Purification and characterization of biologically active recombinant human Eppin expressed in Escherichia coli

作     者：ZHU Qing-yi GU Xiao-jian YANG Jin WANG Jun-hong TANG Bo WU Hong-fei 

作者机构：Department ot Urology First Affiliated Hospital of Nanjing Medical University Nanjing Jiangsu 210029 China Department of Urology Jiangsu Province Hospital of TraditionalChinese Medicine Nanjing Jiangsu 210029 China Department of Biochemistry School of Life Science NanjingUniversity Nanjing Jiangsu 210093 China 

出 版 物：《Chinese Medical Journal》 (中华医学杂志（英文版）)

年 卷 期：2008年第121卷第7期

页      面：620-624页

核心收录：

学科分类：1001[医学-基础医学(可授医学、理学学位)] 10[医学] 

主　　题：epididymis protease inhibitor expression purification biological function 

摘      要：Background Eppin （epididymis protease inhibitor） appears to play an important role in primate fertility. However, the function of Eppin and its antibody in men and its relationship with men＇s infertility are poorly studied. To reveal the significance and possibility of detection of anti-Eppin antibody in clinical infertilty cases, we developed an Escherichia coil expression system for the expression of biologically active human Eppin. Methods The human Eppin gene was cloned into PET-28a（＋） vector after induction with 0.5 mmol/L isopropy-β-Dthiogalactoside （IPTG） at 26℃ for 4 hours, and the expressed fusion protein His6-Eppin was purified by Ni2. affinity chromatography. Afterwards, six female 8-week-old Balb/c mice were immunized with purified His6-Eppin for three weeks. Their sera were collected and polyclonal antibodies against His6-Eppin were purified, all of which were further verified by Western-blot and immunofluorescence analysis. Results About 18.33 mg His6-Eppin was obtained from 1-L flask culture. The produced polyclonal antibodies against His6-Eppin recognized the Eppin protein both in human epididymis and in HEK293T cells by over-expression of the recombinant human Eppin. Conclusion The purified His6-Eppin protein has biological activity, which might be a candidate for clinical diagnosis of infertility and development of male immuno-contraceptive agents.