肝癌生物标志物SOAT1单克隆抗体的制备及评估

Preparation and Evaluation of Monoclonal Antibody to SOAT1, a New Biomarker for Liver Cancer

摘要

肝癌是临床上一种常见的恶性肿瘤, 目前临床上仍缺乏特异有效的诊断标志物。有研究表明, 固醇O-酰基转移酶1 (sterol O-acyltransferase 1, SOAT1)作为胆固醇代谢的关键酶, 与肝癌的发生发展密切相关。因此, SOAT1在作为肝癌生物标志物方面具有巨大潜力。本研究从肝癌细胞Huh-7中获取SOAT1基因, 经原核表达、蛋白质纯化后, 以纯化的重组SOAT1蛋白作为抗原免疫小鼠, 利用杂交瘤等技术共获得5株SOAT1单克隆抗体细胞株, 分别命名为1F3、1G3、1D6、2F8、4D11, 其中4D11能够识别肝癌细胞Huh-7及临床肝癌组织标本中的SOAT1蛋白; 最后, 将4D11送至生物公司进行测序, 获得该抗体可变区的氨基酸序列。本研究利用重组SOAT1蛋白成功制备了抗SOAT1的特异性单克隆抗体, 这不仅为后续应用SOAT1蛋白制备肝癌伴随诊断试剂盒提供了原材料, 而且还为建立基于SOAT1的肝癌诊断方法奠定了基础。

Abstract

Liver cancer is a common malignant tumor in clinic. Studies have shown that sterol O-acyltrans-ferase 1 (SOAT1), a key enzyme in cholesterol metabolism, is closely related to the occurrence and develop-ment of liver cancer. Therefore, SOAT1 has great potential as a biomarker for the cancer. Herein, the gene encoding SOAT1 was obtained from the Huh-7 liver cancer cell line. After prokaryotic expression and pro-tein purification, the purified recombinant SOAT1 protein was used as an antigen to immunize mice. A total of 5 anti-SOAT1 monoclonal antibody cell lines, named 1F3, 1G3, 1D6, 2F8 and 4D11, respectively, were obtained by using hybridoma and other technologies. Among them, 4D11 was able to recognize the SOAT1 protein in Huh-7 cells and clinical liver cancer tissue samples. Subsequently, 4D11 was sent to a biological company for sequencing, and the amino acid sequences of the antibody variable regions were obtained. In conclusion, an anti-SOAT1 monoclonal antibody was successfully prepared by recombinant SOAT1 protein, which would not only provide the raw materials for the subsequent application of SOAT1 protein in develo-ping liver cancer companion diagnostic kits, but also lay a foundation for the establishment of SOAT1-based liver cancer diagnostic methods.

关键词

肝癌 / 固醇O-酰基转移酶1 (SOAT1) / 单克隆抗体

Key words

liver cancer / sterol O-acyltransferase 1 (SOAT1) / monoclonal antibody