三种常见HBV抗原肽-HLA-A*0201复合物四聚体的构建及初步检测应用

Construction and primary application of three kinds of HBV-HLA-A*0201-peptide tetramers

目的体外构建三种常见HBV抗原肽-HLA-A*0201复合物四聚体,并初步用该三种四聚体对抗原肽特异性的细胞毒性T细胞(CTL)进行了初步检测。方法原核高效表达的HLA-A*0201-BSP和β2m蛋白,分别和三种HBV抗原肽(HBVCore18-27,Env335-343,Pol575-583)体外复性折叠成可溶性的HLA-A*0201抗原肽复合物单体,经BirA酶作用并通过凝胶过滤层析法纯化复合物单体,分别将复合物单体与藻红蛋白标记的链霉亲和素按一定比例耦合构建成相应的三种抗原肽四聚体。最后进行流式细胞仪检测。结果Dot-ELISA和ELISA检测显示获得了三种具有天然构象的生物素化的HBV抗原肽-HLA-A*0201复合物单体。构建的三种四聚体均可以检测到相应特异性的CTL,在自限性感染患者体内针对乙肝核心抗原(core18-27)的CTL细胞频数(0.18%)高于针对聚合酶抗原(pol575-583)(0.08%)和包膜抗原(env335-343)(0.06%)。结论成功构建了三种具有完整构象的生物素化HBV抗原肽-HLA-A*0201复合物单体,所构建的三种四聚体都可以检测到抗原特异性的CTL。

Objective To construct three soluble HBV-HLA-A ＊ 0201-peptide complexes and detect the HBV-specific cytotoxic T lymphocytes （CIL） by using the constructed HLA-A ＊ 0201-peptide tetramers. Methods Proteins of HLA-A ＊ 0201-BSP and 132m were obtained by effective prokaryotic expression. After being purified, the proteins were refolded into HLA-A ＊ 0201-β2m-peptide complexes in the presence of three antigenic peptides （Hepatitis B virus Core18-27, Env335-343, and Pol575-583） by dilution method. The complexes then were biotinylated by BirA enzyme and purified via the gel-fdtration chromatography, Tetramers were generated by mixing the complex with PE - Streptavidin at a molar ration of 5 ： 1. Then analysis of stained PBMCs was performed using FACSean and CellQuest software. Results The biotinylated HLA-A ＊ 0201 complexes were identified by Western blotting and were shown to have the natural conformation by Dot-ELISA. The constructed HBV-HLA-A ＊ 0201-peptide tetramers were capable of detecting the HBV-specific CTLs in patients with self-limited acute HBV infection, The tetramer-pesitive ceils specific for the core18-27 epitope were found at a higher frequency than those specific for polymerase 575-583 and envelope 335-343 epitopes. Conclusion The biotinylated HLA-A ＊ 0201-peptide complexes is constructed successfully, which can be used to detect the specific CTLs.