HLA-A^＊2402-BSP在大肠杆菌中的优化表达及其四聚体的制备和鉴定

Improved Expression of HLA-A～*2402-BSP in Escherichia coli and Its Tetram er Preparation

HLA-A*2402是中国人群中最常见的等位基因之一,为研究该基因型人群的人巨细胞病毒(HCMV)特异性细胞毒T细胞(CTL)免疫应答,需要制备负载相应抗原肽的HLA-A*2402四聚体。以RT-PCR方法克隆HLA-A*2402重链基因的cDNA,并构建了羧基端融合生物素化酶BirA底物肽(BSP)的HLA-A*2402重链胞外域融合蛋白(HLA-A*2402-BSP)的表达载体,但该载体不能在大肠杆菌(E.coli)中有效表达HLA-A*2402-BSP融合蛋白;通过对氨基端(N端)区域编码区的密码子进行优化,构建了同义突变的HLA-A*2402-BSP表达载体,融合蛋白在E.coli中获得了高效表达。进而制备了负载HLA-A*2402限制性HCMVpp65341-349抗原肽(QYDPVAALF,QYD)的可溶性HLA-A*2402-QYD单体分子和四聚体,获得的四聚体具有与HLA-A24+供者抗原特异性CTL的结合活性,特异性CTL的频率为总CD8+T细胞的0·09%～0·37%。这些结果为进一步研究HLA-A*2402限制性的特异性CTL免疫应答规律奠定基础。

HLA-A^＊ 2402 is one of the most frequently encountered HLA-A alleles in East Asian populations. In order to study the CD8 ^＋ T cell responses in Chinese populations, we have described the generation and functional test of HLA-A^＊ 2402 tetramer loaded with HCMV pp65 341-349 peptide （QYDPVAALF, QYD）. The cDNA of HLA-A^＊ 2402 heavy chain was cloned by RT-PCR from one of the donors. DNA fragment encoding the ectodomain of HLA-A^＊ 2402 heavy chain fused at its carboxylterminal a BirA substrate peptide （BSP） was amplified by PCR with the cloned heavy chain cDNA as a template. The wild-type geneof HLA-A^＊ 2402-BSP was not expressed in Escherichia coli （E. coli）, while mutant HLA-A^＊ 2402-BSP gene with optimized codons was overexpressed as inclusion bodies in E. coli. Furthermore, the soluble HLA-A^＊ 2402-QYD monomers were generated by in vitro refolding of washed inclusion bodies in the presence of β2-microglobulin and QYD peptide. The tetramer was subsequently formed by mixing HLA-A^＊ 2402-QYD monomers with streptavidin-PE at a molar ratio of 4 ： 1. Flow cytometry analysis indicated that this tetramer possessed binding activity with specific CTL from HLA-A24^＋ donors and the frequencies of tetramer-binding CTL were 0.09% - 0.37% within total CD8^＋ T cells. This tetrameric agent provides a powerful tool to explore the secrets of CTL responses against HCMV antigens in HLA-A^＊ 2402 individuals.