【摘要】 为了制备犬her-2-ECD-4单克隆抗体并验证其识别的抗原序列,本研究利用TMHMM server2. 0、SWISS-MODLE、Prot Param及Sopma等分析软件,对该区域蛋白序列进行了理化性质分析、一级结构分析、二级结构分析以及三级结构建模。综合以上各数据得出犬her-2-ECD-4可能的抗原表位为ecq(56-58)、ykd(80-82)、fade(106-109)、aeqras(134-139),在此基础上针对该区域制备了单克隆抗体,获得了3株能稳定分泌单克隆抗体的杂交瘤细胞株,分别为3-1B10、4-1B2、6-1C7,通过对杂交瘤细胞株的鉴定发现,3-1B10和4-1B2识别同一段抗原多肽,其中包括预测出的抗原表位ykd(80-82),它们的亚类鉴定结果也相同,均为Ig G1。6-1C7识别的抗原多肽含有ecq(56-58)这个预测表位,其亚类鉴定结果为Ig M。另外,通过Western Blot分析,发现本实验制备的单克隆抗体与犬乳腺肿瘤细胞系CHmm中提取的蛋白具有良好的反应性。本研究为下一步开展犬乳腺肿瘤的肿瘤标志物筛查诊断试剂盒研制及靶向治疗提供了基础。更多还原

【Abstract】 In order to develop canine her-2-ECD-4 monoclonal antibody and verify its epitope,physicochemical property,secondary structure and tertiary structure of this protein was predicted with software involving TMHMM server 2. 0,SWISS-MODLE,ProtParam and sopma etc. The results showed that the epitopes of canine her-2-ECD-4 were ecq(56-58),ykd(80-82),fade(106-109)and aeqras(134-139). After screening and subcloning,we obtained 3 hybridoma cell lines(3-1 B10,4-1 B2,and 6-1 C7) that stably secreted McAbs against the her-2. Through the identification of the hybridoma cell lines,it was found that 3-1 B10 and 4-1 B2 recognize the same antigen polypeptide,including the predicted antigenic epitope ykd(80-82),and their subclass identification results were the same. Both were Ig G1. The 6-1 C7 recognized antigenic peptide containing the predicted epitope of ecq(56-58),and the subclass identification result was Ig M. In addition,it was found that the monoclonal antibody prepared in this experiment had good reactivity with the protein extracted from the canine breast tumor cell line CHmm by western blot analysis.更多还原