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中文摘要：
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HCMV pp65341-349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。
英文摘要：
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.
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