight of this fusion protein was 35 000,which is consistent with the theoretical value.Western blotting showed that all of the fusion protein existed in the inclusion bodies,without any products in the supernatant.Conclusion: The prokaryotic expression vector for HLA-A11BSP has been constructed and the fusion protein is expressed in high yield in the form of inclusion bodies in E.coli.更多还原