【摘要】 目的:制备抗TRPC6多肽单克降抗体,并对其生物学特性进行鉴定。方法:采用TRPC6多肽偶联血蓝蛋白(KLH)为抗原,免疫小鼠后经细胞融合,间接ELISA法筛选阳性杂交瘤细胞;单克隆抗体亚类检测试剂盒鉴定Ig亚型;间接ELISA法鉴定该单克隆抗体的特性、效价及其相对亲和力;采用生物信息学进行抗原表位预测,并通过ELISA单抗相加试验分析单抗识别抗原位点的差异性。结果:获得3株能稳定分泌抗TRPC6多肽的单抗杂交瘤细胞株,分别命名为A2、F11和H8;抗体Ig亚类均为IgG1型;染色体鉴定符合杂交瘤细胞的特性;3株单抗相对亲和力比较F11较强,A2和H8接近;抗体识别位点分析结果表明A2与H8、A2与F11的相加指数分别大于50%,可能识别的是不同位点;而F11与H8的相加指数小于50%,可能识别的是同一位点。结论:抗TRPC6多肽单克隆抗体的成功制备及其识别抗原表位分析,为TRPC6蛋白的相关研究提供物质基础。更多还原

【Abstract】 Objective:To prepare the monoclonal antibody(mAb) against TRPC6 peptides and characterize their biological functions.Methods: Female BALB/c mice were immunized with TRPC6 peptides and Keyhole Limpet Hemocyanin(KLH) conjugates,and screen positive hybridoma cell by indirect ELISA after cell fusion;identify subtype of Ig with mAb subclass detection kit;identify characteristics,titer and relative affinity by indirect ELISA;forecast peptides epitope with bioinformatics and analysis the otherness of antigen sites recognized by mAb ELISA addition test.Results: Three hybridoma cells lines(A2,H8 and F11) steadily secreting anti-TRPC6 peptides mAb were obtained;All of their subtype of Ig were IgG1;Appraisal of their chromosome accords with features of hybridoma cells;among the three mAbs,F11 has the stronger relative affinity,A2 and H8 share the similar relative affinity;The analysis shows that the additive index of the antibody recognition epitope A2 and H8,A2 and F11 are both greater than 50%,maybe they are possible to identify the different epitope;While the additive index of F11 and H8 is less than 50%,they are likely to recognize the same epitope.Conclusion: Successful preparation of anti-TRPC6 peptides mAb and analysis of its recognizing peptides epitope provide material basis of research on TRPC6 protein.更多还原