人PIWIL3特异性抗体的制备和PIWIL3蛋白在肿瘤组织中的分布

Preparation and distribution of polyclonal antibodies against human PIWIL3 protein in tumor tissues

目的:制备人Argonaute家族中PIWIL3蛋白的特异性抗体,并检测其在人多种肿瘤组织中的表达和分布。方法:根据序列同源性和多肽免疫性,利用内部多肽选择数据库选择最佳的多肽免疫原合成多肽,再与KLH结合用于免疫;免疫所得的抗血清经多肽包被的凝胶进行亲和层析纯化,酶联免疫吸附分析技术(ELISA)检测纯化后抗体与多肽的结合能力,Westernblot检测抗体对相应蛋白的结合能力。人肿瘤组织芯片检测该蛋白在多种肿瘤组织的表达和定位。结果:成功制备特异性人PIWIL3蛋白多克隆抗体。ELISA和Westernblot检测均表明该抗体具有很好的结合能力。肿瘤组织芯片检测到PIWIL3蛋白在人星形细胞神经胶质瘤和脑脊膜瘤胞质中表达。结论:应用内部多肽选择数据库可以得到最佳的多肽免疫原,以区分亚家族中其他有高度序列同源性的蛋白,成功制备出纯度和结合能力均较好的特异性人PIWIL3抗体,对研究人类特定肿瘤的发病具有潜在的应用价值。

AIM： To prepare specific antibodies against human PIWIL3 protein of Argonaute family and study its expression and distribution in human tumors. METHODS： To synthesize the polypeptide using the optimal polypeptide immunogens based on sequence homology and peptide immunogenicty in the internal polypeptide selecting database. The synthesized polypeptide were combined with KLH, then immunized the experimental rabbits to produce antiserum. The antiserum was purified by gels custodited the polypeptide using affinity chromatograph, and validated its ability to combine with the corresponding proteins by enzyme-linked immunosorbentassay （ELISA） and Western blot. The expression and distribution of PIWIL-3 protein were detected by human tumor tissue biochips. RESULTS： The specific antibodies against human PiWiL3 proteins were prepared and had high purity. PIWIL3 proteins were expressed in the cytoplasma of human astrocytic glioma and meningioma. CONCLUSION： The specific antibodies against human PIWIL3 protein have been successfully prepared by using the internal polypeptide selecting database to obtain the optimal polypeptide immunogens, which are distinguished from other sequence homology proteins in the subfamily. These antibodies will have potential value for human PIWIL3 protein in human specific tumor pathogenesis.