HLA-A*0201-BSP融合基因的构建与原核表达

克隆表达可生物素化的HLA-A＊0201的重链胞外区是制备HLA-A＊0201-肽四聚体的先决条件。本研究构建重组HLA-A＊0201-BSP融合基因的表达载体，以制备HLA—A2四聚体。根据已报道的序列设计特异引物，利用逆转录聚合酶链反应（RT-PCR）方法从已鉴定为HLA—A＊0201基因型的健康人白细胞中克隆HLA—A＊0201重链胞外区的基因．拼接上依赖生物素-蛋白连接酶（biotin-protein ligase，BirA）的可生物素化序列（BirA substrate peptide．BSP），构建HLA—A＊0201-BSP的原核表达载体，在大肠杆菌DH50t中进行表达。结果表明：成功扩增出HLA—A＊0201-BSP融合基因并测序证实，构建的pBV220-HLA—A＊0201-BSP可在大肠杆菌DH50t中高效表达HLA—A＊0201-BSP融合蛋白．表达量占菌体总蛋白的28％，以包涵体的形式表达，经洗涤、变性后，以Sepharcyl S-300HR（S-300）柱层析纯化，纯度可达90％以上。结论：获得了高效表达HLA—A＊0201-BSP的大肠杆菌工程菌株，建立了简便有效的包涵体纯化方法，为制备HLA-A＊0201-肽四聚体奠定了基础。

Cloning and Expression of HLA-A * 0201-BSP

High-yield production of HLA-A*0201 heavy chain is a prerequisite to the preparation of HLA-A2 tetramer. The present study was aimed to construct the expression vector of recombinant HLA-A*0201-BSP fusion gene for preparing HLA-A2 tetramers. The extracellular region HLA*0201 was cloned by RT-PCR from HLA-A2(+) donor, and a 15-amino acid biotin-protein ligase (BirA) substrate peptide (BSP) for BirA-dependent biotinylation was added to the COOH-terminus of HLA-A*0201 heavy chain. Then the fusion gene was cloned into pBV220 vector at EcoRI and Bam HI sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pBV220-HLA-A*0201-BSP was transformed to the competent cells of E.coli DH5alpha. The results showed that the HLA-A*0201-BSP fusion protein was successfully expressed in the form of inclusion body and amounted to over 28% of total cell proteins via induction at 42 degrees C. After washed with triton X-100 and urea, the inclusion body was dissolved with 8 mol/L urea and then purified with Sepharcyl S-300 HR, and the final purity reached above 90%. It is concluded that the HLA-A*0201-BSP fusion gene was cloned successfully and expressed efficiently in E.coli DH5alpha. This work establishes a convenient approach for purification of large quantity of recombinant HLA-A*0201-BSP. This provides the basis for the preparation of HLA-A2 tetramers.