| 鸡EED基因的原核表达与生物信息学分析 |
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| 引用本文： | 郁川,宋敏杰,石胜丽,朱文文,张贺伟,田文静,王聪慧,程相朝.鸡EED基因的原核表达与生物信息学分析[J].中国畜牧兽医,2021,48(10):3545-3553. |
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| 作者姓名： | 郁川 宋敏杰 石胜丽 朱文文 张贺伟 田文静 王聪慧 程相朝 |
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| 作者单位： | 1. 洛阳职业技术学院, 宠物与人类健康工程技术中心, 洛阳 471900;2. 河南科技大学动物科技学院, 洛阳市活载体生物材料与动物疫病防控重点实验室, 洛阳 471023 |
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| 基金项目： | 国家自然科学基金项目（31302059） |
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| 摘 要： | 本研究旨在获得鸡胚胎外胚层发育（embryonic ectoderm development，EED）原核表达蛋白，并利用生物信息学方法探索其潜在生物学功能。以提取的3日龄雏鸡脾脏总RNA为模板，利用RT-PCR技术扩增鸡EED基因，将其克隆至pET-32a （+）载体，构建重组表达载体pET-32a-EED，转化至大肠杆菌Rosetta感受态细胞中进行IPTG诱导表达；Western blotting检测经镍离子亲和层析法纯化的鸡EED蛋白，并利用在线软件对其进行生物信息学分析。结果显示，鸡EED基因序列全长1 341 bp，与参考序列（GenBank登录号：NM_001031376.1）相似性为99.85％；当诱导温度为28 ℃，加入终浓度为1 mmol/L的IPTG诱导剂诱导表达7 h时，可在重组菌裂解上清中纯化得到分子质量约70 ku的鸡EED融合蛋白。在线软件分析表明，鸡EED蛋白相对分子质量为50 377.92，理论等电点（pI）为6.54，为亲水蛋白，无跨膜结构域和信号肽序列，该蛋白序列与人多疏蛋白EED三级结构模板序列（6c23.1）相似，同样具有肿瘤靶点WD40结构域。结果表明，本研究成功获得大肠杆菌表达的鸡EED可溶性蛋白，且该蛋白具有重要的功能元件，可为深入探索鸡EED蛋白的生物学功能与调控机理提供材料。
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| 关 键 词： | 鸡 EED基因 表达 纯化 生物信息学 |
| 收稿时间： | 2021-03-24 |
Prokaryotic Expression and Bioinformatics Analysis of EED Gene in Chicken |
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| YU Chuan,SONG Minjie,SHI Shengli,ZHU Wenwen,ZHANG Hewei,TIAN Wenjing,WANG Conghui,CHENG Xiangchao.Prokaryotic Expression and Bioinformatics Analysis of EED Gene in Chicken[J].China Animal Husbandry & Veterinary Medicine,2021,48(10):3545-3553. |
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| Authors: | YU Chuan SONG Minjie SHI Shengli ZHU Wenwen ZHANG Hewei TIAN Wenjing WANG Conghui CHENG Xiangchao |
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| Institution: | 1. Pet & Human Health Engineering Technology Center, Luoyang Polytechnic, Luoyang 471900, China;2. Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China |
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| Abstract: | This study was aimed to obtain the prokaryotic expression protein of embryonic ectoderm development (EED) in chicken and explore its biological characteristics by bioinformatics. EED gene was amplified from spleen in chicken by RT-PCR, and cloned it into prokaryotic expression vector pET-32a(+) to construct the recombinant expression vector pET-32a-EED. Then pET-32a-EED was transformed into E. coli Rosetta to induce the EED fusion protein expression by IPTG. The recombinant protein was purified, and it was detected by Western blotting. Moreover, the bioinformatics analysis of EED protein in chicken was detected by online software. The results showed that the sequence of EED gene was 1 341 bp, which was 99.85% similarity with the reference sequence (GenBank accession No. :NM_001031376.1). The fusion protein with molecular weight of about 70 ku was obtained when the induction temperature was 28 ℃ and the expression of 1 mmol/L IPTG inducer was added for 7 h. The results of online software analysis showed that the relative molecular weight of EED protein in chicken was 50 377.92, and the isoelectric point was 6.54. It was a hydrophilic protein without transmembrane domain and signal peptide sequence. The tertiary structure prediction of EED protein in chicken was similar to human (6c23.1), and it also had the WD40 domain. In conclusion, the EED soluble protein in chicken was successfully expressed and purified, and EED protein had important functional elementsis, which provided materials for further study on the function of EED protein in chicken. |
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| Keywords: | chicken EED gene expression purification bioinformatics |
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