利用序列特异性DNA亲和层析分离纯化一种前列腺细胞核蛋白  

SEPARATION AND PURIFICATION OF A NUCLEAR PROTEIN OF PROSTATE CELLS BY DNA AFFINITY CHROMATOGRAPHY

作　　者：陈蔚文[1] 张建业[1] 赵健[2] 张莲英[1] 陈留存[1] 张孟业[1] 

机构地区：[1]山东大学医学院生物化学与分子生物学研究所 [2]山东大学齐鲁医院胸外科

出　　处：《山东医科大学学报》2001年第6期491-494,共4页Acta Academiae Medicinae Shandong

基　　金：国家自然科学基金资助课题 ( 39970 80 2 )

摘　　要：目的 :分离纯化RFA结合蛋白并加以鉴定 ,为进一步深入研究RFA及其结合蛋白相互作用和参与AR介导的PSA基因表达奠定基础。方法 :培养人前列腺癌细胞PC 3,提取核蛋白 ,以RFA序列为探针进行亲和层析分离纯化目的蛋白 ,层析前后均用电泳迁移率变动分析 (EMSA)检测目的蛋白的功能。经SDS PAGE测定纯化蛋白的分子量 ,然后切下目的蛋白条带 ,进行蛋白质鉴定。结果 :纯化蛋白在SDS PAGE图谱上 36kD附近呈现单一条带 ,氨基酸组成分析和质谱分析显示纯化蛋白条带含有 2种蛋白 ,与核内不均一核糖核蛋白A1,A2 (hnRNPA1,A2 )高度同源。结论 :①以DynalM 2 80链亲和素磁珠为固相介质的DNA亲和层析 ,具有简便、快速、无毒的优点 ,可在对目的蛋白了解甚少的条件下 ,以较高的纯度将其纯化 ;②RFA结合蛋白与hnRNP超家族成员hnRNPA1,A2高度同源 ,其与RFA相互作用的具体机制和意义有待进一步研究。Objective:To separate and purify the RFA binding protein and identify the protein, which will facilitate the further study of RFA protein interaction and its function in AR mediated expression of PSA gene. Methods:The human prostate cancer cell line PC 3 was incubated in vitro to extract the nuclear proteins, and from which the RFA binding protein was purified by DNA affinity chromatography using RFA as probe. Before and after chromatography, the function of the RFA binding protein was detected by electrophoretic mobility shift assay (EMSA). After determining the molecular weight of the purified protein by SDS PAGE, the band was excised from gel for protein identification.Results:Amino acid sequencing and mass spectrometric analysis indicated that the band contained two proteins, which possessed sequences closely homologous to heterogeneous nuclear ribonucleoprotein A1, A2 (hnRNP A1, A2). Conclusion:①The method using Dynal M 280 magnetic particles coated with streptavidin for affinity chromatography has many advantages such as convenient, fast and nontoxic, which can purify a protein without knowing it in detail;②The RFA binding proteins are closely homologous to hnRNP A1, A2, which are the components of hnRNP superfamily, so the detail mechanism and the significance of RFA interaction with its binding protein is still waiting further study.

关 键 词：亲和层析 核糖核蛋白 前列腺特异抗原 前列腺癌 诊断 治疗 

分 类 号：R737.14[医药卫生—肿瘤]