鸡传染性支气管炎病毒S1蛋白抗原表位多肽的预测、鉴定及免疫效果的初步研究

doi:10.11843/j.issn.0366-6964.2020.09.023

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (9): 2257-2264.doi: 10.11843/j.issn.0366-6964.2020.09.023

鸡传染性支气管炎病毒S1蛋白抗原表位多肽的预测、鉴定及免疫效果的初步研究

范文胜, 朱丹, 张愉, 廖健淇, 王露, 雍璐, 韦平*, 磨美兰*

广西大学动物科学技术学院, 南宁 530005

收稿日期:2020-03-02 出版日期:2020-09-25 发布日期:2020-09-25
通讯作者: 韦平,主要从事禽病与病原分子生物学研究,E-mail:pingwei8@126.com;磨美兰,主要从事禽病与病原分子生物学研究,E-mail:momeilan@163.com
作者简介:范文胜(1983-),男,广东清远人,博士生,主要从事禽病与病原分子生物学研究,E-mail:2003424318@163.com;朱丹(1996-),女,安徽铜陵人,硕士生,主要从事禽病与病原分子生物学研究,E-mail:18956632047@163.com。
基金资助:
广西科技重大专项（桂科AA17204057）；国家自然科学基金项目（31860715）；广西自然科学基金项目（2018GXNSFAA281009；2019GXNSFAA245009）；广西研究生教育创新计划项目（YCBZ2018003）

Prediction, Identification and Preliminary Study on Immune Efficacy of S1 Protein Epitope-polypeptides of Avian Infectious Bronchitis Virus

FAN Wensheng, ZHU Dan, ZHANG Yu, LIAO Jianqi, WANG Lu, YONG Lu, WEI Ping*, MO Meilan*

College of Animal Science and Technology, Guangxi University, Nanning 530005, China

Received:2020-03-02 Online:2020-09-25 Published:2020-09-25

摘要/Abstract

摘要： 旨在对鸡传染性支气管炎病毒（IBV）H120株S1蛋白的B细胞及T细胞可能的表位进行预测并合成相应的多肽，然后免疫小鼠，并分析其免疫效果，以验证候选表位多肽的免疫原性。利用表位预测软件筛选并化学合成针对IBV H120株S1蛋白的5条表位多肽，然后免疫小鼠，通过间接ELISA、中和试验和流式细胞技术检测各表位多肽诱导的特异性抗体、中和抗体和外周血T细胞亚群。ELISA检测结果显示，5条表位多肽均具有良好的反应原性，免疫小鼠的血清效价高低顺序依次为Pep76-106 > Pep240-257 > Pep511-537 > Pep403-421 > Pep135-172；中和试验结果表明，5条多肽免疫小鼠的血清中和滴度均高于空白对照组，其高低顺序依次为Pep240-257=Pep403-421=Pep511-537>Pep76-106=Pep135-172；流式检测结果表明，5条多肽免疫小鼠在CD3⁺、CD4⁺CD8^-、CD8⁺CD4^- T淋巴细胞水平上均极显著高于空白对照组（P<0.01），CD3⁺T及CD4⁺CD8^-T淋巴细胞数大小顺序依次为Pep403-421 > Pep240-257 > Pep76-106 > Pep511-537 > Pep135-172，CD8⁺CD4^-T淋巴细胞数大小顺序依次为Pep403-421 > Pep76-106 > Pep511-537 > Pep240-257 > Pep135-172。合成的5条表位多肽中，Pep240-257、Pep76-106和Pep403-421可以诱导体液免疫，Pep403-421可以诱导细胞免疫，其中，Pep403-421可以同时诱导细胞免疫及体液免疫。本研究结果为深入了解S1蛋白的免疫学特性以及研发诊断试剂和有效表位疫苗奠定了基础。

关键词: 鸡传染性支气管炎病毒, H120株, S1蛋白, 表位多肽, 特异性抗体, 中和抗体, T淋巴细胞亚群

Abstract: For the sake of verifying the immunogenicity of candidate epitope-polypeptide, the B and T cell epitopes of S1 protein of avian infectious bronchitis virus (IBV) H120 strain were predicted and the corresponding epitope-polypeptides were synthesized， and then were used to immunize mice, the immune effect was analyzed. Five epitope-polypeptides against S1 protein of IBV H120 strain were selected by epitope prediction software and acquired by chemical synthesis, then were immunized to mice. The specific antibodies, neutralizing antibodies and T lymphocyte subsets induced by each epitope-polypeptides were analyzed by indirect ELISA, neutralization test and flow cytometry. The ELISA results showed that the five epitope-polypeptides had good reactivity. The antibody titers of antisera induced by the five epitope-polypeptides sorted from high to low as follows: Pep76-106, Pep240-257, Pep511-537, Pep403-421, Pep135-172. The neutralization test results showed that the neutralization titers of antisera induced by the five epitope-polypeptides groups in mice were higher than that of the blank control group, and the order of neutralization titers was Pep240-257 = Pep403-421 = Pep511-537 > Pep76-106 = Pep135-172. The flow cytometry results showed that the percentages of CD3⁺, CD4⁺CD8^- and CD8⁺CD4^- T lymphocytes in all the five epitope-polypeptides groups were significantly higher (P<0.01) than those in the blank control group. The number of the CD3⁺ and CD4⁺CD8^- T lymphocytes sorted from large to small as follows: Pep403-421, Pep240-257, Pep76-106, Pep511-537 and Pep135-172. The number of the CD8⁺CD4^- T lymphocytes sorted from large to small as follows: Pep403-421, Pep76-106, Pep511-537, Pep240-257 and Pep135-172. In conclusion, Pep240-257, Pep76-106 and Pep403-421 could induce humoral immunity among the five epitope-polypeptides, while Pep403-421 could induce cellular immunity. Thus, peptide of Pep403-421 could induce cellular immunity and humoral immunity. This study laid a foundation for further understanding the immunological characteristics of the S1 protein and the development of diagnostic reagents and effective epitope vaccines.

Key words: avian infectious bronchitis virus, H120 strain, S1 protein, epitope-polypeptide, specificity antibody, neutralizing antibody, T lymphocyte subsets

中图分类号:

S852.659.6

范文胜, 朱丹, 张愉, 廖健淇, 王露, 雍璐, 韦平, 磨美兰. 鸡传染性支气管炎病毒S1蛋白抗原表位多肽的预测、鉴定及免疫效果的初步研究[J]. 畜牧兽医学报, 2020, 51(9): 2257-2264.

FAN Wensheng, ZHU Dan, ZHANG Yu, LIAO Jianqi, WANG Lu, YONG Lu, WEI Ping, MO Meilan. Prediction, Identification and Preliminary Study on Immune Efficacy of S1 Protein Epitope-polypeptides of Avian Infectious Bronchitis Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(9): 2257-2264.

参考文献 20

[1]	JACKWOOD M W, LEE D H. Different evolutionary trajectories of vaccine-controlled and non-controlled avian infectious bronchitis viruses in commercial poultry[J]. PLoS One, 2017, 12(5):e0176709.
[2]	CAVANAGH D. Coronavirus avian infectious bronchitis virus[J]. Vet Res, 2007, 38(2):281-297.
[3]	FAN W S, TANG N, DONG Z H, et al. Genetic analysis of avian coronavirus infectious bronchitis virus in yellow chickens in southern China over the past decade:revealing the changes of genetic diversity, dominant genotypes, and selection pressure[J]. Viruses (Basel), 2019, 11(10):898.
[4]	黄梦姣, 张芸, 薛春宜, 等. 应对日益严峻的挑战:中国禽传染性支气管炎研究[J]. 微生物学通报, 2019, 46(7):1837-1849.HUANG M J, ZHANG Y, XUE C Y, et al. To meet the growing challenge:research of avian infectious bronchitis in China[J]. Microbiology China, 2019, 46(7):1837-1849. (in Chinese)
[5]	MO M L, LI M, HUANG B C, et al. Molecular characterization of major structural protein genes of avian coronavirus infectious bronchitis virus isolates in southern China[J]. Viruses (Basel), 2013, 5(12):3007-3020.
[6]	FAN W S, LI H M, HE Y N, et al. Immune protection conferred by three commonly used commercial live attenuated vaccines against the prevalent local strains of avian infectious bronchitis virus in southern China[J]. J Vet Med Sci, 2018, 80(9):1438-1444.
[7]	LI M, WANG X Y, WEI P, et al. Serotype and genotype diversity of infectious bronchitis viruses isolated during 1985-2008 in Guangxi, China[J]. Arch Virol, 2012, 157(3):467-474.
[8]	BANDE F, ARSHAD S S, HAIR BEJO M, et al. Prediction and in silico identification of novel B-Cells and T-Cells epitopes in the S1-spike glycoprotein of M41 and CR88 (793/B) infectious bronchitis virus serotypes for application in peptide vaccines[J]. Adv Bioinformatics, 2016, 2016:5484972.
[9]	YUAN Y, ZHANG Z P, HE Y N, et al. Protection against virulent infectious bronchitis virus challenge conferred by a recombinant baculovirus Co-expressing S1 and N proteins[J]. Viruses (Basel), 2018, 10(7):347.
[10]	TAN L, LIAO Y, FAN J, et al. Prediction and identification of novel IBV S1 protein derived CTL epitopes in chicken[J]. Vaccine, 2016, 34(3):380-386.
[11]	XU P W, WU X, WANG H N, et al. Assembly and immunogenicity of baculovirus-derived infectious bronchitis virus-like particles carrying membrane, envelope and the recombinant spike proteins[J]. Biotechnol Lett, 2016, 38(2):299-304.
[12]	WU X, ZHAI X W, LAI Y, et al. Construction and immunogenicity of novel chimeric virus-like particles bearing antigens of infectious bronchitis virus and Newcastle disease virus[J]. Viruses (Basel), 2019, 11(3):254.
[13]	ARCHETTI I, HORSFALL F L Jr. Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum[J]. J Exp Med, 1950, 92(5):441-462.
[14]	VALASTRO V, HOLMES E C, BRITTON P, et al. S1 gene-based phylogeny of infectious bronchitis virus:an attempt to harmonize virus classification[J]. Infect Genet Evol, 2016, 39:349-364.
[15]	吴翠兰, 何怡宁, 李和鸣, 等. 一株重组鸡传染性支气管炎病毒的分离及结构蛋白基因变异和血清型分析[J]. 畜牧兽医学报, 2015, 46(5):815-823.WU C L, HE Y N, LI H M, et al. Isolation and analysis of structural protein genes variation and serotype of a recombinant strain of chicken infectious bronchitis virus[J]. Acta Veterinaria et Zootechnica Sinica, 2015, 46(5):815-823. (in Chinese)
[16]	刘文华, 王志亮, 吴晓东, 等. 小反刍兽疫病毒化学合成表位多肽对小鼠的免疫效果研究[J]. 畜牧兽医学报, 2015, 46(3):438-444.LIU W H, WANG Z L, WU X D, et al. Research of the immune efficacy of chemical synthetic epitope polypeptide of peste des petits ruminants virus on mice[J]. Acta Veterinaria et Zootechnica Sinica, 2015, 46(3):438-444. (in Chinese)
[17]	PROMKUNTOD N, VAN EIJNDHOVEN R E W, DE VRIEZE G, et al. Mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus[J]. Virology, 2014, 448:26-32.
[18]	WICKRAMASINGHE I N A, VAN BEURDEN S J, WEERTS E A W S, et al. The avian coronavirus spike protein[J]. Virus Res, 2014, 194:37-48.
[19]	IGNJATOVIC J, SAPATS S. Identification of previously unknown antigenic epitopes on the S and N proteins of avian infectious bronchitis virus[J]. Arch Virol, 2005, 150(9):1813-1831.
[20]	ZOU N L, XIA J, WANG F Y, et al. Two novel neutralizing antigenic epitopes of the s1 subunit protein of a QX-like avian infectious bronchitis virus strain Sczy3 as revealed using a phage display peptide library[J]. Vet Immunol Immunopathol, 2015, 168(1-2):49-55.

鸡传染性支气管炎病毒S1蛋白抗原表位多肽的预测、鉴定及免疫效果的初步研究

Prediction, Identification and Preliminary Study on Immune Efficacy of S1 Protein Epitope-polypeptides of Avian Infectious Bronchitis Virus

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 20

相关文章 15

编辑推荐

Metrics

本文评价

[1]	陈璐, 常新宇, 沈嘉忱, 沈瑞廷, 赵振华, 侯晓林. 鸡传染性支气管炎病毒感染HD11细胞的转录组分析[J]. 畜牧兽医学报, 2023, 54(11): 4860-4865.
[2]	刘燕坤, 林焱, 朱伟云. 噬菌体的跨黏膜转位以及噬菌体对机体免疫影响的研究进展[J]. 畜牧兽医学报, 2021, 52(3): 588-595.
[3]	吴艳虹, 赵永强, 韦韬, 章秀婷, 丛丽, 岳志刚, 肖家美, 邵西群. 貉源阿留申病毒VP2和NS1蛋白的抗原性预测[J]. 畜牧兽医学报, 2020, 51(6): 1399-1407.
[4]	侯林杉, 贾敬亮, 顾文源, 刘宝京, 师乾凯, 袁广富, 陈少杰, 范京惠, 左玉柱. 基于猪丁型冠状病毒重组S1蛋白间接ELISA抗体检测方法的建立与应用[J]. 畜牧兽医学报, 2019, 50(8): 1642-1648.
[5]	李闻, 马思续, 李想通, 孙杨杨, 魏凤灵, 许瑞勤, 杨国宇, 夏平安, 张改平. PRRSV VR2332弱毒株经滴鼻、注射途径接种仔猪的病毒血症和抗体产生的差异分析[J]. 畜牧兽医学报, 2019, 50(2): 373-381.
[6]	姚作俊, 郝达仁, 白云, 颜国华, 王海敏, 宋勤叶, 李潭清. 检测猪流行性腹泻病毒S1蛋白抗体的间接ELISA方法[J]. 畜牧兽医学报, 2017, 48(6): 1085-1091.
[7]	赵雪艳, 王彦平, 王怀中, 郭建凤, 齐波, 王继英. TNFRSF1A基因CpG岛甲基化与基因表达及T淋巴细胞亚群指标的相关性分析[J]. 畜牧兽医学报, 2017, 48(10): 1815-1824.
[8]	提金凤，李志杰，李秀丽，张敏敏，张媛媛，刁有祥. 坦布苏病毒NS1蛋白的表达及ELISA检测方法的建立[J]. 畜牧兽医学报, 2016, 47(5): 970-977.
[9]	朱凤珠，鲁梅，黄庆华，杨少华，黄艳艳，吴家强，谭刘刚，张秀美，崔言顺，许传田. 鸡传染性支气管炎病毒S1蛋白MHCⅠ分子限制性T细胞表位的鉴定[J]. 畜牧兽医学报, 2016, 47(3): 559-565.
[10]	胡北侠，许传田，杨少华，黄艳艳，张琳，黄庆华，霍亚飞，张伟，张秀美. 山东地区鸡传染性支气管炎病毒抗原相关性和血清分型[J]. 畜牧兽医学报, 2015, 46(4): 615-623.
[11]	刘文华，王志亮，吴晓东，孙成友. 小反刍兽疫病毒化学合成表位多肽对小鼠的免疫效果研究[J]. 畜牧兽医学报, 2015, 46(3): 438-444.
[12]	陈善真，赵焱，李中圣，罗均，陈克宏，王贵平，李其昌. 含有Th细胞表位的合成PCV2 ORF2 Cap多肽抗原的表达及免疫原性分析[J]. 畜牧兽医学报, 2015, 46(12): 2273-2281.
[13]	胡北侠，杨少华，许传田，张伟，张琳，黄庆华，张秀美，黄艳艳，文心田. 2008—2012年山东省鸡传染性支气管炎病毒遗传演化分析和致病性研究[J]. 畜牧兽医学报, 2014, 45(5): 795-801.
[14]	谭伟，谢芝勋. 甲型流感病毒NS1、NS2和NS3蛋白的研究进展[J]. 畜牧兽医学报, 2014, 45(12): 1911-1916.
[15]	王怀中，林松，王彦平，武英，郭建凤，王继英. 品种和母猪对仔猪血常规和T淋巴细胞亚群性状的效应分析[J]. 畜牧兽医学报, 2014, 45(11): 1760-1766.