【作者】 王黎明；

【导师】 唐波； 华子春；

【作者基本信息】 南京大学 ， 生物化学与分子生物学（专业学位）， 2013， 硕士

【摘要】 肿瘤坏死因子相关的凋亡诱导配体(TNF-related apoptosis-induced ligand, TRAIL)是TNF超家族成员。与以往细胞因子配体相比,TRAIL不仅对多种肿瘤细胞有很强的诱导凋亡作用,而且具有能选择性介导肿瘤细胞凋亡而对大多数正常组织和细胞无损伤的功能。正因为如此,TRAIl已成为肿瘤治疗研究的热点。在TRAIL的研究过程中,高效的单克隆抗体是必不可少的。本论文主要研究了实验室前期构建、纯化得到的抗TRAIL单克隆抗体Abl和Ab2。本论文首先对这两株抗体的基本理化特性进行了鉴定：经SDS-PAGE鉴定可知,两株抗体的相对分子质量为150kd(其中轻链为25kd,重链为55kd)；使用间接ELISA检测表明,Ab1和Ab2腹水的效价为1×10-6；使用Ig亚类ELISA试剂盒测定Abl和Ab2的亚类可知,其Ig亚类为IgGl9(γ)；使用ITC技术对Abl和Ab2的亲和常数检测可知,它们的亲和常数分别为2.61×10-i-M和2.39×10-10M。通过ELISA实验,我们确定所制备的两株都能够特异性地与TRAIL相结合,并且具有较强的中和TRAIL诱导细胞凋亡的能力。上述数据都表明：我们制备的抗TRAIL单克隆抗体具有较高的亲和力、且具有中和TRAIL的性能。本论文还通过噬菌体展示技术确定了与我们所制备TRAIL抗体结合的TRAIL蛋白的抗原决定簇位点。这些为以后TRAIL的检测和临床治疗应用奠定了良好的基础。更多还原

【Abstract】 TRAIL (TNF-related apoptosis-induced ligand) is a new member of TNF superfamily.It contains 281 amino acid residues, and the activity site of TRAIL is the 114-281 amino acid residues which is the extracellular part of this protein (sTRAIL). Similar to other cytokine ligands of TNF superfamily, TRAIL shows a strong capacity to induce tumor cells apoptosis. Moreover, TRAIL could induce most transformed cells apoptosis without inducing most normal cell apoptosis. Thus, TRAIL attracts more and more attentions for cancer therapy. In these research processes of TRAIL, the monoclonal antibody is necessary.In this article, we characterize two anti-TRAILmAbs, named Abland Ab2. Ig subclass and specificity of mAbs were analyzed by ELISA and SDS-PAGE, respectively. The titres of mAbs in ascetic fluid were determined by indirect ELISA. Flow cytometry and MTT test were utilized to analyze the apoptosis-neutralizing activity. The apoptotic function of TRAIL was neutralized significantly by these two anti-TRAIL mAbs detected by flow cytometry and MTT. At last, we determined the epitopes of Abl and Ab2 by Phage display.更多还原