鲢鱼CPIs的三种电泳鉴定方法的比较研究
Comparative study on three electrophoresis methods for identifying silver carp CPIs
-
摘要: 本文对Geltain-substrate-SDS-PAGE反相酶谱法（方法 A）、Native-PAGE反相酶谱法（方法 B）和与papain反应后进行SDS-PAGE法（方法 C）三种电泳方法的鉴定效果、灵敏度进行了对比,确定了鲢鱼卵粗提样中小分子CPIs的最适电泳鉴定方法。结果表明,对于在电泳上活性不稳定的鲢鱼卵小分子CPIs而言,方法 A灵敏度较低,且可能出现假阳性现象;方法 B虽能准确鉴定小分子CPIs的存在,但不能判断分子量。方法 C,即小分子CPIs与papain在37℃下反应5h以上,然后进行SDS-PAGE,抑制剂条带清晰可见,鉴定效果好,且能提供分子量分布信息。此外,尽管荧光底物法和Azocasein法都无法检测到鲢鱼肝胰浓缩粗提液中半胱氨酸蛋白酶抑制活性,但是利用C法,则有效地鉴定了其中CPIs的存在和分子量分布情况。综上所述,方法 C或协同方法 B的电泳鉴定方法,适用于鲢鱼下脚料粗提样品中CPIs的鉴定,效果理想,灵敏度高,操作简便。Abstract: In this study, three methods, the Geltain-substrate-SDS-PAGE reverse zymography ( method A) , Native-PAGE reverse zymography ( method B) and SDS- PAGE after reaction with papain ( method C) , were compared on the aspect of identification effect and sensitivities, so that the optimum way for identifying the CPIs with low molecular weight from silver carp egg extract was determined.The results showed that, for the unstable CPIs liable to inactivation on electrophoresis, method A gave a low sensitivity and false positive phenomenon, and method B could exactly identify the CPIs, but the molecular weight could not be analyzed, whereas method C ( that is, the SDS-PAGE after reaction with papain at 37℃ not less than 5h) obtained effective identification with clear target bands, and provided molecular weight information as well.Although the inhibitive activities of the hepatopancreas extract from silver carp could not be assayed either by fluorescent substrate method or Azocasein method, the existence and molecular weight distribution of the CPIs were identified efficiently by method C. In conclusion, method C or associated with method B was feasible for the identification of CPIs from silver carp wastes.
下载: