摘要
目的:制备TM-TNF-α特异性单克隆抗体,并进行鉴定和初步功能分析。方法:用表位预测软件选定TM-TNF-α特异的20个氨基酸多肽,偶联载体蛋白,免疫BALB/c小鼠;用杂交瘤技术制备单克隆抗体。采用ELISA、流式细胞术和Western blot鉴定单抗的特异性,观察单抗对Jurkat细胞在AICD模型中凋亡率的影响。结果:成功制备了6株抗TM-TNF-α的单克隆抗体。ELISA结果显示6株单抗均特异性结合免疫原多肽,与S-TNF-α等细胞因子无交叉反应;流式细胞术显示6株单抗均能结合细胞膜表面TM-TNF-α分子,并可区分人源和鼠源TM-TNF-α;Western blot结果证实6株单抗只能识别26kD的人TM-TNF-α,而不识别17kD的S-TNF-α。在AICD模型中,单克隆抗体不影响Jurkat细胞凋亡率。结论:成功制备了6株TM-TNF-α特异性单克隆抗体,且单抗不干扰TNF-α与TNFR结合,为进一步研究TM-TNF-α在相关疾病中的作用提供又一研究手段。
Objective：To prepare TM-TNF-α specific monoclonal antibody, identify their specificity and function. Methods：Using antigen anticipation software, the TM-TNF-α segment composing of 20 amino acids residues as antigenic peptide was determined, conjugated with carrier protein and immunized to BALB/c mice. Hyhridoma technique was used to prepare monoclonal antibody. ELISA, flow cytometry and Western blot were performed to identify their specificity. The effect of monoclonal antibody on the apoptosis rate of Jurkat cells was observed in the model of AICD. Results：6 monoclonal antibodies against TM-TNF-α were successfully prepared. The results of ELISA revealed that 6 monoclonal antibodies could all specifically interact with the peptide, but not cross-react with S-TNF-α and other cytokines. It was confirmed by using flow cytometry that 6 monoclonal antibodies could all combine to TM- TNF-α on cell surface and could discriminate human TM-TNF-α from mouse TM-TNF-α. Furthermore, these 6 monoclonal antibodies were found by Western blot to prefer interaction with 26 kD TM-TNF-α rather than 17 kD S-TNF-α. In the model of AICD, monoclonal antibodies were shown no effect on the apoptosis rate of Jurkat cells. Conclusion ：6 of hTM-TNF-α specific monoclonal antibodies have been successfully prepared and shown no interference with the binding of TNF-α and TNF receptor, which may he a useful tool for further explore the roles of TM-TNF-α in related diseases.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2007年第6期488-491,共4页
Chinese Journal of Immunology
基金
国家自然科学基金资助(批准号:30200257)
