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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 a series of polyclonal antibodies against Argonaute (AGO) protein and identify its specificity and its distribution in human normal and tumor tissues. Methods The specific AGO polypeptide was synthesized and the immunogen of polypeptide was screened by using MAH-conjugated keyhole limpet hemocyanin (KLH) as carrier. The rabbit was sensitized to prepare a series of rabbit polyclonal antibody against human AGO and then purified by affinity chromatography antibody. Antibody validation was performed by ELISA and Western blotting, and immunohistochemistry of AGO was performed using human tissue microarray. Results The authors prepared eight rabbit anti-human AGO protein polyclonal antibody, confirmed by ELISA and Western blot rabbit anti-human AGO antibody can be specifically identified AGO polypeptide in human tissue microarray by immunohistochemical staining of the antibody in most normal And tumor tissue epithelial cells derived from the cytoplasm positive staining, in which Piwils antibodies can also make astrocytoma, meningioma and gastric cancer cells staining. Conclusion This study successfully prepared a series of rabbit anti-human AGO polyclonal antibodies, which provided a favorable tool for further study of the role of AGO in miRNA / RNAi pathway and its significance in human diseases.