Native-PAGE染色法用于过氧化氢酶-3酶活性定量分析  

Quantitative analysis for the activity of catalase-3 by Native-PAGE staining method

作　　者：董晴[1,2,3] 韩晓云[4] 王颖[1,2,3] 何群[1,2,3] 

机构地区：[1]中国农业大学生物学院,北京100193 [2]农业生物技术国家重点实验室,北京100193 [3]中国农业大学农业微生物资源及其利用农业部重点实验室,北京100193 [4]黑龙江大学生命科学学院,哈尔滨150080

出　　处：《中国科技论文》2016年第12期1398-1402,1430,共6页China Sciencepaper

基　　金：高等学校博士学科点专项科研基金资助项目(20110008110027);国家基础科学人才培养基金资助项目(J1103520)

摘　　要：通过定量比较过氧化氢酶-3(catalase-3,Cat-3)的酶活性与其蛋白量及mRNA的关系,确定Native-PAGE染色法能够直接反映出Cat-3蛋白含量,进而探究该方法用于定性和定量检测Cat-3活性的准确性。采用同源重组的方法进行cat-3基因敲除;通过大肠杆菌表达重组GST-Cat-3蛋白制备多克隆抗体;通过Western blotting检测、Northern杂交以及Native-PAGE染色法对粗糙脉孢菌Cat-3的酶活性进行定量分析。得到cat-3基因缺失突变体;获得特异性很高的Cat-3蛋白的多克隆抗体,并对H_2O_2胁迫处理和未处理的菌丝体中Cat-3表达量进行分析、比较。与Northern杂交和Western blotting检测相比,NativePAGE染色法能够非常准确地反映出Cat-3蛋白表达量的变化,因而该方法不但能够对粗糙脉孢菌Cat-3的酶活性进行定性分析,还能进行定量分析。The Native-PAGE staining method could precisely reveal the protein level of Cat-3through the quantitative comparision of the enzyme activity,protein level of Cat-3and its mRNA level.Then the accuracy for the qualitative and quantitative determination of Cat-3activities using this method was investigated.The correlation between enzyme activities and cat-3 knockout mutant was created by gene replacement strategy and polyclonal antiserum of Cat-3protein was also generated.Then,the activity of Cat-3was quantified by Western blotting,Northern hybridization and Native-PAGE straining methods.Homokaryotic cat-3 knockout strains and the highly specific Cat-3antibody were obtained and the protein levels of Cat-3were analyzed in WT with or without H_2O_2 treatment.The polyclonal antibody of Cat-3revealed the induced expression of Cat-3under H_2O_2 stress condition in wildtype strain.Compared to Northern blotting and Western analyses,Native-PAGE staining method can precisely reveal the changes of Cat-3activities and its protein level in Neurospora crassa.

关 键 词：生物化学 粗糙脉孢菌 过氧化氢酶-3 NATIVE-PAGE 染色法 过氧化氢酶-3酶活性 

分 类 号：Q5-33[生物学—生物化学]