PIWIL系列蛋白多克隆抗体制备、鉴定及组织分布

Preparation,identification and tissue localization of PIWIL proteins

目的制备PIWIL蛋白多克隆抗体,鉴定其特异性,初步探讨其在人类正常及肿瘤组织中的分布。方法合成特异性PIWIL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PIWIL多克隆抗体,用亲和层析法纯化抗体。用ELISA和Western blot方法进行抗体验证,应用人组织芯片进行PIWIL免疫组化研究。结果通过构建系列PIWIL多肽-KLH载体复合物致敏大白兔,成功制备4个兔抗人PIWIL蛋白多克隆抗体,证实兔抗人PIWIL抗体可特异性识别PIWIL多肽,该抗体在大多数正常及肿瘤组织上皮来源细胞胞质中呈阳性染色。结论成功制备系列兔抗人PIWIL多克隆抗体,为进一步研究PIWIL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 polyclonal antibodies against human PIWIL and to identify their property and tissue distribution of PIWIL. Methods PIWIL polypeptide was synthesized and conjugated to Keyhole limpet hemocyanin （KLH） as an immunogen. Then PIWIL-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 affinity chromatography. PIWIL were then stained on the tissue chip to study their distribution. Results Rabbit antibodies against PIWIL were prepared after injection of PIWIL-KLH conjugation. These antibodies specially recognized PIWIL peptides. Expression of PIWIL was found in the cytoplasm of epithelia cells of varied normal tissues and tumor tissues. Conclusion The successful preparation of the polyclonal antibody against PIWIL 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.