牛病毒性腹泻病毒NS3蛋白的生物信息学分析及多表位筛选

doi:10.16431/j.cnki.1671-7236.2019.12.003

摘要/Abstract

摘要： 本研究旨在对牛病毒性腹泻病毒（BVDV）NS3蛋白进行生物信息学分析及T细胞和B细胞表位的筛选。从GenBank数据库中找到NS3蛋白的氨基酸序列，用Expasy ProtParam在线服务器分析所得序列理化性质。使用TMHMM Server分析跨膜结构域，使用DNAStar软件预测NS3蛋白的二级结构，为了更准确地预测蛋白质的二级结构，使用SOPMA分析软件进行了二次预测。使用Phyre2在线服务器预测NS3蛋白的三级结构，并用Raswin软件标出各个元素所处的位置。使用4种预测软件（BCPREDS、ABCpred、BepiPred和SVMTriP）分析NS3蛋白的线性B细胞表位，使用NetMHCⅡpan预测CD4⁺ T细胞表位，使用NetBoLApan和IEDB预测CD8⁺ T细胞表位，将所得到的结果进行T细胞和B细胞表位筛选。结果表明，NS3蛋白质由683个氨基酸组成，分子质量为75 ku，理论等电点（pI）为8.31，化学式为C₃₃₄₇H₅₃₄₆N₉₁₆O₁₀₀₉S₂₈，不稳定系数为35.93，平均亲水性（GRAVY）值为-0.267，表明NS3为稳定性亲水蛋白质。TMHMM结果显示，NS3蛋白不具有跨膜结构域。SOPMA结果表明，在NS3蛋白的二级结构中，α-螺旋、β-折叠、β-转角和无规则卷曲分别约占30.75%、22.55%、8.35%和38.35%，用DNAStar软件分别标出了α-螺旋、β-折叠、β-转角和无规则卷曲所处的位置。运用4种不同的B细胞预测软件筛选出8个线性表位：12-16、289-298、349-360、394-407、421-432、489-498、515-525和665-679位氨基酸。用NetMHCⅡpan筛选出4个T细胞表位：248-262、315-320、400-414和482-496位氨基酸。本研究为BVDV NS3蛋白优势抗原的筛选提供了理论依据。

关键词: 牛病毒性腹泻病毒(BVDV); NS3蛋白; 生物信息学分析; B细胞; T细胞; 表位筛选

Abstract: This study was aimed to perform bioinformatics analysis and screening of T cell and B cell epitopes for bovine viral diarrhea virus (BVDV) NS3 protein.First,the amino acid sequence of the NS3 protein was found in GenBank database.The resulting sequence was analyzed for physicochemical properties using an Expasy ProtParam online server.Then TMHMM Server was used to analyze the transmembrane domain.The DNAStar software was used to predict the secondary structure of the NS3 protein.To more accurately predict the secondary structure of a protein,the SOPMA analysis software performed a second prediction.The Phyre2 online server was used to predict the tertiary structure of the NS3 protein,and Raswin software was used to identify the location of each element.Linear B cell epitopes of NS3 protein were analyzed by four predictive software (BCPREDS,ABCpred,BepiPred and SVMTriP),CD4⁺ T cell epitopes were predicted by NetMHCⅡpan,NetBoLApan and IEDB were used to predict CD8⁺ T cell epitopes.Finally,the results obtained were screened for T cell and B cell epitopes.The results showed that the NS3 protein consisted of 683 amino acids,the molecular weight was 75 ku,the theoretical pI value was 8.31,chemical formula was C₃₃₄₇H₅₃₄₆N₉₁₆O₁₀₀₉S₂₈,the instability coefficient was 35.93,the average value of hydrophilicity (GRAVY) was -0.267,it was indicated that NS3 was a stable and hydrophilic protein.The TMHMM results showed that the NS3 protein did not have a transmembrane domain.SOPMA results showed that in the secondary structure of NS3 protein,α-helix β-fold,β-turn and random coil accounted for about 30.75%,22.55%,8.35% and 38.35%,respectively.DNAStar software marked their respective position.Eight linear epitopes were screened using four different B cell prediction software:12-16,289-298,349-360,394-407,421-432,489-498,515-525 and 665-679 amino acids.Four T cell epitopes were screened using NetMHCⅡpan:248-262,315-320,400-414 and 482-496 amino acids.This study provided a theoretical basis for the screening of the dominant antigen of NS3 protein in BVDV.

Key words: bovine viral diarrhea virus (BVDV); NS3 protein; bioinformatics analysis; B cells; T cells; epitope screening

中图分类号:

S852.65⁺3

何金科, 杨亚军, 邓肖玉, 何延华, 张超, 马忠臣, 王勇, 陈创夫. 牛病毒性腹泻病毒NS3蛋白的生物信息学分析及多表位筛选[J]. 中国畜牧兽医, 2019, 46(12): 3475-3485.

HE Jinke, YANG Yajun, DENG Xiaoyu, HE Yanhua, ZHANG Chao, MA Zhongchen, WANG Yong, CHEN Chuangfu. Bioinformatics Analysis and Multi-epitope Screening of NS3 Protein of Bovine Viral Diarrhea Virus[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(12): 3475-3485.

参考文献

[1] MACHADO G,EGOCHEAGA R M F,HEIN H E,et al.Bovine viral diarrhoea virus (BVDV) in dairy cattle:A matched case-control study[J].Transboundary & Emerging Diseases,2016,63(1):e1-13.
[2] 陈新诺,张斌.牛病毒性腹泻病毒的分子生物学研究进展[J].中国畜牧兽医,2017,44(11):32-37. CHEN X N,ZHANG B.Progress in molecular biology of bovine viral diarrhea virus[J].China Animal Husbandry & Veterinary Medicine,2017,44(11):32-37.(in Chinese)
[3] 陈备娟,高全新,朱毅兴,等.牛病毒性腹泻病的流行病学调查[J].上海畜牧兽医通讯,2010,5:43-44. CHEN B J,GAO Q X,ZHU Y X,et al.Epidemiological investigation of bovine viral diarrhea[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2010,5:43-44.(in Chinese)
[4] VILTROP A,ALAOTS J,PARN M,et al.Natural changes in the spread of bovine viral diarrhoea virus (BVDV) among Estonian cattle[J].Journal of Veterinary Medicine B Infectious Diseases & Veterinary Public Health,2010,49(6):263-269.
[5] HOUE H.Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections[J].Veterinary microbiology,1999,64(2-3):89-107.
[6] FU Q,SHI H,REN Y,et al.Bovine viral diarrhea virus infection induces autophagy in MDBK cells[J].Journal of Microbiology, 2014,52(7):619-625.
[7] TAUTZ N,TEWS B A,MEYERS G.The molecular biology of pestiviruses[J].Advances in Virus Research,2015,93:47-160.
[8] BOLIN S R.Immunogens of bovine viral diarrhea virus[J].Veterinary Microbiology,1993,37(3-4):263-271.
[9] ZOTH S C,TABOGA O.Multiple recombinant ELISA for the detection of bovine viral diarrhoea virus antibodies in cattle sera[J].Journal of Virological Methods,2006,138(1-2):99-108.
[10] WISKERCHEN M,COLLETT M S.Pestivirus gene expression:Protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing[J].Virology,1991,184(1):341-350.
[11] AGAPOV E V,MURRAY CL,FROLOV I,et al.Uncleaved NS2-3 is required for production of infectious bovine viral diarrhea virus[J].Journal of Virology,2004,78(5):2414-2425.
[12] 侯佩莉,孙涛,何洪彬.牛病毒性腹泻病毒非结构蛋白NS3的研究进展[J].中国畜牧兽医,2012,39(5):68-72. HOU P L,SUN T,HE H B.Research progress of non-structural protein NS3 of bovine viral diarrhea virus[J].China Animal Husbandry & Veterinary Medicine,2012,39(5):68-72.(in Chinese)
[13] 贾莹,李岩,尹鑫,等.牛病毒性腹泻病病毒NS3表位串联蛋白表达及抗体间接ELISA方法的建立[J].畜牧兽医学报,2011,42(8):1120-1125. JIA Y,LI Y,YIN X,et al.Establishment of NS3 epitope tandem protein expression and indirect ELISA for bovine viral diarrhea virus[J].Chinese Journal of Animal and Veterinary Sciences,2011,42(8):1120-1125.(in Chinese)
[14] COLLEN T,MORRISON W I.CD4⁺ T-cell responses to bovine viral diarrhoea virus in cattle[J].Virus Research,2000,67(1):67-80.
[15] 李岩,聂明非,魏伟,等.牛病毒性腹泻病毒NS3基因的序列分析、表达与抗原性鉴定[J].生物工程学报,2010,26(3):311-316. LI Y,NIE M F,WEI W,et al.Sequence analysis,expression and antigenic identification of NS3 gene of bovine viral diarrhea virus[J].Chinese Journal of Biotechnology,2010,26(3):311-316.(in Chinese)
[16] KHATOON N,PANDEY R K,PRAJAPATI V K.Exploring Leishmania secretory proteins to design B and T cell multi-epitope subunit vaccine using immunoinformatics approach[J].Scientific Reports,2017,7(1):8285.
[17] POURSEIF M M,MOGHADDAM G,SAEEDI N,et al.Current status and future prospective of vaccine development against Echinococcus granulosus[J].Biologicals,2018,51:1-11.
[18] GUIMARÃES-PEIXOTO R P M,PINTO P S A,SANTOS M R,et al.Development of the multi-epitope chimeric antigen rqTSA-25 from Taenia saginata for serological diagnosis of bovine cysticercosis[J].PLoS Neglected Tropical Diseases,2018,12(4):e0006371.
[19] THOMASINI R L,SOUZA H G A,BRUNA-ROMERO O,et al.Evaluation of a recombinant multiepitope antigen for diagnosis of hepatitis C virus:A lower cost alternative for antigen production[J].Journal of Clinical Laboratory Analysis,2018,32(6):e22410.
[20] LV C,FU Z,LU K,et al.A perspective for improving the sensitivity of detection:The application of multi-epitope recombinant antigen in serological analysis of buffalo schistosomiasis[J].Acta Tropica,2018,183:14-18.
[21] REHMAN I,BOTELHO S.Biochemistry,Tertiary Structure,Protein[M].StatPearls Publishing,2018.
[22] 李江英,白雪娟,梁艳,等.用DNAStar软件预测Rv1410c结核分枝杆菌蛋白抗原表位[J].细胞与分子免疫学杂志,2015,31(4):474-477. LI J Y,BAI X J,LIANG Y,et al.Prediction of Rv1410c Mycobacterium tuberculosis protein epitopes by DNAStar software[J].Chinese Journal of Cellular and Molecular Immunology,2015,31(4):474-477.(in Chinese)
[23] WATERHOUSE A,BERTONI M,BIENERT S,et al.SWISS-MODEL:Homology modelling of protein structures and complexes[J].Nucleic Acids Research,2018,46(W1):W296-303.
[24] 王艳华,张德林,殷宏,等.抗原表位预测方法的研究进展[J].中国兽医科学,2009,39(10):938-940. WANG Y H,ZHANG D L,YIN H,et al.Research progress in antigen epitope prediction methods[J].Chinese Veterinary Science,2009,39(10):938-940.(in Chinese)
[25] NIELSEN M,CONNELLEY T,TERNETTE N.Improved prediction of bovine leucocyte antigens (BoLA) presented ligands by use of mass-spectrometry-determined ligand and in vitro binding data[J].Journal of Proteome Research,2018,17(1):559-567.
[26] ELLIS S A,MORRISON W I,MACHUGH N D,et al.Serological and molecular diversity in the cattle MHC class Ⅰ region[J].Immunogenetics,2005,57(8):601-606.
[27] KRISHNA S,ANDERSON K S.T-cell Epitope Discovery for Therapeutic Cancer Vaccines[M].New York:Humana Press,2016.
[28] FARRELL D,JONES G,PIRSON C,et al.Integrated computational prediction and experimental validation identifies promiscuous T cell epitopes in the proteome of Mycobacterium bovis[J].Microbial Genomics,2016,2(8):e000071.