PC-1基因功能研究:PC-1蛋白兔多克隆抗体制备的改进和应用  

Function of PC-1 gene: Improvement and application of the preparation of polyclonal antibody against PC-1 protein in rabbits

作　　者：高雪松[1] 张慧[1] 孙玉龙[1] 周立权[2] 梁瑞霞[1] 陈伟[1] 洪宝发[2] 周建光[1] 

机构地区：[1]解放军军事医学科学院生物工程研究所,北京市100850 [2]解放军军医进修学院泌尿外科,北京市100853

出　　处：《中国临床康复》2005年第43期70-71,i0001,共3页Chinese Journal of Clinical Rehabilitation

摘　　要：目的:获得高效价PC-1蛋白的兔多克隆抗体,用于PC-1功能的实验。方法:实验于2004-02/08在解放军军事医学科学院生物工程研究所完成。以纯化后的PC-1蛋白及其N端46个氨基酸与GST的融合蛋白(GST-PC-1和GST-PC-1-46)为抗原,通过一种经淋巴结注射、免疫家兔的改进的免疫方法制备兔多克隆抗体。应用ProteinA亲和层析纯化抗体。以酶联免疫吸附和Western-blot及免疫组化法鉴定所得抗体。结果:①2个兔多克隆抗体的效价分别为1∶12800和1∶51200。②PC-1在高恶性前列腺癌细胞C4-2中高表达,在低恶性前列腺癌细胞LNCaP中有少量表达。③经免疫组化染色后,含有PC-1蛋白的C4-2及LNCaP细胞均呈阳性反应,细胞质中可见明显着色,而空泡状的胞核则没有着色,提示PC-1蛋白主要分布在细胞浆中。结论:用一种改进的免疫方法,获得高效价的、可应用于酶联免疫吸附和Western-blot及免疫组化实验的PC-1蛋白兔多克隆抗体。AIM： To produce high titer PC-1 protein rabbit polyclonal antibody and explore PC-1 function. METHODS： The experiment was carried out in the Chinese PLA Academy of Military Medical Sciences between February and August 2004. Rabbit polyclonal antibody were produced by utilizing GST fusion proteins of PC-1 Protein and its N-terminal 46 amino acids （GST- PC-1 and GST-PC- 1-46）, as antigen and an improved immune method. The antisera were purified with protein A affinity chromatography. PC-1 Protein expression was detected by enzyme-linked immunoabsordent assay （ELISA）, Western blot and immunohistochemical method. RESULTS： （1） The titers of two rabbits＇ polyclonal antibody were 1：12 800 and 1：51 200. （2） PC-1 was highly expressed in high malignant prostate cancer cell C4-2, but only expressed by a small amount in low malignant prostate cancer cell LNCaP. （3） After immunohistochemical staining, the C4-2 and LNCaP cells containing PC-1 protein showed positive reaction, obvious pigmenting could be observed in cytoplasm, but no pigmenting was observed in vacuole-like nuclei, indicating that PC-1 protein mainly distributed in cytoplasm. CONCLUSION： By means of improved immunological method, the PC-1 protein rabbit polyclonal antibodies have been obtained, which can be used in ELISA analysis, Western blot and immunohistochemical experiments.

关 键 词：前列腺肿瘤 兔 免疫法 

分 类 号：R394.33[医药卫生—医学遗传学]