河南华溪蟹金属硫蛋白单克隆抗体的制备与鉴定  

Preparation and characterization of monoclonal antibodies against metallothionein of freshwater crab( Sinopotamon henanense)

作　　者：何永吉[1] 刘进平[1] 王兰[1] 

机构地区：[1]山西大学生命科学学院,山西太原030006

出　　处：《细胞与分子免疫学杂志》2014年第8期844-847,855,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(30970361);高等学校博士学科点专项科研基金(20111401110010);山西省自然科学基金(2010011043-2)

摘　　要：目的制备小鼠抗河南华溪蟹金属硫蛋白(MT)单克隆抗体(mAb),并鉴定其免疫学特性。方法分别利用小泛素样修饰蛋白(SUMO)融合表达系统和碱性磷酸酶(phoA)分泌表达载体制备河南华溪蟹重组蛋白SUMO-MT和His-MT,以SUMO-MT为免疫抗原、His-MT为检测抗原,利用杂交瘤技术建立抗河南华溪蟹MT mAb杂交瘤细胞株。采用间接ELISA测定mAb腹水效价,Western blot法、Dot-ELISA分析mAb的特异性。结果成功建立了2株稳定分泌抗MT蛋白的mAb杂交瘤细胞株,分别命名为mAb-MT2和mAb-MT3,均属于IgG1亚类。腹水效价分别为1∶500 000、1∶1000 000,Western blot和Dot-ELISA结果证实2株mAb均能特异性识别重组MT和内源性MT。结论成功制备了具有较高特异性的河南华溪蟹MT mAb。Objective To prepare a monoclonal antibody （mAb） against metallothionein （MT） of freshwater crab （ Sinopotamon henanense） and characterize its immunologic properties. Methods Two recombinant MT of S. henanense, namely SUMO-MT and His-MT, were produced by SUMO fusion system and phoA secretion expression system in E. coil SUMO-MT was used as an antigen to immunize BALB/c mice. By means of the cell fusion technique, multiple cell subcloning, repeated screening with His-MT as detecting antigen, the hybridomas specifically secreting mouse mAb against the MT of S. henanense were generated. The titers of mAbs were measured by indirect ELISA and the specificity of the mAbs was evaluated by Western blotting and Dot-ELISA. Results Two hybridoma cell lines designated mAb-MT2 and mAb-MT3 with the property of secreting mAb against the MT continuously and steadily were successfully obtained. Their immunoglobulin subclass was IgG1. The titers of the ascites fluid were 1：500 000 and 1：1000 000, respectively. Western blot analysis confirmed that the two mAbs both reacted with recombinant SUMO-MT and His-MT with good sensitivity. The Dot-ELISA demonstrated that the two mAbs reacted specifically not only with recombinant MT but also natural MT. Conclusion The mAbs against MT of S. henanense with high specificity were successfully prepared.

关 键 词：河南华溪蟹 金属硫蛋白 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]