鸡ELAVL1基因克隆、原核表达及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2023.05.008

摘要/Abstract

摘要： 【目的】试验旨在对鸡胚胎致死性异常视觉蛋白1(embryonic lethal abnormal vision like 1,ELAVL1)基因的CDS区进行克隆、表达和生物信息学分析。【方法】以鸡胚成纤维细胞(chick embryo fibroblast,CEF)cDNA为模板,通过PCR方法扩增ELAVL1基因CDS区序列,构建pMD19-T-ELAVL1克隆质粒。以pMD19-T-ELAVL1为模板进一步构建pET-32a-ELAVL1原核重组表达质粒,使用IPTG对重组菌株进行诱导表达,利用SDS-PAGE和Western blotting检测重组蛋白表达情况。通过在线软件对ELAVL1蛋白进行生物信息学分析。【结果】鸡ELAVL1基因CDS区长981 bp,编码326个氨基酸,与鸭的亲缘关系最近,与斑马鱼亲缘关系最远。SDS-PAGE和Western blotting结果表明,诱导表达重组蛋白主要以可溶性形式表达,其分子质量大小约55 ku。生物信息学分析显示,ELAVL1蛋白分子质量为36.10 ku,分子式为C₁₅₈₇H₂₄₉₄N₄₅₈O₄₇₉S₁₄,为非脂溶性、弱碱性的亲水稳定蛋白。ELAVL1蛋白无信号肽与跨膜区,含有1个N-糖基化位点、3个O-糖基化位点、32个磷酸化位点和10个线性B细胞抗原表位结合位点。该蛋白主要位于细胞核中,二级结构及三级结构主要由α-螺旋和无规则卷曲组成,含有3个RNA识别基序(RNA recognition motif,RRM),预测可能与多种RNA结合蛋白和蛋白激酶存在相互作用。【结论】本试验成功克隆了鸡ELAVL1基因CDS区,其编码蛋白为非脂溶性、弱碱性的亲水稳定蛋白,经诱导表达后成功获得了可溶性表达的ELAVL1重组蛋白。本研究结果为进一步探究鸡ELAVL1基因功能提供科学依据。

关键词: 鸡; ELAVL1基因; 克隆; 生物信息学分析

Abstract: 【Objective】 This study was aimed to clone,express and bioinformatics analyze the CDS region of chicken embryonic lethal abnormal vision like 1 (ELAVL1) gene.【Method】 The CDS region of ELAVL1 gene was amplified by PCR using the cDNA of chicken embryonic fibroblast (CEF) as a template to construct pMD19-T-ELAVL1 clone plasmid.The pET-32a-ELAVL1 prokaryotic recombinant expression plasmid was constructed using pMD19-T-ELAVL1 template.IPTG was used to induce the expression of the recombinant strain,and the recombinant protein expression were detected by SDS-PAGE and Western blotting. Bioinformatics analysis of ELAVL1 protein was performed by online software. 【Results】 The length of ELAVL1 gene CDS in chicken was 981 bp and encoded 326 amino acids, which had the closet relationship with duck and the furthest relationship with zebrafish.SDS-PAGE and Western blotting results showed that the recombinant protein was mainly expressed in soluble form,and its molecular weight was about 55 ku.Bioinformatics analysis showed that the molecular weight of ELAVL1 protein was 36.10 ku,which was a non-fat soluble,weakly alkaline hydrophilic stable protein.The ELAVL1 protein contained 1 N-glycosylation site,3 O-glycosylation sites,32 phosphorylation sites and 10 linear B cell epitope binding site.Subcellular localization revealed that the protein was located in the nucleus.The secondary and tertiary structures were mainly composed of alpha helix and random coil. The ELAVL1 protein contained 3 RNA recognition motif (RRM), which were predicted to interact with various RNA-binding proteins and protein kinases.【Conclusion】 This study successfully cloned the CDS region of ELAVL1 gene in chicken,and ELAVL1 was a non-fat soluble and weakly basic hydrophilic stable protein.After induced expression,soluble expression of ELAVL1 recombinant protein was successfully obtained.The results provided scientific basis for further function of chicken ELAVL1 gene.

Key words: chicken; ELAVL1 gene; clone; bioinformatics analysis

中图分类号:

刘佳隆, 张译文, 张悦然, 孙奇娟, 陈建, 何雷, 贾艳艳, 丁轲, 余祖华. 鸡ELAVL1基因克隆、原核表达及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(5): 1807-1817.

LIU Jialong, ZHANG Yiwen, ZHANG Yueran, SUN Qijuan, CHEN Jian, HE Lei, JIA Yanyan, DING Ke, YU Zuhua. Cloning,Prokaryotic Expression and Bioinformatics Analysis of ELAVL1 Gene in Chicken[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1807-1817.

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