加载HPV6E7抗原肽的HLA-A2单体及其四聚体的制备和鉴定  

Preparation and Identification of The Soluble HLA-A2 Monomer and Tetramer Loaded with HPV6E7 Antigenic Peptide

作　　者：张彩娥[1] 邓云华[2] 吴雄文[3] 梁智辉[3] 翁秀芳[3] 卢小玲[3] 夏芳珍[3] 钟茂华[3] 陆盛军[3] 

机构地区：[1]武汉工业学院医学院免疫学教研究,湖北武汉430030 [2]华中科技大学同济医学院附属同济医院皮肤科,湖北武汉430030 [3]华中科技大学同济医学院免疫学系,湖北武汉430030

出　　处：《中国皮肤性病学杂志》2006年第9期519-522,共4页The Chinese Journal of Dermatovenereology

基　　金：国家自然科学基金资助项目(编号:30271201);科技部973计划课题资助项目(编号:2001CB510008)

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。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.

关 键 词：HLA-A2 sHLA-A2/HPV6E7四聚体 尖锐湿疣 细胞毒性T细胞 

分 类 号：R392.11[医药卫生—免疫学]