人类天冬胺酰基β-羟化酶的表达及其单克隆抗体的制备

Expression of human aspartyl β-hydroxylase and preparation of its monoclonal antibody

旨在深入研究人类天冬胺酰基β-羟化酶(HAAH)在肿瘤早期诊断中的作用机制。应用RT-PCR方法从肝癌组织中获得人类天冬胺酰基β-羟化酶HAAH编码基因,并在原核表达载体pBV-IL1中进行融合表达,将Ni柱纯化的融合蛋白免疫Balb/c小鼠,获得了3株稳定的阳性单克隆细胞株(H3/E10、E4/F12、G4/D8),间接ELISA和Western blotting鉴定单抗的特异性和灵敏度,以单抗H3/E10介导的间接免疫荧光检测HAAH在各种肿瘤细胞系中的表达,可见特异的荧光。试验中成功构建了可表达HAAH基因的原核表达载体pBV-IL1-HAAH,并制备了3株抗HAAH的单克隆抗体,为HAAH的结构和功能的研究奠定了基础,同时也为进一步将该抗体应用于肿瘤的早期诊断以及研究HAAH在肿瘤发生转移中的机理提供了重要工具。

We investigated the mechanism of human aspartyl β-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 Ni2＋-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.