人颗粒蛋白前体F片段结构域单克隆抗体的制备与鉴定  

Preparation and characterization of monoclonal antibodies against F domain of human progranulin

作　　者：李彦青[1] 任东妮 夏海滨[1] 

机构地区：[1]陕西师范大学生命科学学院秦巴山区可持续发展协同创新中心,陕西西安710119

出　　处：《细胞与分子免疫学杂志》2015年第4期540-543,552,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81272543;81301957;81471772);陕西省自然科学基金重点项目(2014JZ005);中央高校基金项目(GK201301010)

摘　　要：目的制备抗人颗粒蛋白前体F片段(GrnF)的单克隆抗体(mAb),并初步鉴定其生物学特性。方法通过分子生物学技术构建含人GrnF编码序列的酵母表达载体,表达并纯化酵母来源的融合人血清白蛋白(HSA)的GrnF片段HSA-GrnF。用纯化的HSA-GrnF免疫BALB/c小鼠,然后将免疫小鼠的脾细胞与小鼠骨髓瘤细胞Sp2/0进行融合,用ELISA筛选阳性克隆,并采用有限稀释法进行亚克隆;采用免疫荧光染色及Westernblot法对所获得的mAb的特异性进行鉴定,并用抗体亚类测定试剂盒检测所获得单克隆抗体亚类。结果成功获得2株可分泌特异性抗GrnF的杂交瘤细胞株(1G6及4E8),经鉴定这2株抗体重链亚类均为IgG1亚类。结论成功制备了抗人GrnF片段结构域的单克隆抗体。Objective To prepare and characterize the monoclonal antibody（mAb） against F domain of human progranulin（GrnF）.Methods Yeast expression vector containing GrnF gene was constructed using molecular biological technology.Then eukaryotic fusion protein human serum albumin（HSA）-GrnF from yeast vector was expressed and purified.BALB / c mice were immunized by the purified HSA-GrnF fusion protein.The splenocytes of the BALB / c mice were isolated from spleen and fused with Sp2 /0 myeloma cells.Indirect ELISA and limiting dilution assay were used for screening hybridoma cell lines.The specificity of monoclonal antibodies against GrnF was evaluated with indirect ELISA,Western blotting and immunohistochemistry.The immunoglobulin subclass was identified with mouse monoclonal antibody isotyping reagents.Results Two hybridoma cell lines,1G6 and 4E8,were obtained and heavy chain subclasses of the two hybridoma cell lines were Ig G1.Conclusion The m Abs that can specifically recognize GrnF have been successfully prepared.

关 键 词：颗粒前体蛋白 Grn F 酵母表达载体 杂交瘤 单克隆抗体 

分 类 号：Q813.2[生物学—生物工程] R392.11[医药卫生—免疫学]