Argonaute系列蛋白多克隆抗体制备和鉴定及组织定位  

Preparation,identification and tissue localization of polyclonal antibodies against argonaute proteins

作　　者：卢峪霞[1] 黄志刚[1] 陈锡美[1] 郜恒骏[1,2] 胡莺[2] 张小燕[2] 

机构地区：[1]同济大学附属同济医院消化内科,上海200065 [2]生物芯片上海国家工程研究中心,上海201203

出　　处：《同济大学学报（医学版）》2009年第1期23-26,31,共5页Journal of Tongji University(Medical Science)

基　　金：国家863计划功能基因组与生物芯片重大专项基金资助项目(2002AA2Z2021)

摘　　要：目的制备一系列Argonaute(简称AGO)蛋白多克隆抗体,并鉴定其特异性,应用组织芯片初步探讨其在人类组织中的分布。方法合成特异性AGO多肽,以马来酰胺活化匙孔血蓝蛋白(keyhole limpet hemocyanin,KLH)作为载体构建多肽免疫原,通过致敏大白兔,制备系列兔抗人AGO多克隆抗体,然后用亲和层析法纯化抗体。用ELISA和western方法进行抗体验证,并应用人组织芯片进行AGO的免疫组化研究。结果通过构建系列AGO多肽-KLH载体复合物致敏大白兔,成功制备了3个兔抗人AGO蛋白多克隆抗体,经ELISA及Western blot证实兔抗人AGO抗体可特异性识别AGO多肽,在人组织芯片中通过免疫组化染色显示该抗体在部分肿瘤组织上皮来源细胞胞浆中呈阳性染色。结论本项研究成功制备系列兔抗人AGO多克隆抗体,为进一步研究AGO在miRNA/RNAi通路中的作用及在人类疾病中的意义提供了有利工具。Objective To study the preparation and identification of polyclonal antibodies against human argonautes （AGOs）, and to investigate the distribution of AGOs in tissues by tissue chip. Methods AGOs polypeptide was synthesized by chemical method, and conjugated to Keyhole limpet hemocyanin （KLH） as immunogen. Then AGO-KLH conjugations were injected into rabbits subcutaneously to produce polyclonal antibodies. The specificity and sensitivity of antibodies were identified by ELISA and Western blot after purification by using affinity chromatography. AGOs were then immunologically stained on the tissue chip to study their distribution by immunohistochemistry. Results Rabbit antibodies against AGOs were prepared after injection of AGO-KLH conjugation. These antibodies were confirmed to specially recognize AGO peptides analyzed by ELISA and Western blot. Expression of AGOs was found in the cytoplasm of epithelia cells of varied normal tissue. Conclusion The successful preparation of polyclonal antibody against AGOs will provide an efficient reagent for further study of its role in the pathway of miRNA and RNA interference and in the pathogenesis of human disease.

关 键 词：Argonaute蛋白 多克隆抗体 组织芯片 

分 类 号：R392-33[医药卫生—免疫学] R996[医药卫生—基础医学]