兔抗人DR5分子多克隆抗体的制备与初步应用  

Generation and preliminary application of rabbit antihuman polyclonal antibodies against DR5

作　　者：白慧玲[1] 裴景堂[2] 杜耀武[1] 李淑莲[1] 刘广超[1] 马远方[1] 

机构地区：[1]河南大学免疫学研究所河南省天然药物与免疫工程重点实验室,河南开封475004 [2]河南大学附属淮河医院,河南开封475004

出　　处：《免疫学杂志》2008年第6期634-637,共4页Immunological Journal

基　　金：河南省教育厅自然科学基础研究(200510475039);国家自然科学基金项目(30571697)

摘　　要：目的获得兔抗人DR5的多克隆抗体,并对多克隆抗体进行初步鉴定。方法皮下注射DR5免疫日本大耳白兔,加强免疫得到抗血清,应用蛋白G纯化获得多克隆抗体。对纯化的抗体进行ELISA、免疫印迹、细胞凋亡活性等初步鉴定。结果经纯化后的抗体效价为1∶32000;Western blot分析该抗体能够识别相对分子质量26000的sDR5;可以明显导致肿瘤细胞Jurkat凋亡,Western blot检测到随着抗体诱导时间的延长Jurkat细胞Bcl-2蛋白表达减少,Cyt-C、Bax、有活性的caspase3和caspase9蛋白的表达增加。结论所制备的多克隆抗体可应用于ELISA、免疫印迹和诱导肿瘤细胞凋亡活性及机制等实验,为确定DR5分子的组织及细胞分布和定位等提供了有力的工具。Objective To generate and identify the rabbit polyclonal antibodies aganst sDR5. Methods Rabbits were immunized with sDR5 for 3 times. The polyclonal antibodies from the collected antiserum were purified with protein G. Specialty of the polyclonal antibodies was analyzed by indirect-ELISA; apoptotic effects of the polyclonal antibodies on Jurkat cells were detected by FCM analysis; Bcl-2, CytC, caspase3, Bax, and caspase9 were detected with Western blotting. Results The titers of purified polyclonal antibodies were 1 ： 32 000. Western blot results showed that the antibodies could recognize the protein with molecular weight of 26 000 （sDR5）. Apoptosis of Jurkat ceils treated by the polyclonal antibodies was increased obviously. Specificity of Bcl 2, CytC, caspase3, Bax, and caspase9 were detected, and they changed with the time of polyclonal antibodies treatment. Conclusion Polyclonal antibodies against sDR5 has been prepared successfully. The polyclonal antibodies may be a useful tool in investigating tumor＇ s therapy by using DR5 as target molecule and exploring the functional domain of DR5.

关 键 词：DR5 多克隆抗体 凋亡 

分 类 号：R392.1[医药卫生—免疫学]