小鼠H-2D~b-BSP融合基因的构建与表达

目的:构建小鼠H-2Db-BSP融合基因,并诱导其在大肠杆菌中表达.方法:采用RT-PCR方法从C57BL/6小鼠淋巴细胞中扩增出H-2Db链的胞外段基因,经双酶切置换已构建的HLA-A*0201-BSP重组体中的HLA-A*0201胞外段序列,使H-2Db与BirA酶底物肽(BSP)序列融合,构建H-2Db-BSP融合基因表达载体,转化大肠杆菌BL21(DE3)菌株,筛选重组子,并经IPTG诱导融合蛋白表达.结果:构建的H-2Db-BSP融合基因插入正确且序列与GenBank一致;经1mmol/L浓度的IPTG诱导后4h蛋白表达达到高峰,且融合蛋白以包涵体形式存在.结论:成功构建H-2Db-BSP融合基因,并诱导其在大肠杆菌得以表达,为进一步构建H-2Db四聚体,研究T细胞应答提供了物质基础.

Construction and Expression of Mouse H-2Db-BSP Fusion Gene

Objective：To construct the fusion gene of mouse H-2Db-BSP, and investigate its expression in E.coli. Methods：The H-2Db was amplified by RT-PCR method from total RNA of leukomonocyte which isolated from C57BL/6 （B6） mouse （H-2b）. The sequence of H-2Db was used to replace the HLA-A＊0201 sequence in a pre-existing HLA-A＊0201-BSP vector, to generate an H-2Db-BSP expressing vector. After transformation of E.coli BL21 （ DE3） cells with the vectors, the expression of fusion protein was induced by isopropyl -D-thiogalactoside （IPTG）. Results： The sequence of the H-2Db-BSP was matched with that of Genbank. Upon induction with 1mmol/ L IPTG 4 hours, the fusion protein was expressed in E.coli BL21 cells, and large amounts of recombinant protein accumulated in intracellular inclusion bodies. Conclusion：We have successfully constructed the fusion gene of mouse H-2Db-BSP, and confirmed that the fusion gene can be expressed in E.coli BL21 cells. High efficient expression of H-2Db-BSP fusion protein lays the foundation for the constructing H-2Db tetramers to explore corresponding of T cell recognition.