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 Tetramer Preparation

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 pp65_341-349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。

Abstract:

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 carboxyl-terminal a BirA substrate peptide (BSP) was amplified by PCR with the cloned heavy chain cDNA as a template. The wild-type gene of 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.