人Tamm-Horsfall片段蛋白的表达及单克隆抗体的制备和鉴定  

Expression of human Tamm-Horsfall fragment protein and its monoclonal antibody preparation and identification

作　　者：马红雨[1] 叶龙[2] 祁术元[3] 全首祯[1] 朱美财[1] 

机构地区：[1]空军总医院临床检验中心,北京100142 [2]大连医学院检验系 [3]空军总医院输血科,北京100142

出　　处：《中华临床医师杂志（电子版）》2013年第3期130-132,共3页Chinese Journal of Clinicians(Electronic Edition)

基　　金：北京首都医学发展基金(2009-2056)

摘　　要：目的构建人Tamm-Horfall蛋白(THP)抗原决定簇的原核表达载体,表达纯化重组人Tamm-Horfall片段蛋白并制备单克隆抗体。方法将人Tamm-Horfall蛋白片段cDNA克隆至原核表达载体pET28a,经大肠杆菌表达、纯化,获得的Tamm-Horfall蛋白片段免疫小鼠,取其脾淋巴细胞与sp2/0细胞融合,制备能产生THP蛋白单克隆抗体的杂交瘤细胞株,进一步采用Western blot、ELISA、免疫组化等技术对制备的单克隆抗体进行鉴定。结果原核表达重组质粒在大肠杆菌中能高效表达THP蛋白抗原决定簌的蛋白片段,用纯化的THP蛋白片段制备了鼠抗THP单克隆抗体。结论成功制备了9株THP单克隆抗体,为进一步研究THP蛋白的分布、结构、功能及检测试剂盒的研发奠定了基础。Objective To construct expression vectors of human Tamm-Horfall protein antigenic determinant,to purify recombinant human Tamm-Horfall protein and to prepare monoclonal antibodies.Methods cDNA of human Tamm-Horfall protein fragment was cloned into pET28a expression vector and was further transfected into DE3.The recombinant protein fragment of human Tamm-Horsfall was used immuned Balb/c mouse.The spleen lymphocyte was fused with sp2/0 hybridoma to produce monoclonal antibody against THP protein.Western blot,ELISA and immunohistochemistry were carried out to identify the monoclonal antibody produced by these hybridomas.Results Protein fragment of THP protein antigenic determinant was purified from DE3 transfected with THP protein antigenic determinant cDNA prokaryotic expression vector,and purified THP protein fragment was used to prepare THP monoclonal antibody against rats.Conclusions We successfully prepare nine hybridomas which can stably produce monoclonal antibodies against THP,and these work has laid foundation for further study regarding THP proteins distribution,structure,function and for detection kits.

关 键 词：抗体 单克隆 原核细胞 人Tamm-Horfall蛋白 纯化 

分 类 号：R392[医药卫生—免疫学]