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

  • 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e IF4A1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e IF4A1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。

     

    Abstract: 【Objective】To clone the Kunming mouse eukaryotic translation initiation factor 4A1 gene(e IF4A1) and construct its prokaryotic/eukaryotic expression vector to explore its structure and biological characteristics, and lay a foundation for the identification of eIF4A1 interacting protein network in vitro and in vivo. 【Method】Using the cDNA synthesized by reverse transcription of total RNA of Kunming mouse brain tissue as template, mouse e IF4A1 gene coding region(CDS) sequence was amplified by PCR, and then cloned into pGEX-4T-1 and pcDNA3.0 vectors to construct prokaryotic/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 expression vector, and the expression and distribution of eIF4A1 protein in HEK-293T cells were detected by Western blotting and indirect immunofluorescence(IFA). The biological information of mouse eIF4A1 protein was analyzed by ProtParam, 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 constructed by cloning pGEX-4T-1 vector. Fusion protein eIF4A1-Flag could be expressed in large quantities under the induction of 0.5 mmol/L IPTG at 25 and 30 ℃, and it mainly existed in the form of inclusion bodies. The mouse e IF4A1 gene was cloned into pcDNA3.0 vector and the eukaryotic expression vector pcDNA3.0-eIF4A1-Flag was successfully constructed. After transfecting HEK-293T cells with pcDNA3.0-e IF4A1-Flag, e IF4A1 protein was successfully expressed in HEK-293T cells and was mainly distributed in the cytoplasm. The results of bioinformatics analysis showed that the relative molecular weight of eIF4A1 protein was 46.15408 kD, the theoretical isoelectric point(pI) was 5.267, and it contained 407 amino acid residues. It was an unstable hydrophilic non-secretory protein with no transmembrane structure and no signal peptide, and its secondary structure was dominated by α-helix and random coil. There were 10 main interacting proteins of mouse eIF4A1 protein, including Paip1 and Pdcd4 interacting proteins in addition to eIFs family proteins.【Conclusion】eIF4A1 is an unstable hydrophilic non-secreted protein which distributes mainly in cytoplasm, without transmembrane structure and signal peptide. The fusion protein eIF4A1-Flag obtained by constructing prokaryotic/eukaryotic expression vector can be used to screen and identify eIF4A1 interacting proteins. To provide technical support for further investigation of the structure and biological function of eIFs.

     

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