鸡EED基因的原核表达与生物信息学分析

doi:10.16431/j.cnki.1671-7236.2021.10.005

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (10): 3545-3553.doi: 10.16431/j.cnki.1671-7236.2021.10.005

鸡EED基因的原核表达与生物信息学分析

郁川^1,2, 宋敏杰¹, 石胜丽², 朱文文¹, 张贺伟¹, 田文静¹, 王聪慧¹, 程相朝^1,2

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

收稿日期:2021-03-24 出版日期:2021-10-20 发布日期:2021-09-30
通讯作者: 程相朝 E-mail:chengxch@126.com
作者简介:郁川(1985-),男,河南安阳人,博士,研究方向:动物疫病与分子免疫学,E-mail:yu_chuan1985@163.com;宋敏杰(1990-),女,河南杞县人,硕士生,研究方向:动物疫病及抗肿瘤机制,E-mail:songmj9005@163.com
基金资助:
国家自然科学基金项目（31302059）

Prokaryotic Expression and Bioinformatics Analysis of EED Gene in Chicken

YU Chuan^1,2, SONG Minjie¹, SHI Shengli², ZHU Wenwen¹, ZHANG Hewei¹, TIAN Wenjing¹, WANG Conghui¹, CHENG Xiangchao^1,2

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

Received:2021-03-24 Online:2021-10-20 Published:2021-09-30

摘要/Abstract

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。

关键词: 鸡; EED基因; 表达; 纯化; 生物信息学

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.

Key words: chicken; EED gene; expression; purification; bioinformatics

中图分类号:

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.

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 and Veterinary Medicine, 2021, 48(10): 3545-3553.

参考文献

[1] MARGUERON R, JUSTIN N, OHNO K, et al.Role of the polycomb protein EED in the propagation of repressive histone marks[J]. Nature, 2009, 461(7265):762-767.
[2] CAO R, WANG L, WANG H, et al.Role of histone H3 lysine 27 methylation in polycomb-group silencing[J]. Science, 2002, 298(5595):1039-1043.
[3] YU W, ZHANG F, WANG S, et al.Depletion of polycomb repressive complex 2 core component EED impairs fetal hematopoiesis[J]. Cell Death & Disease, 2017, 8(4):e2744.
[4] KOUZNETSOVA V L, TCHEKANOV A, LI X M, et al.Polycomb repressive 2 complex-molecular mechanisms of function[J]. Protein Science, 2019, 28:1387-1399.
[5] YU H, SIMONS D L, ILANA S, et al.PRC2/EED-EZH2 complex is up-regulated in breast cancer lymph node metastasis compared to primary tumor and correlates with tumor proliferation in situ[J]. PLoS One, 2012, 7(12):e51239.
[6] SEO G S, YU J I, CHAE S C, et al.EED gene polymorphism in patients with colorectal cancer[J]. International Journal of Biological Markers, 2013, 28(3):274-279.
[7] YING X, PAN R, ZHONG J, et al.Significant association of EED promoter hypomethylation with colorectal cancer[J]. Oncology Letters, 2019, 18(2):1564-1570.
[8] QI W, ZHAO K, GU J, et al.An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED[J]. Nature Chemical Biology, 2017, 13(4):381-388.
[9] 韩威.文昌鸡性成熟启动前后下丘脑部分功能基因和miRNA表达谱的变化[D].南京:南京农业大学, 2015. HAN W.Global changes of hypothalamic gene transcription and microRNA profile before and after puberty onset in Wenchang chicken[D].Nanjing:Nanjing Agricultrual University, 2015.(in Chinese)
[10] LIU Q, WANG G, LI Q, et al.Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer[J]. International Journal of Cancer, 2019, 145(2):415-426.
[11] HUANG Y, ZHANG J, YU Z, et al.Discovery of first-in-class, potent, and orally bioavailable embryonic ectoderm development (EED) inhibitor with robust anticancer efficacy[J]. Journal of Medicinal Chemistry, 2017, 60(6):2215-2226.
[12] 高祥, 李辉, 张礼洲, 等.传染性法氏囊病病毒超强毒株衣壳蛋白的可溶性表达、纯化与活性分析[J]. 中国畜牧兽医, 2017, 44(7):2096-2102. GAO X, LI H, ZHANG L Z, et al.Soluble expression, purification and activity analysis of capsid protein of very virulent infectious Bursal disease virus[J]. China Animal Husbandry & Veterinary Medicine, 2017, 44(7):2096-2102.(in Chinese)
[13] 李芳芳, 龙开旭, 俸祥仁, 等.河流型水牛热休克蛋白70的原核表达及其对精液抗冻性的影响[J]. 中国畜牧兽医, 2018, 45(9):2417-2424. LI F F, LONG K X, FENG X R, et al.Prokaryotic expression of HSP70 and it's effect on the freezing resistance of semen in river buffalo[J]. China Animal Husbandry & Veterinary Medicine, 2018, 45(9):2417-2424.(in Chinese)
[14] 李明珠, 曲哲会, 宋志峰, 等.牛副流感病毒3型HNex蛋白表达及其多克隆抗体的制备[J]. 中国畜牧兽医, 2021, 48(1):265-272. LI M Z, QU Z H, SONG Z F, et al.Expression of Bovine parainfluenza virus type 3 HNex protein and preparation of its polyclonal antibodies[J]. China Animal Husbandry & Veterinary Medicine, 2021, 48(1):265-272.(in Chinese)
[15] 张孝忠, 刘一凡, 谷思颖, 等.流行性乙型脑炎病毒NS1基因克隆及其蛋白表达纯化[J]. 中国畜牧兽医, 2018, 45(8):2320-2326. ZHANG X Z, LIU Y F, GU S Y, et al.Cloning, expression and purification of NS1 gene of epidemic encephalitis type B[J]. China Animal Husbandry & Veterinary Medicine, 2018, 45(8):2320-2326.(in Chinese)
[16] POTJEWYD F, TURNER A W, BERI J, et al.Degradation of polycomb repressive complex 2 with an EED-targeted bivalent chemical degrader[J]. Cell Chemical Biology, 2020, 27(1):47-56.
[17] XU C, MIN J.Structure and function of WD40 domain proteins[J]. Protein & Cell, 2011, 2(3):202-214.
[18] ZHANG C, ZHANG F.The multifunctions of WD40 proteins in genome integrity and cell cycle progression[J]. Journal of Genomics, 2015, 3:40-50.
[19] MIGLIORI V, MAPELLI M, GUCCIONE E.On WD40 proteins:Propelling our knowledge of transcriptional control?[J]. Epigenetics, 2012, 7(8):815-822.
[20] HAN Z, XING X, HU M, et al.Structural basis of EZH2 recognition by EED[J]. Structure, 2007, 15(10):1306-1315.
[21] JUSTIN N, ZHANG Y, TARRICONE C, et al.Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2[J]. Nature Communications, 2016, 7:11316.
[22] MARGUERON R, REINBERG D.The polycomb complex PRC2 and its mark in life[J]. Nature, 469(7330):343-349.
[23] MARTIN M C, ZENG G, YU J, et al.Small molecule approaches for targeting the polycomb repressive complex 2(PRC2) in cancer[J]. Journal of Medicinal Chemistry, 2020, 63(24):15344-15370.

鸡EED基因的原核表达与生物信息学分析

Prokaryotic Expression and Bioinformatics Analysis of EED Gene in Chicken

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	王赛桥, 赵路, 翟振翰, 张丙雷, 贾万航, 王玉琴. 绵羊miR-449a/b前体序列组织表达及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(6): 2175-2184.
[2]	韦明邦, 边巴琼达, 肖青青, 段梦琪, 强巴央宗, 商鹏. 藏猪CDKN1B基因克隆、生物信息学及表达分析[J]. 中国畜牧兽医, 2023, 50(6): 2196-2206.
[3]	吴旋, 曾申明. 血清白蛋白的功能、应用及纯化工艺研究进展[J]. 中国畜牧兽医, 2023, 50(6): 2265-2275.
[4]	杜元军, 段国超, 刘玮, 李大鹏, 韩海霞, 雷秋霞, 周艳, 刘杰, 王杰, 曹顶国, 李福伟, 陈甫. 暗斑对蛋鸡蛋品质的影响及其与肠道功能的关系[J]. 中国畜牧兽医, 2023, 50(6): 2305-2311.
[5]	吴立婷, 陆睿, 刘半红, 包红朵, 王永娟, 周艳, 王冉, 张辉. 产气荚膜梭菌噬菌体及乳酸杆菌联用改善肉鸡生长性能和调节肠道菌群功能研究[J]. 中国畜牧兽医, 2023, 50(6): 2321-2332.
[6]	肖鹏, 尚江华, 杨春艳, 李孟琪, 段安琴, 马小娅, 冯超, 黄晨茜, 张博, 周金陈, 韦科龙, 郑威, 郑海英. α-亚麻酸对水牛卵巢颗粒细胞体外培养的影响[J]. 中国畜牧兽医, 2023, 50(6): 2380-2387.
[7]	焦琳琳, 成玉婷, 吴庆国, 吴双, 吴植, 朱善元, 钱莺娟. 鸭坦布苏病毒Capsid蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2395-2402.
[8]	李灿, 李楚楚, 曾维, 张宁, 纪春晓, 陈韬. 猪RegⅢγ蛋白的重组表达及功能研究[J]. 中国畜牧兽医, 2023, 50(6): 2414-2426.
[9]	李永彬, 杨惠琳, 宋子豪, 张昱, 杨瑾, 孟佳, 崔小珍, 吕晓玲, 郑明学, 白瑞. 柔嫩艾美耳球虫MIF基因克隆及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(6): 2450-2459.
[10]	宋越, 苏胜杰, 张帆, 白帆, 戴伶俐, 王娜, 王大伟, 杨茜雯, 赵世华, 张月梅. 溶血性曼氏杆菌截短型白细胞毒素的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2479-2486.
[11]	欧云文, 潘琴, 汪洋, 代军飞, 任绍科, 张洋, 张杰. 猪细小病毒6型ORF2基因的截短表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2487-2495.
[12]	刘鹏, 李静静, 乔彦良, 朱连勤, 朱风华. 黄芩多糖的提取及结构表征[J]. 中国畜牧兽医, 2023, 50(6): 2518-2530.
[13]	卢玉葵, 刘佳琪, 钟嘉诚, 陈济铛, 朱婉君, 张溢珊, 张济培. 鹅源副鸡禽杆菌的分离鉴定及致病性研究[J]. 中国畜牧兽医, 2023, 50(6): 2562-2572.
[14]	张殿琦, 马鑫浩, 杨志梅, 梅楚刚, 昝林森. 秦川肉牛PAI1基因生物信息学分析及其对肌内脂肪细胞增殖和分化的影响[J]. 中国畜牧兽医, 2023, 50(5): 1729-1741.
[15]	李佳富, 莫小兵. 多杀性巴氏杆菌CRISPR-Cas系统生物信息学分析[J]. 中国畜牧兽医, 2023, 50(5): 1742-1753.