HLA-A*0206-BSP和-A*0207-BSP融合基因的构建与表达

Construction and expression of HLA-A*0206-BSP and-A*0207-BSP fusion genes

采用RT-PCR技术从HLA-A*0206和-A*0207阳性个体的PBMC中分别克隆出HLA-A*0206和-A*0207基因的全长cDNA序列,构建HLA-A*0206和-A*0207克隆载体。再利用PCR技术从构建的克隆载体中扩增HLA-A*0206和-A*0207的α链(重链)胞外段序列,分别经双酶切置换本室保存的HLA-A*0201-BSP重组体中的HLA-A*0201胞外段序列,使HLA-A*0206和-A*0207与BirA酶底物肽(BirA substrate peptide,BSP)序列融合,构建HLA-A*0206-BSP和-A*0207-BSP融合基因的表达载体,经限制性酶切和DNA测序证实。然后将该表达载体转化E.coliBL21(DE3)后获得表达产物,通过体外稀释复性,初步纯化的表达产物通过ELISA和Western blot检测证明能够与β2微球蛋白(HLA I类分子轻链)及HLA-A2限制性抗原肽(HBV core 18-27)折叠形成具有HLA I类分子天然构象的抗原肽/HLA-A2复合物单体。为进一步构建HLA-A*0206和-A*0207四聚体,探讨相应HLA-A2亚型的功能特点提供了物质基础。

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. I 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 pcDNAHLA-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 ＊ 020I sequence in a pre-existing HLA-A ＊ 020I-BSP vector, to generate an HLA-A ＊ 0206-BSP and an HLA-A ＊ 0207-BSP expressing vectors. After transformation of E. coli BL2I （DE3） with the vectors, the fusion protein was expressed and purified. The fusion protein was refolded in vitro by limiting dilution with β2 and 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 antibod y. Restriction enzyme assay and DNA sequencing showed the HLA -A ＊ 0206-BSP and -A ＊ 0207-13SP was constructed, which could be expressed in E. coli after transformation. The HI.A-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.