Preparation And Identification Of HLA-A*1101-GPI Tetramer Loading With HCMV Pp65 Antigenic Peptide

Aim: To construct the prokaryotic expression vector for the ectodomain of HLA-A*1101 fused with a BirA substrate peptide(BSP) at its carboxyl terminus and express the fusion protein in Escherichia coli(E. coli) and to prepare HLA-A*1101-GPI tetramers loading with human cytomegalovirus(HCMV) pp65 peptide.Methods: The cDNA of the heavy chain gene of HLA-A* 1101 was cloned by RT-PCR from HLA-A2^- donors and verified by DNA sequencing. An expression vector, which encoded the ectodomain of HLA-A*1101 fused with BSP at the carboxyl terminus, was constructed by PCR with the cloned cDNA as a template. The recombinant protein was expressed in E. coli. The expression of HLA-A* 1101 heavy chain fused with biotinylation enzyme (BirA) substrate (HLA-A11-BSP) in E. coli was optimized in the inducing temperature, time and the concentration of IPTG, and identified by immuno-blotting with anti-HLA-A*0201 antiserum. In the present study, HLA-A11-BSP was used as heavy chain andβ₂m as light chain to refold in the presence of an HCMV antigenic peptide pp65_16-24 (GPISGHVLK, GPI) with dilution method. The HLA-A*1101 tetramers were formed by mixing the monomer with streptavidin-PE and were vertifed by flow cytometry.Results: The cDNA of HLA-A*1101 heavy-chain gene was cloned from peripheral blood mononuclear cells of HLA-A2^- donors. The expression vector for the fusion protein of the ectodomain(residues 1—276) of HLA-A*1101 fused with BSP at the carboxyl terminus was constructed and the recombinant plasmid was verified to be correct by DNA sequencing. The fusion protein was over-expressed in E. coli BL21(DE3), which accounted for 20ï¼…of total bacterial proteins. The relative molecular weight of this fusion protein was 35 000, which is consistent with the theoretical value. Western blotting showed that all of the fusion protein existed in the inclusion bodies, without any products in the supernatant. Soluble HLA-A*1101-GPI monomers were obtained by in vitro refolding of heavy chain in the presence of light chainβ₂m and antigenic peptide GPI. The biotinylated monomers were purified by anion exchanger chromatography and combined in 4: 1 molar ratio with Streptavidin-PE to form the tetramers. Flow cytometry analysis showed that HLA-A*1101-GPI tetramers had the activity to bind to specific CD8⁺ T cells, suggesting soluble HLA-A*1101-GPI tetramers were successfully prepared.Conclusion: The prokaryotic expression vector for HLA-A11-BSP has been constructed and the fusion protein is expressed in high yield in the form of inclusion bodies in E. coli. Moreover, soluble HLA-A*1101 monomers and tetramers were prepared successfully. This provides the basis for further study of HLA-A*1101-restricted cellular immune responses against HCMV infection.