摘要
为制备Cancertestis55(CT55)单克隆抗体,需构建带有人源CT55片段的原核表达质粒,把该质粒转化Rosetta感受态进行原核表达并得到目的蛋白,蛋白质被纯化后免疫6周雌性BALB/c小鼠。按传统的单克隆抗体的制备方法,取小鼠脾细胞与骨髓瘤细胞(sp2/0)进行融合,经ELISA方法筛选及两次连续亚克隆,共获得多株能稳定分泌抗CT55蛋白单克隆抗体的杂交瘤细胞,如3D8B7B12、4C8E1C9、3D8C10G9等。ELISA及Westernblot(WB)分析结果表明,筛选的细胞株均能产生单克隆抗体,且该抗体均分别能与原核表达及真核表达的CT55蛋白发生特异性结合。单克隆抗体可用于免疫荧光试验,且与P53发生互作的荧光主要位于细胞核边缘。结果表明,成功制备了针对人源CT55蛋白的单克隆抗体。CT55蛋白单克隆抗体的制备为今后肝癌、胃癌、结肠癌等癌症的快速的病原学诊断以及CT55蛋白的结构和功能研究奠定了物质基础。
In order to prepare the monoclonal antibody of Cancer testis 55( CT55),the prokaryotic expression plasmid with human CT55 fragment was constructed first,and the plasmid was expressed in the Rosetta competent cell. The target protein was purified and immunized for 6 weeks young female BALB/c mice.According to the preparation method of the conventional monoclonal antibody,the spleen and myeloma cells( sp2/0) of mice was prepared to fuse together. The hybridoma cell lines were obtained by indirect ELISA method and 2 successive subclones,such as 3D8 B7B12,4C8E1C9 and 3D8C10G9. The results of ELISA and Western blot analysis showed that the screened cell lines could produce monoclonal antibodies,and the antibody could specifically recognize the prokaryotic expression and eukaryotic expression of CT55 protein. Specific antibodies can be used in immunofluorescence test,and fluorescence is mainly located at the edge of nucleus.The results showed that monoclonal antibodies against human CT55 protein were successfully prepared. The preparation of CT55 protein monoclonal antibody has laid the material foundation for the rapid etiological diagnosis of cancer of liver cancer,gastric cancer and colon cancer,and the structure and function of CT55 protein.
作者
任建委
李军
李尚泽
REN Jian-wei;LI Jun;LI Shang-ze(Tibet University Medical College,Lasha 85000,China;ABclonal,Wuhan430072,China;Zhongnan Hospital of Wuhan University,Wuhan 430071,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2018年第11期1-8,共8页
China Biotechnology
基金
武汉大学青年教师(2042018kf0076)资助项目
关键词
CT55
原核表达
单克隆抗体
CT55
Prokaryotic expression
Monoclonal antibody
