【作者】 李剑；

【导师】 吴坚平； 杨巍；

【作者基本信息】 浙江大学 ， 生物工程（专业学位）， 2016， 硕士

【摘要】 背景抗体从多克隆发展到单克隆抗体药物,已被广泛应用于生物科学及医药研发技术产品中。相比鼠单克隆抗体而言,兔单克隆抗体具有高亲和力,特异性强,易于人源化等优点。TRPM2(Transient receptor potential channels melastatin,瞬时受体电位通道M型2)是Ca2+非选择性阳离子通道,受ADPR(Adenosine diphosphate ribose,二磷酸腺苷核糖)和H202激活调控胞内Ca2+浓度,从而对细胞的各项生理功能进行调控,与能量代谢修复和氧化应激相关的病理性疾病有关。目前市面上主要是TRPM2多克隆抗体,其特异性和亲和力并不是十分理想。目的制备TRPM2兔单克隆抗体。方法本实验设计合成了三段TRPM2多肽,YCA-10987, YCA-10988和YCA-10989,每段多肽免疫三只实验兔。经过血清blot实验初步筛选,将可能免疫了TRPM2兔脾细胞与240E兔骨髓瘤样细胞株融合,产生能分泌TRPM2抗体的兔-兔杂交瘤细胞,用HAT作为选择性培养基对融合后的细胞进行培养,运用ELISA法和WB法,筛选获得阳性克隆,选取其中WB信号最强的克隆进入抗体质粒重组阶段,再进一步筛选,选取分泌单克隆抗体最强的配对进入单克隆抗体大转生产。结果YCA-10988免疫失败。YCA-10987和YCA-10989在WB应用上有差异,YCA-10987在过表达裂解液中能检测出强信号,但在细胞裂解液中检测出很弱的信号,而YCA-10989与YCA-10987相反。结论本实验设计合成了三段TRPM2多肽,通过杂交瘤和重组技术,获得YCA-10987和YCA-10989两个不同位点TRPM2兔单克隆抗体,两者在WB应用上有差异,但可满足检测需要,有潜在应用前景。更多还原

【Abstract】 Background Developing from polyclonal antibodies to monoclonal antibody drugs, antibodies had been widely applied in biological science and medical technological products. Compared with mouse monoclonal antibodies, rabbit monoclonal antibodies (RabMAbs) had the advantages of higher binding affinity and specificity, and was easier to be humanized. Transient receptor potential channels melastatin-2 (TRPM2) was a nonselective Ca2+-permeable cation channel. It was regulated by ADPR(Adenosine diphosphate ribose) and H2O2, when TRPM2 was activated by ADPR and H2O2, it could regulate Ca2+ channel, it was related to the pathological disease by energy metabolism repair and oxidative stress. But there were mainly TRPM2 polyclonal antibodies on the market. Objective To prepare TRPM2 RabMAb. Methods In our experiment, we synthesized three peptides of TRPM2, YCA-10987, YCA-10988 and YCA-10989, immuned three experimental rabbits for each peptide. After preliminary screening by serum blot experiment, the splenocytes were fused with rabbit myeloma-like cell line of 240E (Epitomics Inc., USA), then we acquired rabbit-rabbit hybridoma cells which secreted TRPM2 antibodies. The fused cells were maintained in HAT selection medium. To acquire positive clones, ELISA and WB assays were used for screening. The one with the strongest signal of WB was chosen to plasmid recombinant phase. After further screening, the best plasmid pair was used for large-scale transfection (production). Results YCA-10988 immune failed in immunization. YCA-10987 and YCA-10989 displayed different effects in WB application, in which YCA-10987 was detected with a strong signal in over-expression lysate but a weak one in cell lysate, while YCA-10989 was opposite.Conclusion In our experiment, we synthesized three peptides of TRPM2 by hybridoma and recombinant technology. YCA-10987 and YCA-10989 displayed different effects in WB application, but can meet the different test requirements and have potential application prospects.更多还原