摘要
目的 观察附睾蛋白酶抑制剂（Eppin）是否抑制前列腺特异性抗原（PSA）酶活性.方法 利用分子克隆技术体外构建、表达、纯化重组Eppin和PSA.利用PSA水解变色底物S-2586的颜色变化,于分光光度计下测定其吸光度值,代表PSA酶反应速度,反应体系是在缓冲液0.1 mol/LTris-HCl,pH8.3,1.0 mol/L NaCl中进行,观察不同浓度Eppin的加入对PSA酶反应速度的影响.结果通过体外重组技术获得较高纯度和生理活性的Eppin和PSA,通过PSA水解变色底物S-2586的检测,重组PSA具有酶活性,0.3 μmol/L PSA与底物S-2586反应的饱和曲线分析显示Km（反应速度为最大反应速度一半时的底物浓度）值是0.5 μmol,底物饱和浓度0.2 mmol/L,分别加入4中不同浓度的重组Eppin 0、0.56、1.16、2.32 μmol/L,随着Eppin浓度的增加,PSA水解其变色底物S-2586速度明显减慢,PSA活性越来越受到抑制.结论 附睾蛋白酶抑制剂（Eppin）是前列腺特异性抗原（PSA）一种新的特异性抑制剂.
Objective To investigate the inhibitory effects of the epididymal protease inhibitor （Eppin） on the activity of prostate specific antigen （PSA）.Methods Recombinant Eppin and recombinant PSA were produced by molecular cloning technique in vitro.The enzymatical analysis of Eppin inhibiting PSA was done in the reaction buffer 0.1 mol/L Tris-HC1,pH 8.3,1.0 mol/L NaCl.The hydrolysis of velocity of PSA to the chromogenic substrate S-2586 was detected by spectrometer.Results Recombinant Eppin and PSA with high purity were produced by molecular cloning technique.The recombinant PSA had the enzymatical activity by hydrolyzing its substrate S-2586.0.3μmol/L PSA and the substate S-2586 reaction to the saturation curve analysis showed that Km（ response rate of half the maximum reaction rate when the substrate concentration） value was 0.5μmol,and substrate saturated concentration was 0.2 mmol/L.With the increases of Eppin （0,0.56,1.16,2.32μmol/L）,the hydrolysis velocity of PSA to S-2586 was slowed down.Conclusion Eppin,as a new inhibitor,specifically inhibits the activity of PSA.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2010年第12期1793-1795,共3页
Chinese Journal of Experimental Surgery
基金
国家＂十一五＂科技支撑计划资助项目（2006BAI03B12）
国家自然科学基金资助项目（30973199）
江苏省卫生厅兴卫工程开放课题资助项目（XK17200903）
