酿酒酵母钙通道膜蛋白单克隆抗体制备及鉴定

Preparation and identification of monoclonal antibodies of calcium channel membrane proteins in Saccharomyces cerevisiae

以酿酒酵母电压门控钙通道膜蛋白(Cch1p)、牵张敏感性钙通道膜蛋白(Mid1p)和瞬时受体电位钙通道膜蛋白(Yvc1p)为研究材料,制备其单克隆抗体。采用生物信息学方法确定3种膜蛋白抗原表位,根据分析结果克隆抗原基因,并进行原核表达和表达产物分析鉴定,通过Ni2+-NTA树脂亲和层析技术获得重组抗原蛋白,免疫小鼠后细胞融合技术制备单克隆抗体,酶联免疫吸附测定(ELISA)检测抗体效价,免疫印迹技术检测单克隆抗体对重组纯化抗原和天然酿酒酵母钙通道膜蛋白的反应性和特异性。生物信息学分析结果表明,Cch1p、Mid1p和Yvc1p抗原表位可能分别位于1~300位氨基酸残基、359~548位氨基酸残基、1~236位氨基酸残基;克隆目的基因条带大小分别为926bp、570bp和708bp,与预期结果一致;原核表达抗原蛋白分子量分别为60000、25000和30000,Western blot检测条带正确;重组纯化抗原免疫BALB/c小鼠,细胞融合技术制备单克隆抗体,ELISA检测显示单克隆抗体效价分别高达1∶256000、1∶128000和1∶64000,Western blot检测到3种重组纯化抗原和天然酿酒酵母钙通道膜蛋白Cch1p、Mid1p和Yvc1p。这些结果说明本文制备的单克隆抗体可以成功用于检测酿酒酵母钙通道膜蛋白Cch1p、Mid1p和Yvc1p表达的相关研究。

The monoclonal antibodies against three calcium channel membrane proteins were successfully prepared by using voltage-gated calcium channel membrane protein(Cch1p),stretch-activated calcium channel membrane protein(Mid1p),and transient receptor potential calcium channel membrane protein(Yvc1p)in Saccharomyces cerevisiae as the study materials.The epitopes of three membrane proteins were analyzed by bioinformatics methods.Based on the analysis results,the genes of three epitopes were cloned,and recombinant proteins were expressed and identified.The BALB/c mice were immunized with three recombinant antigens which were purified by Ni2+-NTA resin affinity chromatography,respectively.The monoclonal antibodies were generated by the cell fusion method and their titers were detected by ELISA.The reactivity and specificity of three monoclonal antibodies were tested by Western blot with three purified recombinant antigens and natural Cch1p,Mid1p and Yvc1p.The results showed that three antigen epitopes region may be located in 1—300 amino-acid residues,359—548 amino-acid residues,and 1—236 amino-acid residues.The purpose band sizes by gene cloning were 926bp,570bp and 708bp,which were consistent with the expected results.Furthermore,the molecular weight of three antigen proteins from prokaryotic expression systems were 60000,25000,30000,respectively and three bands were proved to be correct by Western blot.The ELISA results showed that three monoclonal antibodies prepared by cell fusion method presented high titers of 1∶256000,1∶128000,and 1∶64000.These antibodies could detect the purified recombinant antigens and natural proteins of Cch1,Mid1 and Yvc1 in Western blot.These results indicated that the prepared monoclonal antibodies against Cch1p,Mid1p and Yvc1p could be successfully used for the related research about the detection of the expression of calcium channel membrane proteins in S.cerevisiae.