HLA-A＊0206-BSP和-A＊0207-BSP融合基因的构建与表达

To construct and express fusion protein of HLA-A*0206-BSP and -A*0207-BSP, the full-length HLA-A*0206, -A*0207 cDNA was amplified from peripheral blood mononuclear cells of an HLA-A*0206 positive donor and an HLA-A*0207 positive donor, respectively, by RT-PCR with HLA-A2 locus-specific primers. The cDNA was recombined into plasmid pcDNA3.1 to form pcDNA-HLA-A*0206 and pcDNA-HLA-A*0207. The extracellular fractions of HLA-A*0206, -A*0207 (without encoding signal peptide and the fragment of transmembrane) were cloned by PCR using pcDNA-HLA-A *0206 or pcDNA-HLA-A*0207 as template. These sequences of HLA-A*0206(extra) and -A*0207(extra) were used to replace the HLA-A*0201 sequence in a pre-existing HLA-A*0201-BSP vector, to generate an HLA-A*0206-BSP and an HLA-A*0207-BSP expressing vectors. After transformation of E. coli BL21(DE3) with the vectors, the fusion protein was expressed and purified. The fusion protein was refolded in vitro by limiting dilution with β2 m arid HLA-A2 restricted peptide (HBc18-27 NH2-FLPSDFFPSV-COOH) to produce the sHLA-A*0206-peptide complex monomer. The conformation of the monomer was verified by ELISA and Western blotting using HLA class Ⅰ specific mAb w6/32 or/and β2m-specific antibody. Restriction enzyme assay and DNA sequencing showed the HLA-A*0206-BSP and -A*0207-BSP was constructed, which could be expressed in E. coli after transformation. The HLA-A*0206-BSP and -A*0207-BSP fusion protein can be refolded with β2m and peptide in vitro into peptide/HLA-A*0206 or HLA-A*0207 complex monomer, as the ELISA and Western blotting showed the monomer share the natural conformation with classical HLA class Ⅰ molecule. High efficient expression of HLA-A*0206 or HLA-A*0207 fusion protein lays the foundation for the constructing MHC-peptide tetramers and artificial antigen presenting cells to explore the allelic feature of corresponding HLA-A2 subtype in T-cell recognition.