应用dtSCT技术制备低亲和力抗原肽InsB_(15-23)肽的MHCⅠ四聚体

Construction of dtSCT MHCⅠ tetramer of low affinity InsB_(15-23) antigenic peptide

目的制备低亲和力InsB15-23抗原肽的MHCⅠ四聚体。方法应用二硫键捕获型单链三聚体(disulfide-trap singlechain trimer,dtSCT)技术,将InsB15-23抗原肽、小鼠β2微球蛋白(β2-microglobulin,β2m)和H-2Kd分子以柔性接头(linker,L)依次连接,并在抗原肽的C末端与H-2Kd分子间引入了二硫键,以提高抗原肽与H-2Kd分子结合的稳定性。融合基因克隆入pET-22b原核表达载体,经过诱导表达、洗涤包涵体、稀释复性等步骤获得单体,生物素化后制成InsB15-23dtSCT型四聚体。结果我们比较了自制InsB15-23dtSCT四聚体和商品化G9V传统五聚体对小鼠外周血特异性CTLs的检出率。2种多聚体对阴性对照Balb/c小鼠都显示出很低的非特异性标记水平,都能检出4周龄NOD母鼠外周血中低水平的InsB15-23特异性CTLs,且二者的检出率无统计学差异。结论我们成功制备出稳定的InsB15-23dtSCT型四聚体。与商品化的传统五聚体相比,我们自制的四聚体具有类似的检出率和低非特异性标记水平。

In order to construct MHC I tetramer of low affinity InsBl5-23 antigenic peptide, we connected lnsB15-23 antigenic peptide, β2-microglobulin and H-2Ka covalently by two short flexible linkers and introduced a disulfide bond between the linker 1 and H-2Kd to trap antigenic peptides into the binding groove. The fusion product was digested by Nde I and Xho I , and then inserted into plasmid pET-22b and transformed into E. coli BL21（DE3）. The purified inclusion bodies refolded in a refolding buffer containing 2 mol/L urea. After biotinylated by BirA, tetramer was derived by mixing the biotinylated monomers with PE-streptavidin at a molar ratio of 4：1. We compared our tetramer with customed G9V pentamer, and they show similar low non-specific marking level and detection rate of InsB15-23 specific CTLs in the peripheral blood of 4 weeks female NOD mice.