一组miRNA/RNAi通路中关键蛋白-Argo-naute系列多克隆抗体制备、鉴定及组织定位初步研究  

Development and validation of series of antibodies against human argonautes-key protein in the pathway of miRNA/RNAi and localization of Argonautes in human normal and tumor tissue using tissue array

作　　者：王韶英 陈锡美[1] 黄志刚[1] 郜恒骏[1,2] 胡莺[2] 张小燕[2] 孟逊 

机构地区：[1]上海同济大学附属同济医院消化内科,200065 [2]生物芯片上海国家工程研究中心 [3]Dragonfly Sciences

出　　处：《中国实用医药》2009年第6期1-3,共3页China Practical Medicine

基　　金：国家863计划功能基因组与生物芯片重大专项(项目编号:2002AA2Z2021)

摘　　要：目的制备一系列Argonaute(AGO)蛋白多克隆抗体,并鉴定其特异性及其在人类正常及肿瘤组织中的分布。方法合成特异性AGO多肽,以马来酰胺活化匙孔血蓝蛋白(KLH)作为载体构建多肽免疫原,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。结果笔者制备了8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大多数正常及肿瘤组织上皮来源细胞胞浆中呈阳性染色,其中Piwils抗体还可使星状胶质细胞瘤、脑脊膜瘤及胃癌癌细胞染色。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 prepare rabbit polyclonal antibodies against human argonautes（AGOs）,identify their property and investigate the tissue distribution of AGOs using tissue chip. Methods AGO peptides including two sub-classes：resemble Arabidopsis AGO1 and resemble Drosophila Piwi were synthesized using chemical method,and conjugated to Keyhole limpet hemocyanin （KLH） as immunogen. And 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 using affinity chromatography. AGOs were then immune stained on the tissue chip to study their distribution. 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 and tumor tissue,and Piwis also expressed in tumor cells of astrocytoma,meningioma and gastric cancer. Conclusions The successful preparation of the polyclonal antibody against AGOs will provide 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[医药卫生—免疫学]