Chen Hai-Xia, Cao Jiang, Shen Jian-Gen, Zheng Shu
2nd Affiliated Hospital, Zhejiang University College of Medicine, Hangzhou 310009, China.
Sheng Wu Gong Cheng Xue Bao. 2004 Jul;20(4):496-500.
The aim of this study is to develop monoclonal antibody against human hepatocyte growth factor activator inhibitor 1 (HAI-1) for future study of HAI-1. The cDNA fragments of human hepatocyte growth factor activator inhibitor 1 (HAI-1) were subcloned to construct GST-HAI-1 fusion protein expression vectors. The vectors were transformed into E. coli and fusion protein expression was induced by IPTG. The GST-HAI-1 fusion proteins were separated on preparative SDS-PAGE and recovered by electroelution, and used to immunize BALB/c mice. Hybridomas producing monoclonal antibodies against human HAI-1 were prepared by cell fusion technique and characterized by ELISA, Western Blot and immunohistochemical staining. One hybridoma cell line, ZMC6, was obtained, which produces specific antibody against the expressed GST-HAI-1 fusion protein. The monoclonal antibody recognizes both the membrane-type and secretory-type HAI-1 proteins of colorectal tissue. The successful development of anti-HAI-1 antibody provides a powerful tool for further investigation on HAI-1's function.
本研究的目的是开发针对人肝细胞生长因子激活剂抑制剂1（HAI-1）的单克隆抗体，以便未来对HAI-1进行研究。将人肝细胞生长因子激活剂抑制剂1（HAI-1）的cDNA片段进行亚克隆，构建GST-HAI-1融合蛋白表达载体。将载体转化到大肠杆菌中，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导融合蛋白表达。GST-HAI-1融合蛋白在制备性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上分离，通过电洗脱回收，并用于免疫BALB/c小鼠。通过细胞融合技术制备产生抗人HAI-1单克隆抗体的杂交瘤，并通过酶联免疫吸附测定（ELISA）、蛋白质免疫印迹法（Western Blot）和免疫组织化学染色进行鉴定。获得了一个杂交瘤细胞系ZMC6，它产生针对表达的GST-HAI-1融合蛋白的特异性抗体。该单克隆抗体可识别结肠直肠组织的膜型和分泌型HAI-1蛋白。抗HAI-1抗体的成功开发为进一步研究HAI-1的功能提供了有力工具。
