鼠源eIF4A1基因克隆及其原核/真核表达鉴定  

Cloning of mouse eIF4A1 gene and its prokaryotic/eukaryotic expression identification

作　　者：李梦茹 张文 段辰星 马良 栗朵朵 彭璟 梁晶晶[1,2] 罗廷荣 李晓宁[1,2] LI Meng-ru;ZHANG Wen;DUAN Chen-xing;MA Liang;LI Duo-duo;PENG Jing;LIANG Jing-jing;LUO Ting-rong;LI Xiao-ning(College of Animal Science and Technology,Guangxi University/Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease,Nanning,Guangxi 530004,China;Guangxi Engineering Research Center of Veterinary Biologics,Nanning,Guangxi 530004,China)

机构地区：[1]广西大学动物科学技术学院/广西高校动物疫病预防与控制重点实验室,广西南宁530004 [2]广西兽用生物制品工程研究中心,广西南宁530004

出　　处：《南方农业学报》2023年第10期3073-3082,共10页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31902311)。

摘　　要：【目的】克隆昆明小鼠真核生物翻译起始因子4A1基因(eIF4A1)并构建其原核/真核表达载体,探究其结构和生物学特性,为在体内外鉴定eIF4A1互作蛋白网络打下基础。【方法】以昆明小鼠脑组织总RNA反转录合成的cDNA为模板,PCR扩增鼠源eIF4A1基因编码区(CDS)序列,然后克隆到pGEX-4T-1和pcDNA3.0载体上分别构建原核/真核表达载体;利用大肠杆菌BL21(DE3)感受态细胞对原核表达载体进行诱导表达、纯化及鉴定;同时以真核表达载体转染HEK-293T细胞,通过Western blotting和间接免疫荧光(IFA)检测eIF4A1蛋白在HEK-293T细胞内的表达及分布情况;并以ProtParam、ProtScale、TMHMM-2.0、SignalP-5.0、SOPMA和SWISS-MODEL等在线软件对鼠源eIF4A1蛋白进行生物学信息分析。【结果】鼠源eIF4A1基因CDS序列长1221 bp,克隆到pGEX-4T-1载体能成功构建原核表达载体pGEX-4T-eIF4A1-Flag,在25和30℃下经0.5 mmol/L IPTG诱导均能大量表达出融合蛋白eIF4A1-Flag,且主要以包涵体形式存在。将鼠源eIF4A1基因克隆至pcDNA3.0载体成功构建获得真核表达载体pcDNA3.0-eIF4A1-Flag,以其转染HEK-293T细胞后,eIF4A1蛋白在HEK-293T细胞中成功表达,且主要分布在细胞质中。生物信息学分析结果表明,eIF4A1蛋白相对分子量为46.15408 kD,理论等电点(pI)为5.267,含有407个氨基酸残基;属于不稳定的亲水性非分泌蛋白,无跨膜结构,也没有信号肽,其二级结构以α-螺旋和无规则卷曲为主。鼠源eIF4A1蛋白的主要互作蛋白有10个,除eIFs家族蛋白外,还包括Paip1和Pdcd4互作蛋白。【结论】eIF4A1主要是分布在细胞质,为不稳定的亲水性非分泌蛋白,无跨膜结构和信号肽,通过构建原核/真核表达载体表达获得的融合蛋白eIF4A1-Flag可用于筛选和鉴定eIF4A1互作蛋白,为深入探究eIFs的结构及生物学功能提供技术支持。【Objective】To clone the Kunming mouse eukaryotic translation initiation factor 4A1 gene(eIF4A1)and construct its prokaryotic/eukaryotic expression vector to explore its structure and biological characteristics,and lay a foun-dation for the identification of eIF4A1 interacting protein network in vitro and in vivo.【Method】Using the cDNA synthe-sized by reverse transcription of total RNA of Kunming mouse brain tissue as template,mouse eIF4A1 gene coding region(CDS)sequence was amplified by PCR,and then cloned into pGEX-4T-1 and pcDNA3.0 vectors to construct prokaryo-tic/eukaryotic expression vectors,respectively.The prokaryotic expression vector was induced,purified and identified by Escherichia coli BL21(DE3)receptive cells.At the same time,HEK-293T cells were transfected with eukaryotic ex-pression vector,and the expression and distribution of eIF4A1 protein in HEK-293T cells were detected by Western blot-ting and indirect immunofluorescence(IFA).The biological information of mouse eIF4A1 protein was analyzed by Prot-Param,ProtScale,TMHMM-2.0,SignalP-5.0,SOPMA and SWISS-MODEL.【Result】The CDS sequence length of mouse eIF4A1 gene was 1221 bp,and the prokaryotic expression vector pGEX-4T-eIF4A1-Flag could be successfully con-structed by cloning pGEX-4T-1 vector.Fusion protein eIF4A1-Flag could be expressed in large quantities under the induc-tion of 0.5 mmol/L IPTG at 25 and 30℃,and it mainly existed in the form of inclusion bodies.The mouse eIF4A1 gene was cloned into pcDNA3.0 vector and the eukaryotic expression vector pcDNA3.0-eIF4A1-Flag was successfully con-structed.After transfecting HEK-293T cells with pcDNA3.0-eIF4A1-Flag,eIF4A1 protein was successfully expressed in HEK-293T cells and was mainly distributed in the cytoplasm.The results of bioinformatics analysis showed that the rela-tive molecular weight of eIF4A1 protein was 46.15408 kD,the theoretical isoelectric point(pI)was 5.267,and it con-tained 407 amino acid residues.It was an unstable hydrophilic non-secretory protein with no transmembr

关 键 词：鼠源 真核生物翻译起始因子(eIFs) eIF4A1基因 原核表达 真核表达 生物信息学分析 

分 类 号：S865.13[农业科学—野生动物驯养]