人可溶性单链β2m-HLA-A2-BSP融合蛋白表达载体的构建及其表达

Construction and Expression of Human Soluble Single Chain β2m-HLA-A2-BSP Fusion Protein Expression Vector

目的构建并表达了人可溶性单链β2m-HLA-A2-BSP(scHLA-A2)融合蛋白表达载体,为进一步制备HLA-A2四聚体及其功能研究奠定了基础。方法应用重叠延伸PCR(overlap-PCR)对编码HLA-A2的cDNA序列进行一个碱基的同义突变,并将β2m和HLA-A2胞外段亚单位两段编码序列的cDNA通过一疏水性多肽接头(Gly4Ser)3的DNA序列进行前后拼接,并利用引物所带的BSP编码序列,构建单链β2m-HLA-A2-BSP融合基因,将测序正确的β2m-HLA-A2-BSP融合基因插入原核表达载体pET-22b中,转化BL21(DE3)细菌,SDS-PAGE检测其表达,融合蛋白于体外抗原肽存在条件下稀释复性后由Western blot鉴定其空间构象。结果经DNA序列分析表明:同义突变与预期结果一致,β2m和HLA-A2-BSPl、inker的连接顺序、方向及序列完全正确,表达载体转化BL21(DE3)细菌后可表达β2m-HLA-A2-BSP融合蛋白,SDS-PAGE显示该融合蛋白分子量为45 kD,与理论值一致,将融合蛋白与特异性抗原肽在体外进行稀释复性后经Western blot鉴定表明该复合物能与HLA-Ⅰ类分子特异性结合的单克隆抗体W6/32结合。结论人可溶性单链β2m-HLA-A2-BSP融合蛋白的构建及其表达获得成功,经体外复性可获得HLA-Ⅰ类分子天然构象。

Objective To construct and express the human soluble single chain β2m-HLA-A2-BSP fusion protein expression vector. Methods A synonymous mutation and a hybrophobic polypeptide linker （Gly1Ser）3 were used to splice three different gene fragments of β2m and HLA-A2 extracellular region subunits by overlap-PCR for constructing single chain β2m- HLA-A2-BSP fusion gene in vitra. After sequencing,β2m-HLA-A2-BSP fusion gene was inserted into pET22b bacterial expression vector and then transformed into BL21（DE3）. The fusion protein was analyzed by using SDS-PAGE. In the presence of the corresponding antigenic peptide, the fusion protein was refolded to form the monomer by in vitro dilution method and identified by Western blot. Results Sequence analysis showed that the anticipated synonymous mutation, splicing order, orientation and sequence of β2m-HLA-A2-BSP fusion gene were completely correct. The expressed β2m-HLA-A2-BSP fusion protein in BL21 cells presented as a band at 45 kD which was in accordance with its theoretical molecular weight. Western blot with mAb W6/32 demonstrated that scHI.A-A2-peptide complex monomer shared the antigenic determinant with the native HI.A class I molecules. Conclusion The constructed human soluble single chain β2m-HLA-A2-BSP fusion protein was exressed in prokaryotic cells successful on as native HI.A class Ⅰ . and the complex with corresponding antigenic peptide had the correct spatial construeion as native HLAclass Ⅰ.