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本研究利用RT-PCR的方法扩增出人ICF45基因片段并将其构建至原核表达载体p ET-30a,随后将此质粒转化,用IPTG诱导大量表达ICF45蛋白。利用纯化的ICF45蛋白免疫BALB/c小鼠后,取其脾细胞与骨髓瘤细胞SP2/0进行融合,采用间接酶联免疫ELISA筛选阳性克隆,最终获得了2株分泌ICF45抗体的杂交瘤细胞。Western Blot、免疫荧光和免疫组化结果均显示其分泌的抗体能特异性识别ICF45蛋白,并且该抗体灵敏度高、特异性强。本研究获得的ICF45单克隆抗体,为深入阐明ICF45在人类细胞周期调控和肿瘤发生发展中的作用机制提供了重要的工具。
In this study, human ICF45 gene fragment was amplified by RT-PCR and cloned into prokaryotic expression vector p ET-30a. Subsequently, this plasmid was transformed into IPTG to induce large expression of ICF45 protein. BALB / c mice were immunized with purified ICF45 protein. The spleen cells were fused with myeloma cells SP2 / 0. The positive clones were screened by indirect enzyme-linked immunosorbent ELISA. Two hybridoma cells secreting ICF45 antibody were obtained. Western Blot, immunofluorescence and immunohistochemistry results showed that the secreted antibodies could specifically recognize ICF45 protein, and the antibody was highly sensitive and specific. The ICF45 monoclonal antibody obtained in this study provides an important tool for further elucidating the mechanism of action of ICF45 in the regulation of human cell cycle and tumorigenesis.