利用非天然氨基酸代谢掺入法检测新生成蛋白  

Detection of Newly Synthesized Proteins via Metabolic Incorporation of Non-Natural Amino Acid

作　　者：崔秀雲 孙宁宁[1] 谢小娜 孙万春 赵晴[3] 刘宁[1] CUI Xiu-Yun;SUN Ning-Ning;XIE Xiao-Na;SUN Wan-Chun;ZHAO Qing;LIU Ning(Central Laboratory,The Second Hospital,Key Laboratory of Zoonosis, Ministry of Education,Jilin University,Changchun 130041,China;Department of Endocrinology,The First Hospital of Jilin University,Changchun 130012,China;Department of Neurology,China-Japan Union Hospital,Changchun 130033,China)

机构地区：[1]吉林大学第二医院研究中心,教育部人兽共患病研究重点实验室,长春130041 [2]吉林大学第一医院内分泌科,长春130012 [3]吉林大学中日联谊医院神经内科,长春130033

出　　处：《分析化学》2018年第11期1808-1813,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.81472030);吉林省科技厅项目(Nos.20110739,20180101267JC);吉林大学白求恩计划B(No.2012210);吉林省卫生和计划生育委员会项目(No.2015Z041)资助。

摘　　要：以脂多糖(Lipopolysaccharide,LPS)刺激Raw264.7细胞产生肿瘤坏死因子α(Tumor necrosis factorα,TNF-α)为模型体系,建立了非天然氨基酸代谢掺入法检测特定新生成蛋白的方法。通过代谢掺入的方法,使细胞中的蛋白质,特别是新生成的蛋白质一级结构中掺入非天然氨基酸,即带有叠氮基团的甲硫氨酸类似物(Azidohomoalanine,AHA)。考察了在不同浓度的LPS和FBS,以及不同的刺激时间等条件下,LPS刺激Raw264.7细胞产生TNF-α的实验参数,确定了最优的实验条件为:在含有1%胎牛血清的无甲硫氨酸(Met)的DMEM培养基中,分别在不加LPS和加10 ng/m L的LPS条件下刺激Raw264.7细胞4 h,在刺激细胞的同时掺入AHA。利用Cu+催化的叠氮基团与带有生物素(Biotin)标签的炔烃基团的环加成反应,使蛋白质标记上Biotin标签。利用吸附在固相载体上的抗体特异性捕获TNF-α分子,再用耦联辣根过氧化物酶(Horseradish peroxidase,HRP)的链霉亲和素(Streptavidin)对TNF-α分子上的Biotin进行识别,实现对特定的新生成蛋白质(TNF-α)进行检测。本方法为检测特定微量新生成蛋白、表征特定蛋白质的周转等研究提供了新的思路法。A method for detection of newly synthesized protein via metabolic incorporation of non-natural amino acid was developed,in which Raw264.7 cells were treated by lipopolysaccharide(LPS)to generate tumor necrosis factor alpha(TNF-α).Experimental parameters including the concentration of LPS and the duration of LPS treatment were investigated by measuring the LPS stimulated TNF-αfrom Raw264.7 cells.The optimal experimental conditions were determined as follows.Raw264.7 cells were cultured in Met-free DMEM containing 1%fetal bovine serum(FBS),and then treated for 4 h with LPS at concentration of 10 ng/mL in presence of AHA.The biotin tags were introduced into proteins containing azidohomoalanine(AHA)by copper-catalyzed alkyne-azidecycloaddition(CuAAC).Then TNF-αwas specifically captured by the antibody adsorbed on the solid support,on which the biotin tag could be detected by streptavidin coupled with horseradish peroxidase(HRP).This study provided a method for the detection of a specific newly synthesized protein,and characterization of specific protein turnover.

关 键 词：新生成蛋白 非天然氨基酸 肿瘤坏死因子Α 点击化学 

分 类 号：R446.1[医药卫生—诊断学] O652[医药卫生—临床医学]