中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2010年
12期
1793-1795
,共3页
蒋荣江%王增军%牛晓兵%王鹏%贾跃军%苏仕峰%张炜%王心如
蔣榮江%王增軍%牛曉兵%王鵬%賈躍軍%囌仕峰%張煒%王心如
장영강%왕증군%우효병%왕붕%가약군%소사봉%장위%왕심여
附睾%蛋白酶抑制剂%前列腺特异性抗原
附睪%蛋白酶抑製劑%前列腺特異性抗原
부고%단백매억제제%전렬선특이성항원
Epididymal%Protease inhibitor%Prostate specific antigen
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.
目的 觀察附睪蛋白酶抑製劑(Eppin)是否抑製前列腺特異性抗原(PSA)酶活性.方法 利用分子剋隆技術體外構建、錶達、純化重組Eppin和PSA.利用PSA水解變色底物S-2586的顏色變化,于分光光度計下測定其吸光度值,代錶PSA酶反應速度,反應體繫是在緩遲液0.1 mol/LTris-HCl,pH8.3,1.0 mol/L NaCl中進行,觀察不同濃度Eppin的加入對PSA酶反應速度的影響.結果通過體外重組技術穫得較高純度和生理活性的Eppin和PSA,通過PSA水解變色底物S-2586的檢測,重組PSA具有酶活性,0.3 μmol/L PSA與底物S-2586反應的飽和麯線分析顯示Km(反應速度為最大反應速度一半時的底物濃度)值是0.5 μmol,底物飽和濃度0.2 mmol/L,分彆加入4中不同濃度的重組Eppin 0、0.56、1.16、2.32 μmol/L,隨著Eppin濃度的增加,PSA水解其變色底物S-2586速度明顯減慢,PSA活性越來越受到抑製.結論 附睪蛋白酶抑製劑(Eppin)是前列腺特異性抗原(PSA)一種新的特異性抑製劑.
목적 관찰부고단백매억제제(Eppin)시부억제전렬선특이성항원(PSA)매활성.방법 이용분자극륭기술체외구건、표체、순화중조Eppin화PSA.이용PSA수해변색저물S-2586적안색변화,우분광광도계하측정기흡광도치,대표PSA매반응속도,반응체계시재완충액0.1 mol/LTris-HCl,pH8.3,1.0 mol/L NaCl중진행,관찰불동농도Eppin적가입대PSA매반응속도적영향.결과통과체외중조기술획득교고순도화생리활성적Eppin화PSA,통과PSA수해변색저물S-2586적검측,중조PSA구유매활성,0.3 μmol/L PSA여저물S-2586반응적포화곡선분석현시Km(반응속도위최대반응속도일반시적저물농도)치시0.5 μmol,저물포화농도0.2 mmol/L,분별가입4중불동농도적중조Eppin 0、0.56、1.16、2.32 μmol/L,수착Eppin농도적증가,PSA수해기변색저물S-2586속도명현감만,PSA활성월래월수도억제.결론 부고단백매억제제(Eppin)시전렬선특이성항원(PSA)일충신적특이성억제제.
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.
