Huyan Ting, Yin Dachuan, Wang Wei, Song Kai, Wang Yan, Lu Huimeng, Yang Hui, Xue Xiaoping
Faculty of Life Science, Northwestern Polytechnical University, Xi'an 710072, China.
Sheng Wu Gong Cheng Xue Bao. 2011 Apr;27(4):659-66.
We investigated the mechanism of human aspartyl beta-hydroxylase (HAAH) in early diagnosis of tumors. The encoding gene of HAAH was cloned from the hepatic carcinoma by RT-PCR and expressed as a fused protein in the prokaryotic vector pBV-IL1. The expressed HAAH was purified by Ni(2+)-NTA purification column and the purified protein was then used to immunize Balb/c mice. Three hybridoma cell lines (respectively designated H3/E10, E4/F12 and G4/D8) stably expressing the monoclonal antibody specific to HAAH fusion protein were obtained. The specificity and sensitivity of the monoclonal antibody were assessed by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Finally, the monoclonal antibody expressed by H3/E10 cell line was used to detect the expression of HAAH in several tumor cell lines by indirect immuno-fluorescence, and the specific fluorescence was observed. In conclusion, this study successfully constructed the recombinant prokaryotic vector pBV-IL1-HAAH and prepared HAAH-specific monoclonal antibody for further study of the structure and function of the protein. The result may also lay solid foundation for the research of the molecular mechanism of HAAH in early diagnosis of tumors.
我们研究了人天冬氨酰β-羟化酶（HAAH）在肿瘤早期诊断中的机制。通过逆转录聚合酶链反应（RT-PCR）从肝癌组织中克隆出HAAH的编码基因，并在原核表达载体pBV-IL1中表达为融合蛋白。表达的HAAH经镍离子-亚氨基二乙酸（Ni(2+)-NTA）纯化柱纯化，然后用纯化后的蛋白免疫Balb/c小鼠。获得了三株稳定分泌特异性抗HAAH融合蛋白单克隆抗体的杂交瘤细胞系（分别命名为H3/E10、E4/F12和G4/D8）。通过间接酶联免疫吸附测定（ELISA）和蛋白质免疫印迹分析（Western blot）评估单克隆抗体的特异性和敏感性。最后，用H3/E10细胞系分泌的单克隆抗体通过间接免疫荧光法检测几种肿瘤细胞系中HAAH的表达，观察到特异性荧光。综上所述，本研究成功构建了重组原核表达载体pBV-IL1-HAAH并制备了HAAH特异性单克隆抗体，为进一步研究该蛋白的结构和功能奠定了基础，该结果也可能为HAAH在肿瘤早期诊断中的分子机制研究奠定坚实基础。
