鸡MHCⅡ类分子部分基因的原核表达及单克隆抗体的制备与鉴定

目的]制备鸡MHCⅡ类分子的单克隆抗体。方法]在分析鸡MHCII类分子蛋白序列基础上,选取鸡MHCⅡα链基因第2～6外显子和β链基因第3～6外显子进行原核表达,以纯化的融合蛋白免疫Balb/c小鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合,经克隆和间接ELISA筛选阳性细胞株。结果]共得到1株分泌鸡MHCⅡα链抗体和2株MHCⅡβ链抗体的杂交瘤细胞,分别命名为MHCⅡα-4、Ⅱβ6-2和Ⅱβ6-31,其腹水效价分别为1∶256000、1∶256000和1∶1280000。Western blotting分析表明其特异性强,能与相应蛋白结合。结论]成功获得了3株稳定分泌抗鸡MHCⅡ类分子抗体的杂交瘤细胞。

Prokaryotic Expression of the Section Gene of Chicken MHC ClassⅡ Molecules and Preparation of Its Monoclonal Antibodies

Objective]To prepare the monoclonal antibody against chicken MHC class Ⅱ molecules.Method]The prokaryotic expression of the gene fragments of exons 2-6 in MHCⅡ encoding α chain and exons 3-6 in MHCⅡ encoding β chain were performed based on analyzing its protein sequences.After Balb/c mice were immunized with the purified protein antigen,a fusion with mouse spleen cells and SP2/0 was carried out.Result]One strain secreting monoclonal antibody against α chain and two strains secreting monoclonal antibody against β chain were obtained by cloning and ELISA measuring.They were named as MHCⅡα-4,Ⅱβ6-2 andⅡβ6-31,and their titers of ascites were 1∶256 000,1∶256 000 和 1∶1 280 000,respectively in ELISA.These antibodies could specifically recognize MHCⅡ α chain or β chain in Western blotting.Conclusion] Three obtained hybridoma stains can stably product the monoclonal antibody against chicken MHC Ⅱclass molecules.