抗人骨桥蛋白单克隆抗体制备及其特异性研究  

Preparation of Monoclonal Antibody Against Human Ospteopontin And Analysis of Its Specifity

作　　者：姚辉[1,2] 聂新民 黄作良[2] 

机构地区：[1]湘雅附三医院检验科,湖南长沙410000 [2]邵阳医学高等专科学校附属医院检验科,湖南邵阳422000

出　　处：《邵阳学院学报（自然科学版）》2010年第2期58-63,共6页Journal of Shaoyang University：Natural Science Edition

摘　　要：利用RT-PCR技术克隆人骨桥蛋白(hOPN)基因,构建OPN原核表达质粒pET-32a(+)-hOPN,转化BL21菌株,经IPTG诱导表达重组人骨桥蛋白(rhOPN).以纯化的rhOPN为免疫原,免疫BABL/c小鼠,取其脾细胞与小鼠骨髓瘤细胞NS1融合.通过有限稀释法进行克隆和间接ELISA筛选,获得抗人OPN蛋白单克隆抗体杂交瘤细胞株,以ELISA、Western blot对抗体特异性进行鉴定.通过竞争抑制试验对单克隆抗体识别抗原位点进行分析.结果共获得2株抗人OPN单克隆抗体,分别命名为8F1和2B5,亚型测定皆为IgG1.通过细胞侵袭抑制试验检测,2株抗人OPN mAb皆能很好地抑制细胞迁移.本研究成功获得了抗人骨桥蛋白的特异性单克隆抗体,为进一步研究OPN蛋白在自身免疫病和肿瘤中的功能提供了重要的工具.BABL/c mice were immunized with recombinant human ospteopontin（rhOPN） and splenocytes of the immunized mice were fused with myeloma NS1 cells to produce hybridoma cell lines.ELISA assay was used to identify the monoclonal antibody against ospteopontin protein and western blotting were applied to identify the specificity of this antibody.It was demonstrated that 2 monoclonal antibodies recognizing 2 different epitopes on OPN were obtained.These antibodies showed specific binding power to t rhOPN.The subtype of 2 antibodies was proved to be IgG1.These antibodies showed specific binding activity to rhOPN.It is evident that monoclonal antibodies against OPN-mediated cells migration.these results may provide a solid basis for the further studies on the biological characteristics of ospteopontin.

关 键 词：骨桥蛋白 原核表达 单克隆抗体 

分 类 号：Q51[生物学—生物化学]