人MAPK3基因原核表达载体的构建及鉴定  

Construction of recombinant prokaryotic expressional plasmid of human MAPK3 gene and its identification

作　　者：石松 薛殿婷 张培 石静 孙达权 SHI Song;XUE Dianting;ZHANG Pei;SHI Jing;SUN Daquan(Department of Critical Medicine,the People's Hospital of Southeast Guizhou,Kaili 556000,Guizhou,China;School of Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]黔东南苗族侗族自治州人民医院重症医学科,贵州凯里556000 [2]贵州医科大学基础医学院生化与分子生物学教研室,贵州贵阳550025

出　　处：《贵州医科大学学报》2021年第10期1145-1150,共6页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81560390);贵州省科学技术基金[黔科合基础(2017)1147]。

摘　　要：目的探讨人丝裂原激活蛋白激酶3(MAPK3)基因原核表达载体的构建及鉴定。方法取对数生长期正常人肝细胞系HL-7702细胞,采用TRIzol法抽提细胞总RNA,以此为模板用反转录聚合酶链式反应(RT-PCR)反转录扩增MAPK3基因的蛋白编码区,脱氧核糖核苷酸(DNA)测序鉴定后插入原核表达载体pGEX-4t-1中构建重组原核表达质粒(命名为pGEX-MAPK3_(GST));将pGEX-MAPK3_(GST)导入细菌Rosetta(DE3)中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达MAPK3融合蛋白(GST-MAPK3),采用Sepharose 4B亲和层析法纯化MAPK3融合蛋白(GST-MAPK3),采用考马斯亮蓝染色和蛋白免疫印迹分析GST-MAPK3的蛋白纯度和验证目的蛋白。结果RT-PCR法成功克隆了人MAPK3基因的蛋白编码区,DNA测序结果显示该片段无任何突变;用克隆获得的基因片段成功构建了pGEX-MAPK3_(GST),通过原核表达的方式获得了GST-MAPK3,并通过亲和层析纯化了GST-MAPK3。结论成功克隆人MAPK3基因并获得MAPK3蛋白,可用于体外研究MAPK3对细胞角蛋白18(CK18)的磷酸化作用。Objective To explore the construction of human mitogen-activated protein kinase 3(MAPK3)gene and to detect its identification.Methods RNA of human hepatocytes of HL-7702 cells in exponential growth period were extracted by TRIzol method,as the template,and the protein coding region of MAPK3 gene was amplified by reverse transcription polymerase chain reaction(RT-PCR)and identified by deoxyribonucleic acid(DNA)sequencing.Then,the verified sequence was inserted into prokaryotic expressional vector pGEX-4t-1 to construct recombinant prokaryotic expressional plasmid,named pGEX-MAPK3_(GST).The pGEX-MAPK3_(GST)was introduced into Rosetta(DE3)by heat shock method,and the MAPK3 fusion protein(GST-MAPK3)was induced by IPTG.The bacteria were broken by homogenizer and the GST-MAPK3 was purified by Sepharose 4B through affinity chromatography.The protein purity was analyzed and the target protein was confirmed by Coomassie brilliant blue R-250 staining and Western blot.Results The protein encoding region of human MAPK3 gene was cloned successfully by RT-PCR,and no mutation was found in the DNA region detected by DNA sequencing.Moreover,the recombinant prokaryotic expression plasmid pGEX-MAPK3_(GST)was constructed using the cloned DNA fragment,and the fusion protein GST-MAPK3 was expressed by prokaryotic expression and purified by affinity chromatography.Conclusion Human MAPK3 gene is successfully obtained and its protein MAPK3 is purified,which can be employed to study the phosphorylation of MAPK3 on cytokeratin 18(CK18)in vitro.

关 键 词：丝裂原激活蛋白激酶类 丝裂原激活蛋白激酶3 基因克隆 载体构建 原核表达 亲和层析 蛋白纯化 

分 类 号：R34[医药卫生—基础医学]