抗第二线粒体凋亡蛋白SmacDiablo单克隆抗体的筛选

摘要:第二线粒体凋亡蛋白 Smac/ Diablo 是近年来新发现的一种线粒体蛋白，Smac/Diablo 可以有效抑制细胞的增值，促进细胞的凋亡，对于治疗肿瘤有显著效果，因此，抗 Smac/Diablo 单克隆抗体的筛选对 Smac/Diablo 蛋白的检测具有重要意义。本实验用 Western Blot 方法对抗 Smac/Diablo 兔单克隆抗体进行初筛和复筛，从众多单克隆抗体中筛选出与蛋白 Smac/Diablo 结合效果最好的一株。在实验中，我们筛选出了 Diablo-R006，用 Diablo-R006 抗体对抗原检测限进行鉴定，结果显示，Smac/Diablo 蛋白含量在 0.0005ug/ul 时即可被检测出来，灵敏度比较高，说明该株抗体与蛋白Smac/Diablo 特异性结合效果比较好，可以用于后续的医药研究及开发。37736
毕业论文关键词：Smac/Diablo；单克隆抗体；Western Blot；凋亡
The Selection of Anti-Smac/Diablo Monoclonal Antibodies
Abstract:The cysteine proteinase activator ( Smac/Diablo) ,which is derived from thesecond mitochondria, is a new mitochondrial protein discovered in recent years.Smac/Diablo has a significant effect in treating tumors as it can inhibit theproliferation and promote the apoptosis of the cell, so the screening of smac/Diablomonoclonal antibodies is is very important for detecting cellular Smac/Diablo protein.This experiment will use Western Blot method to screen and re-screen anti-Smac/Diablo rabbit monoclonal antibodies, to selected from several monoclonal antibodyand protein Smac/Diablo combining effect is the best one. In the experiments, finallywe selected the Diablo-R006，Diablo-R006 combines with Smac/Diablo protein is thebest. Furthermore, we used the Diablo-R006 to identify the Smac/Diablo antigendetection limit and found that the protein can be detected when its concentration was0.005ug/ul and the sensitivity was high. It is concluded that the strain of Smac/Diablospecificity of antibody and protein combined effect is the best, so it can be used forsubsequent pharmaceutical research and development.
Key words: Smac/Diablo; Monoclonal Antibody; Western Blot;Apoptosis
目录
摘要 1
引言 2
1材料与方法.3
1.1材料.3
1.2抗 Diablo 兔单克隆抗体的初筛4
1.3抗 Diablo 兔单克隆抗体的复筛6
1.4抗 Diablo 兔单克隆抗体的抗原检测限7
2结果与分析.8
2.1抗 Diablo 兔单克隆抗体的初筛8
2.2抗 Diablo 兔单克隆抗体的复筛8
2.3抗 Diablo 兔单克隆抗体的抗原检测限9
2.4抗 Diablo 单克隆抗体的细胞裂解实验10
3讨论与结论. 11
参考文献. 12
致谢 13
引言细胞凋亡是主动的由基因决定的自主性细胞有序死亡，对保持机体的自身稳定和正常生长有十分重要的意义[1]。细胞凋亡受抑，将导致细胞生存期延长，使突变的细胞快速繁殖，最终可致肿瘤和多种疾病的产生。例如：增生性瘢痕、胃癌、肾癌、胰腺癌等癌症[2]。细胞凋亡存在内源和外源两条通路，分别称为死亡受体通路和线粒体通路。这两条凋亡途径的起始和终止都需要细胞凋亡蛋白酶（caspase）家族的参与[3]。在细胞受到各种凋亡刺激(包括抗癌药物、紫外线照射、化学信号、DNA 损伤)时，第二线粒体凋亡蛋白 Smac/Diablo(The second mitochondrial activator ofcaspase/direct IAP binding protein with low PI)的线粒体定位信号肽被切除，形成有活性的 Smac/Diablo 蛋白释放到细胞质中，这时的 Smac/Diablo 蛋白可以特异结合细胞凋亡抑制因子（IAP）[4]，解除凋亡因子 IAP 对于 caspase3、caspase9 等的抑制作用，从而促进细胞凋亡。凋亡的线粒体通路是经典的内源性通路，不同的促凋亡信号聚集在线粒体内，诱导线粒体膜通透性的改变，此时线粒体释放可溶的、可促进凋亡的蛋白(intermem branespaces，IMP)[5]。经典的膜间蛋白有细胞色素 C(cytoC)、凋亡诱导因子（Apoptosis Iducing Factor,AIF)等， Smac/Diablo 是新发现的线粒体膜间蛋白，是 cytoC、AIF 一样，是最新发现的线粒体膜间促凋亡蛋白，其促凋亡活性与细胞定位有关[6]。抗第二线粒体凋亡蛋白SmacDiablo单克隆抗体的筛选:http://www.youerw.com/shengwu/lunwen_36581.html