【摘要】 目的制备可生物素化的可溶性HLA-A2/HPV6E7单体,进一步组装成PE标记的可溶性HLA-A2/HPV6E7四聚体。方法以原核表达的sHLA-A2BSP为重链,β2m为轻链,与人工合成的HPV6E7(22-30)抗原肽(GLH-CYEQLV)利用稀释法进行共折叠复性得到单体。利用HLAI类分子单抗(W6/3)和抗Bzm抗体进行Westernblot和ELISA鉴定折叠产物的构象。以BirA酶对其进行生物素化,经过超滤离心后得到纯化的sHLA-A2/HPV6E7单体,再与Streptavidin-PE按4:1比例混合形成四聚体。结果该四聚体具有与HLA-A2阳性HPV6感染的CA患者的抗原特异性CTL结合活性。结论成功获得了天然构象的sHLA-A2/HPV6E7单体及PE标记的HLA-A2/HPV6E7四聚体。更多还原

【Abstract】 Objective To acquire the soluble HLA-A2 monomer loaded HPV6E7 peptide and tetramer labeled by Streptavidin-PE.Methods The heavy chain of soluble HLA-A2 and β2m were expressed highly as insoluble aggregates in E.coli,and then the two subunits were refolded to form a sHLA-A0201-monomer by dilution method in the presence of Human Papillomavirus E7 peptide(GLHCYEQLV).The refolded product was detected by ELISA and Western blot.Refolded A2-GLH monomer was biotinylated with a commercial BirA.The tetramer was then formed by mixing A2-GLH monomer with Streptavidin-PE in a ratio of 4:1.Results Flow cytometry analysis indicated that this tetramer could bind to specific CTL from HLA-A2+ Condyloma Acuminata(CA).Conclusion The HLA-A2/HPV6E7 monomer and tetramer loaded HPV6E7 were successfully gained.更多还原