Improved Expression of HLA-A*2402-BSP in Escherichia coli and Its Tetramer Preparation

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.