CAJ | 학술논문

目的建立一种快速、相对定量检测乙酰化蛋白方法,并初步应用其检测药物作用后Jurkat细胞乙酰化总蛋白水平.方法用不同浓度的曲古菌素A(TSA)诱导Jurkat细胞蛋白高乙酰化后,利用抗乙酰化赖氨酸抗体亲和层析柱富集纯化乙酰化总蛋白,经酸洗脱后固定于酶标板,ELISA法检测乙酰化蛋白相对总量,并用基质辅助激光解吸电离串联飞行时间质谱仪(MALDI-TOF/TOF)分析验证其成分；同法检测不同浓度的没食子酸、大黄素、单乙酰化大黄素A三种药物作用Jurkat细胞后乙酰化总蛋白水平的变化,筛查诱导Jurkat细胞蛋白乙酰化药物.结果 1μmol/L TSA作用于4×105Jurkat细胞24 h,乙酰化总蛋白相对水平最高.MALDI-TOF/TOF分析显示,TSA诱导Jurkat细胞产生的乙酰化蛋白共有22种,其中15种为乙酰化组蛋白.没食子酸、大黄素、单乙酰化大黄素A作用Jurkat细胞后所导致的乙酰化程度不同,以1μmol/L浓度TSA为阳性对照组,无药物为空白对照组,35.09 μmol/L和17.54 μmol/L大黄素处理的Jurkat细胞乙酰化蛋白相对水平分别为4.3％和14.2％；1.47 μmol/L和2.94 μmol/L没食子酸处理组乙酰化蛋白相对水平分别为28.7％和11.5％；152.91 μmol/L和30.58 μmoL/L单乙酰化大黄素组分别为22.0％和3.6％；其中1.47 μmol/L没食子酸所诱导的乙酰化蛋白水平最高.结论初步建立了活细胞基础上纯化富集并检测乙酰化总蛋白水平的方法,该法可快速、简便的筛选以组蛋白去乙酰化酶为靶点的抗癌药物.
목적 건립일충쾌속、상대정량검측을선화단백방법,병초보응용기검측약물작용후Jurkat세포을선화총단백수평.방법 용불동농도적곡고균소A(TSA)유도Jurkat세포단백고을선화후,이용항을선화뢰안산항체친화층석주부집순화을선화총단백,경산세탈후고정우매표판,ELISA법검측을선화단백상대총량,병용기질보조격광해흡전리천련비행시간질보의(MALDI-TOF/TOF)분석험증기성분；동법검측불동농도적몰식자산、대황소、단을선화대황소A삼충약물작용Jurkat세포후을선화총단백수평적변화,사사유도Jurkat세포단백을선화약물.결과 1μmol/L TSA작용우4×105Jurkat세포24 h,을선화총단백상대수평최고.MALDI-TOF/TOF분석현시,TSA유도Jurkat세포산생적을선화단백공유22충,기중15충위을선화조단백.몰식자산、대황소、단을선화대황소A작용Jurkat세포후소도치적을선화정도불동,이1μmol/L농도TSA위양성대조조,무약물위공백대조조,35.09 μmol/L화17.54 μmol/L대황소처리적Jurkat세포을선화단백상대수평분별위4.3％화14.2％；1.47 μmol/L화2.94 μmol/L몰식자산처리조을선화단백상대수평분별위28.7％화11.5％；152.91 μmol/L화30.58 μmoL/L단을선화대황소조분별위22.0％화3.6％；기중1.47 μmol/L몰식자산소유도적을선화단백수평최고.결론 초보건립료활세포기출상순화부집병검측을선화총단백수평적방법,해법가쾌속、간편적사선이조단백거을선화매위파점적항암약물.
Objective To establish a rapid,relatively quantitative method of detecting acetylated proteins.Methods The proteins of Jurkat cells were acetylated by Trichostatin A (TSA) at different concentrations,then enriched and purified by anti-acetylated lysine antibodies affinity chromatography colum.The components eluted by acid were fixed on the microplate,the levels of acetylated proteins were tested by ELISA,and their components were identified by MALDI-TOF-TOF mass spectrometry.Also the above-mentioned methods were applied to the other three agents (gallic acid,emodin and monoacetylated emodin A).Results That 4 × 10s Jurkat cells treated with 1 μmol/L TSA produced the optimal acetylated effect,up to 22 acetylated proteins were identified by MALDI-TOF-TOF,of them 15 were acetylated histones.The other three agents also induced acetylation,the relative values of acetylated proteins of Jurkat cells treated with 35.09 μmol/L and 17.54 μmol/L gallic acid were 4.3％ and 14.2％ respectively; those as of 28.7％ and 11.5％ treated with 1.47 μmol/L and 2.94 μmol/L emodin; those as of 22.0％ and 3.6％ treated with 152.91 μmol/L and 30.58 μmol/L monoacetylated emodin A.Conclusion The method based on affinity chromatography colum may be useful for the detection of acytylated proteins,and could be used to screen agents which target to histone deacetylase.