肿瘤坏死因子受体相关因子6泛素化位点突变载体的构建及其功能位泛素化位点的鉴定  

Construction of TRAF6 ubiquitination site mutation vectors and identification of its functional ubiquitination sites

作　　者：王琴 林春霖[1,2] 程志彬 何若凡 林鹏航 陈辉 叶建新[1,2] 朱广伟[1,2] WANG Qin;LIN Chunlin;CHENG Zhibin;HE Ruofan;LIN Penghang;CHEN Hui;YE Jianxin;ZHU Guangwei(Department of Gastrointestinal Surgery,Fujian Abdominal Surgery Research Institute,First Affiliated Hospital,Fujian Medical University,Fuzhou 350005,China;Key Laboratory of Gastrointestinal Cancer Ministry of Education,Fujian Medical University,Fuzhou 350005,China)

机构地区：[1]福建医科大学附属第一医院胃肠外科二区腹部外科研究所,福建福州350005 [2]福建医科大学消化道恶性肿瘤教育部重点实验室,福建福州350005

出　　处：《吉林大学学报（医学版）》2021年第3期551-558,共8页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金项目(81702424,81872364);福建省科技厅自然科学基金项目(2018J05127);福建省卫健委中青年骨干项目(2018-ZQN-46)。

摘　　要：目的:预测人肿瘤坏死因子受体相关因子6(TRAF6)基因上的泛素化位点,并构建泛素化位点突变质粒,探讨突变质粒对人结直肠癌HCT116和SW480细胞中核因子κB(NF-κB)和激活蛋白1(AP-1)相对荧光素酶活性的影响。方法:采用UbPred、UbiSite和BDM-PUB软件预测TRAF6基因的泛素化位点,采用CE Design V1.04软件设计突变引物,采用突变试剂盒进行定点突变,采用PCR法扩增获得突变后的目的片段,扩增产物经Dpnl酶消化,去除甲基化模板质粒,消化产物在ExnaseⅡ催化作用下发生同源重组获得重组质粒;将重组质粒转化至DH5α大肠杆菌感受态细胞,对重组质粒进行DNA测序。采用双荧光素酶报告基因系统检测转染突变质粒后HCT116和SW480细胞中NF-κB和AP-1相对荧光素酶活性。结果:经过DNA测序,泛素化突变位点已经成功突变,泛素化突变质粒构建成功。与TRAF6野生型基因比较,转染第124、319和331位突变质粒后,结直肠癌HCT116和SW480细胞中NF-κB和AP-1相对荧光素酶活性降低(P<0.05或P<0.01),其中转染第124位突变质粒后,结直肠癌HCT116和SW480细胞中NF-κB和AP-1相对荧光素酶活性降低最明显(P<0.01)。结论:成功构建人TRAF6基因的泛素化突变质粒,TRAF6的第124位氨基酸是其最重要的泛素化位点,可能影响下游信号通路中NF-κB和AP-1的活性。Objective:To predict the ubiquitination sites of human tumor necrosis factor receptorassociated factor 6(TRAF6)gene and construct the ubiquitination mutant plasmid,and to explore the effect of mutant plasmid on the relative luciferase activity of nuclear factor kappa-B(NF-κB)and activator protein-1(AP-1)in the human colorectal cancer HCT116 and SW480 cells.Methods:UbPred,UbiSite,and BDMPUB softwares were used to predict the ubiquitination sites of TRAF6 gene;the mutation primers were designed by CE Design V1.04 software,and the mutation kits were used for site-directed mutation;the mutated target fragment was amplified by PCR method;the amplified products were digested by Dpnl enzyme to remove the methylated template plasmids;the digested products were recombined under the catalysis of ExnaseⅡto obtain the recombinant plasmids;the recombinant plasmids were transformed into the competent cells of DH5αE.coli and the sequence was sequenced.The relative luciferase activities of NF-κB and AP-1 in the colorectal cancer HCT116 and SW480 cells were detected by dual-luciferase reporter gene system.Results:After DNA sequencing,the ubiquitination mutation site was successfully mutated,and the ubiquitination mutant plasmid was successfully constructed.Compared with TRAF6 wildtype gene strain,the relative luciferase activities of NF-κB and AP-1 in the colorectal cancer HCT116 and SW480 cells were decreased after transfected with 124 mut,319 mut,and 331 mut plasmids(P<0.05 or P<0.01),and the relative luciferase activities of NF-κB and AP-1 in the colorectal cancer HCT116 and SW480 cells were the most significantly decreased after transfected with 124 mut plasmid(P<0.01).Conclusion:The ubiquitination mutant plasmids are successfully constructed.The 124 th amino acid of TRAF6 is the most important ubiquitination site,which may affect the activities of NF-κB and AP-1 factors in downstream signaling pathways.

关 键 词：肿瘤坏死因子受体相关因子6 突变质粒构建 泛素化 荧光素酶报告系统 核因子κB 激活蛋白1 

分 类 号：R735.3[医药卫生—肿瘤]