抗人线粒体核糖体小亚基蛋白17单克隆抗体的制备和鉴定

以纯化人线粒体核糖体小亚基蛋白17(MRPS17)免疫BALB/c小鼠,经细胞融合和ELISA法筛选成功获得1株抗MRPS17杂交瘤细胞。以所获特异性单抗作为一抗,使用Western印迹、免疫组化和免疫荧光等方法检测标本中MRPS17。结果显示:Western印迹检测人骨骼肌组织、黑素瘤组织和体外培养HeLa细胞提取蛋白质,在分子量约13kDa处有一特异性条带,与阳性对照纯化MRPS17相一致;免疫组化检测石蜡切片标本显示人骨骼肌细胞和恶性黑素瘤细胞胞浆中强阳性着色;细胞免疫荧光检测于培养的HeLa细胞,可见细胞核周围胞浆部位颗粒状绿色荧光,其分布与线粒体特异性荧光探针(MitoTrackerRedCM-H2XRos)的荧光分布一致。说明成功制备了具有高度特异性并可适用于多种检测方法的抗人MRPS17单抗,应用该单克隆抗体对人MRPS17进行了亚细胞水平定位,为线粒体生物学相关研究提供了新的研究工具。

Preparation and Characterization of Monoclonal Antibody against Human Mitochondrial Ribosomal Protein S17

BALB/c mice were immunized with purified mitochondrial ribosomal protein S17(MRPS17).Spleen cells were fused with SP2/0 cells using polyethylene glycol and hybridoma cells were selected by HATmedium and screened with ELISA. After rounds of cloning with limited dilution method, one hybridoma cell line wasobtained which stably secret monoclonal antibody of IgG2a. The obtained antibody was used for samples assay byWestern blot, immunohistochemical and immunofluorescence staining. Western blot showed that the antibody spe-cifically recognized about 13 kDa protein. Immunohistochemical staining showed strong positive signals locatedin the cytoplasm of muscle cells and melanoma cells. Immunofluorescence staining showed green fluorescencemainly in the formation of a spotted distribution inside the cytoplasm of HeLa cells, which was identical to thedistribution of mitochondria shown by mitochondrion-selective stains (MitoTracker Red CM-H2XRos). These re-sults indicated that a MRPS17 monoclonal antibody with high specificity was developed, which could be used inWestern blot, immunohistochemical and immunofluorescent techniques.