牛干扰素调节因子9基因的克隆、序列分析与抗原优势区表达

doi:10.16431/j.cnki.1671-7236.2020.02.002

摘要/Abstract

摘要： 本研究旨在克隆牛干扰素调节因子9（bovine interferon regulatory factor 9，BoIRF9）基因，分析其生物学信息，并表达其抗原优势区。从感染水疱性口炎病毒（vesicular stomatitis virus，VSV）的EBK细胞中提取总RNA，以反转录得到的cDNA第一条链为模板，参考GenBank中牛IRF9基因序列设计特异性引物进行BoIRF9基因的扩增，对克隆得到的BoIRF9基因进行分子特征、高级结构与分子进化等生物学分析。根据BoIRF9的亲水性、抗原指数和表面可及性，设计1对特异性引物克隆BoIRF9的抗原优势区域片段，将其连接至pET30a（+）载体，构建重组表达质粒pET30a-IRF9-Part。将鉴定正确的质粒转化大肠杆菌Rosetta感受态细胞，经IPTG诱导表达抗原优势区域蛋白，运用SDS-PAGE和Western blotting方法进行蛋白质鉴定。结果显示，本研究克隆得到BoIRF9基因全长1 684 bp，CDS区长1 320 bp，编码439个氨基酸；BoIRF9氨基酸序列与人、马、猪、山羊相似性为80.0%~96.2%；进化树分析结果显示，荷斯坦奶牛与山羊亲缘关系最近，其次为猪、马、非洲象和人；BoIRF9蛋白的分子式为C₂₁₃₉H₃₃₄₂N₅₉₄O₆₅₈S₁₈，分子质量为48.5 ku，理论等电点（pI）为5.71；BoIRF9基因编码蛋白不存在跨膜结构，无信号肽，存在1个潜在的N-糖基化位点和多个磷酸化位点；BoIRF9蛋白二级结构含有115个α-螺旋、22个β-转角、68个延伸链和234个无规则卷曲；BoIRF9蛋白包含2个保守结构域，选择包含第一个保守结构域的第12－221位氨基酸作为抗原优势区域，克隆出648 bp的基因片段，并表达出分子质量为27 ku的重组蛋白。本研究结果可为干扰素调节因子的深入研究提供参考。

关键词: BoIRF9基因; 克隆; 原核表达; 生物信息学分析

Abstract: The aim of this study was to clone bovine interferon regulatory factor 9 (BoIRF9) gene,analyze its biological information,and express its antigenic dominant region.In this study,total RNA was extracted from EBK cells infected with vesicular stomatitis virus (VSV).Using the first chain of retroviral DNA as template,the specific primers were designed to amplify BoIRF9 gene according to bovine IRF9 gene sequence in GenBank.The molecular characteristics,advanced structure and molecular evolution of the cloned BoIRF9 gene were analyzed.According to the hydrophilicity,antigen index and surface accessibility of BoIRF9,a pair of specific primers were designed to clone the dominant antigen region fragment of BoIRF9 and connected it to pET30a (+) vector to construct the recombinant expression plasmid pET30a-IRF9-Part.The correct plasmid was transformed into E.coli Rosetta competent cells,and the dominant region protein was induced by IPTG.The protein was identified by SDS-PAGE and Western blotting.The results showed that the full-length of BoIRF9 gene was 1 684 bp,the length of BoIRF9 gene CDS was 1 320 bp,incoding 439 amino acids.The amino acids sequence similarity of BoIRF9 was 80.0% to 96.2% with Homo sapiens,Equus caballus,Sus scrofa and Capra hircus.Phylogenetic tree analysis showed that Holstein dairy cows had the closest relationship with Capra hircus,followed by Sus scrofa,Equus caballus,Loxodonta africana and Homo sapiens.The molecular formula of BoIRF9 protein was C₂₁₃₉H₃₃₄₂N₅₉₄O₆₅₈S₁₈,the molecular weight was 48.5 ku,and the theoretical isoelectric point (pI) was 5.71.There was no transmembrane structure and no signal peptide in BoIRF9 protein,and there was a potential N-glycosylation site and multiple phosphorylation sites.The secondary structure of BoIRF9 protein contained 115 alpha helix,22 beta turn,68 extend chain and 234 random coil,respectively.There were two conserved domains of BoIRF9 protein,a 648 bp gene fragment was cloned from the 12 to 221 amino acids containing the first conserved domain,and the recombinant protein with a molecular weight of 27 ku was expressed.This results could provide a reference for the further study of interferon regulatory factors.

Key words: BoIRF9 gene; cloning; prokaryotic expression; bioinformatics analysis

中图分类号:

卢盈妃, 黄春正, 戴海越, 郭永丽, 张忆蕾, 张博洋, 鲁兆祥, 徐玮程, 王君伟, 高明春. 牛干扰素调节因子9基因的克隆、序列分析与抗原优势区表达[J]. 中国畜牧兽医, 2020, 47(2): 326-335.

LU Yingfei, HUANG Chunzheng, DAI Haiyue, GUO Yongli, ZHANG Yilei, ZHANG Boyang, LU Zhaoxiang, XU Weicheng, WANG Junwei, GAO Mingchun. A Cloning,Sequence Analysis and Antigen Dominant Region Expression of Bovine Interferon Regulatory Factor 9 Gene[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(2): 326-335.

参考文献

[1] RICHEZ C,BARNETCHE T,MICELI-RICHARD C,et al.Role for interferon regulatory factors in autoimmunity[J].Joint Bone Spine,2010,77(6):525-531.
[2] ROMEO G,FIORUCCI G,CHIANTORE M V,et al.IRF-1 as a negative regulator of cell proliferation[J].Journal of Interferon & Cytokine Research,2002,22(1):39-47.
[3] LUKER K E,PICA C M,SCHREIBER R D,et al.Overexpression of IRF9 confers resistance to antimicrotubule agents in breast cancer cells[J].Cancer Research,2001,61(17):6540-6547.
[4] JIANG D S,LUO Y X,ZHANG R,et al.Interferon regulatory factor 9 protects against cardiac hypertrophy by targeting myocardin[J].Hypertension,2014,63(1):119-127.
[5] RAUCH I,ROSEBROCK F,HAINZL E,et al.Noncanonical effects of IRF9 in intestinal inflammation:More than type Ⅰ and type Ⅲ interferons[J].Molecular and Cellular Biology,2015,35(13):2332-2343.
[6] 沈敏.干扰素介导的自身炎症性疾病[J].中华临床免疫和变态反应杂志,2015,9(4):311-317. SHEN M.Interferon-mediated auto-inflammatory diseases[J].Chinese Journal of Allergy & Clinical Immunology,2015,9(4):311-317.(in Chinese)
[7] HUBER M,SUPRUNENKO T,ASHHURST T,et al.IRF9 prevents CD8⁽⁺⁾ T cell exhaustion in an extrinsic manner during acute lymphocytic choriomeningitis virus infection[J].Journal of Virology,2017,91(22):e01219-17.
[8] TAMURA T,YANAI H,SAVITSKY D,et al.The IRF family transcription factors in immunity and oncogenesis[J].Annual Review of Immunology,2008,26:535-584.
[9] 王玳玮,邓学梅.干扰素调节因子-1分子结构与生物学功能[J].中国畜牧兽医,2010,37(11):22-24. WANG D W,DENG X M.Molecular structure and function of interferon regulatory fector-1[J].China Animal Husbandry & Veterinary Medicine,2010,37(11):22-24.(in Chinese)
[10] 王建举,夏平安,尹彦涛,等.鸡干扰素的研究进展[J].中国畜牧兽医,2008,35(5):152-154. WANG J J,XIA P A,YIN Y T,et al.Research progress of chicken interferon[J].China Animal Husbandry & Veterinary Medicine,2008,35(5):152-154.(in Chinese)
[11] GARCIA-MORATO M B,CALVO A A,BRAVO-GALLEGO L Y,et al.Impaired control of multiple viral infections in a family with complete IRF9 deficiency[J].The Journal of Allergy and Clinical Immunology,2019,144(1):309-312.
[12] 李小玮,陈泽婷,智绪亭.IFN-α诱导的信号传导通路的研究进展[J].中国现代普通外科进展,2012,15(7):548-551. LI X W,CHEN Z T,ZHI X T.Advances in signal transduction pathways induced by IFN-α[J].Chinese Journal of Current Advances in General Surgery,2012,15(7):548-551.(in Chinese)
[13] POAT B,HAZARI S,CHANDRA P K,et al.Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication[J].Virology Journal,2010,7:265.
[14] WEIHUA X,LINDNER D J,KALVAKOLANU D V.The interferon-inducible murine p48(ISGF3gamma) gene is regulated by protooncogene c-myc[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(14):7227-7232.
[15] PAUN A,PITHA P M.The IRF family,revisited[J].Biochimie,2007,89(6-7):744-753.
[16] RENGACHARI S,GROISS S,DEVOS J M,et al.Structural basis of STAT2 recognition by IRF9 reveals molecular insights into ISGF3 function[J].Proceedings of the National Academy of Sciences of the United States of America,2018,115(4):E601-E609.
[17] HONDA K,TANIGUCHI T.IRFs:Master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors[J].Nature Reviews Immunology,2006,6(9):644-658.
[18] ESCALANTE C R,YIE J,THANOS D,et al.Structure of IRF-1 with bound DNA reveals determinants of interferon regulation[J].Nature,1998,391(6662):103-106.
[19] OZATO K,TAILOR P,KUBOTA T.The interferon regulatory factor family in host defense:Mechanism of action[J].The Journal of Biological Chemistry,2007,282(28):20065-20069.
[20] EROSHKIN A,MUSHEGIAN A.Conserved transactivation domain shared by interferon regulatory factors and Smad morphogens[J].Journal of Molecular Medicine,1999,77(5):403-405.
[21] SHI J,ZHANG Y,ZHANG J,et al.Expression regulation of zebrafish interferon regulatory factor 9 by promoter analysis[J].Developmental and Comparative Immunology,2013,41(4):534-543.
[22] LAU J F,PARISIEN J P,HORVATH C M.Interferon regulatory factor subcellular localization is determined by a bipartite nuclear localization signal in the DNA-binding domain and interaction with cytoplasmic retention factors[J].Proceedings of the National Academy of Sciences of the United States of America,2000,97(13):7278-7283.
[23] 王玉姣,郭永丽,罗修鑫,等.牛Ⅰ型干扰素受体α链与其配体结合活性的鉴定[J].中国畜牧兽医,2018,45(9):2610-2619. WANG Y J,GUO Y L,LUO X X,et al.Identification of ligand binding activity between bovine type Ⅰ interferon receptor α chain and its ligand[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2610-2619.(in Chinese)