兔抗人Argonaute2蛋白多克隆抗体制备及鉴定

Preparation and characterization of the rabbit anti-human polyclonal antibody against argonaute 2 protein and its location in tumor tissues

为了制备兔抗人Argonaute_2(AGO_2)的多克隆抗体,并鉴定其特异性,应用组织芯片初步探讨其在人类正常及肿瘤组织中的分布。采用合成特异性AGO_2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,通过致敏大白兔,制备兔抗人AGO_2多克隆抗体,然后用亲和层析法纯化抗体。用ELISA和Western blot方法进行抗体验证,并应用人组织芯片进行AGO_2的免疫组化研究。结果显示,通过构建AGO_2多肽-KLH载体复合物致敏大白兔,我们制备了兔抗人AGO_2蛋白多克隆抗体,经ELISA及Western blot证实兔抗人AGO_2抗体可特异性识别AGO_2多肽,在人组织芯片中通过免疫组化染色显示该抗体在很多正常组织上皮细胞及肿瘤细胞胞浆中呈阳性染色。成功制备兔抗人AGO_2多克隆抗体,为进一步研究AGO_2在微小RNA/RNA干扰通路中的作用及在人类疾病中的意义提供了有利工具。

To prepare rabbit polyclonal antibody against human argonaute 2（AGO2） protein, and investigate the tissue distribution of AGO2 using tissue chip. The AGO2 peptide was synthesized using chemical method, and conjugated to keyhole limpet hemocyanin （KLH） as immunogen. Then the AGO2 KLH conjugate was injected into rabbits subcutaneously to produce polyclonal antibodies. The specificity and sensitivity of antibodies were identified by ELISA and Western blot assay after purification using affinity chromatography. AGO2 was then stained on the tissue chip to study its distribution. Rabbit antibodies a gainst AGO2 were prepared after injection with AGO2-KLH conjugate. These antibodies were confirmed to specially recognize AGO2 peptides analyzed by ELISA and Western blot assay. AGO2 protein was stained positively in the cytoplasm of tumor cells and epithelial cells in many normal tissues. It is evident that the successful preparation of the polyclonal antibody against AGO2 protein will provide an efficient reagent for the further study of its role in the miRNA and RNA interference and in the patho genesis of human disease.