兔抗人PIWIL4蛋白多克隆抗体的制备和鉴定

目的 制备兔抗人PIWIL4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.方法 合成特异性PIWIL 4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PIWIL4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PIWIL4的免疫组化研究.结果 通过构建P IWIL4多肽-KLH载体复合物致敏大白兔,我们制备了兔抗人PIWIL4蛋白多克隆抗体,经ELIS A 及Western blot证实兔抗人PIWIL4抗体可特异性识别PIWIL4多肽,在人组织芯片中通过免疫组化染色显示该抗体在BU部分正常组织和肿瘤组织中呈阳性染色.结论 本项研究成功制备兔抗人PIWIL4多克隆抗体,为进一步研究PIWIL4在人类疾病中的意义提供了有利工具.

Preparation and identification of rabbit antibody against human PIWIL4 protein

Objective To prepare rabbit polyclonal antibody against human PIWIL4 protein,identify the properties of PIWIL4,and investigate the distribution of PIWIL4 in tissues using tissue chip.Methods PIWIL4 peptide was synthesized using chemical method,and conjugated to Keyhole limpet hemocyanin(KLH)as immunogen.And then PIWIL4-KLH conjugation was injected into rabbits subcutaneously to produce polyclonal antibodies.The specificity and sensitivity of the antibodies were identified by ELISA and Western blotting after purification using affinity chromatography.The tissue chip was used to study the distribution of PIWIL4.Results Rabbit antibodies against PIWIL4 were prepared after injection of PIWIL4-KLH conjugation.These antibodies could specially recognize PIWIL4 peptides confirmed to by ELISA and Western blotting.PIWIL4 protein was stained positively in some normal and tumor tissue.Conclusion The successful preparation of the polyclonal antibody against PIWIL4 protein will provide an efficient reagent for further study of PIWIL4 role in the pathogenesis of human disease.