H9N2禽流感病毒血凝素蛋白的主要特性及B/T细胞抗原表位预测分析
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摘要
目的应用生物信息学方法分析人源和禽源H9N2禽流感病毒血凝素蛋白(hemagglutinin,HA)的主要特性,预测可能的B/T细胞抗原表位。方法以H9N2禽流感病毒HA蛋白的氨基酸序列一级结构为基础分析其保守性,采用ProtParam预测H9N2禽流感病毒HA蛋白的理化特性,SOPMA预测其二级结构,MotifScan预测翻译后修饰位点,DNAStar分析其序列的亲水性指数、柔韧性指数、可及性参数以及抗原指数并推测B细胞抗原表位的可能位置,采用SYFPEITHI的T细胞表位预测工具分别预测CTL和Th细胞表位。结果人源和禽源H9N2禽流感病毒HA蛋白氨基酸序列分别有90和75个变异位点,其中有38个突变位点与氨基酸置换均相同,有14个突变位点相同但氨基酸置换不同。人源和禽源毒株中还各有38和23个特异性突变位点。HA蛋白存在多个糖基化、酰胺化及磷酸化等翻译后修饰位点。除了蛋白激酶C磷酸化位点外,其他类型的翻译后修饰位点在人源和禽源毒株中均有一些变化。经综合各种单参数,HA蛋白均为亲水性蛋白。人源和禽源H9N2禽流感病毒HA蛋白含有4个B细胞表位,8个CTL细胞表位和6个Th细胞表位,以人源和禽源的B细胞和Th细胞表位差异较大,而CTL细胞表位有部分序列相同。结论人源和禽源H9N2禽流感病毒HA蛋白的氨基酸序列有一定差异,其B/T细胞抗原表位的不同,可为H9N2禽流感病毒疫苗的研制提参考。
Objective To predict and compare the main characteristics and B-and T-cell epitopes of the hemagglutinin(HA)protein from avian influenza A(H9N2)infecting humans and birds. Methods Analysis of the extent to which the HA protein of influenza A(H9N2)was conserved performed based on its primary structure,the physical and chemical properties of which were predicted using ProtParam,and its secondary structure was predicted using the SOPMA online server.Post translation sites were predicted using MotifScan software.B-cell epitopes were analyzed using DNAStar software,which determined the hydrophilicity,flexible regions,surface probability,and antigenic index of the protein.T-cell epitopes were analyzed using SYFPEITHI. Results The H9N2 virus infecting humans had 90 mutations while the H9N2 virus infecting birds had 75 mutations,including 38 mutations causing the same amino acid substitution,and 14 mutations causing different amino acid substitutions.Thirty-eight mutations were specific to the H9N2 virus infecting humans and 23 were specific to the H9N2 virus infecting birds.In addition,the HA protein was found to have multiple post-translational modification sites.With the exception of 13 protein kinase C phosphorylation sites,other post-translational modification sites differed between the two types of H9N2 virus,such as 9 N-glycosylation sites in the H9N2 virus infecting humans and 7 N-glycosylation sites in the H9N2 virus infecting birds,6 casein kinase II phosphorylation sites in the H9N2 virus infecting humans and 7 casein kinase II phosphorylation sites in the H9N2 virus infecting birds,1 kinase phosphorylation site in the H9N2 virus infecting humans and 2 kinase phosphorylation sites in the H9N2 virus infecting birds,and 10 N-myristoylation sites in both the H9N2 virus infecting humans and the H9N2 virus infecting birds.However,mutations at these sites differed.In addition,4 potential B-cell epitopes,8 potential CTL cell epitopes,and 6 Thcell epitopes of the HA protein were predicted for the H9N2virus infecting humans and the H9N2virus infecting birds.The predicted potential B-cell epitopes and potential CTL cell epitopes of the HA protein differed widely between the two types of H9N2virus,though the Th cell epitopes of the HA protein were similar.Conclusion The amino acid sequences of the H9N2virus infecting humans and the H9N2virus infecting differ somewhat.Specific prediction of the characteristics and B-and T-cell epitopes of the HA protein might help to develop vaccines against H9N2based on peptides of human and avian strains of the H9N2virus.
Objective To predict and compare the main characteristics and B-and T-cell epitopes of the hemagglutinin(HA)protein from avian influenza A(H9N2)infecting humans and birds. Methods Analysis of the extent to which the HA protein of influenza A(H9N2)was conserved performed based on its primary structure,the physical and chemical properties of which were predicted using ProtParam,and its secondary structure was predicted using the SOPMA online server.Post translation sites were predicted using MotifScan software.B-cell epitopes were analyzed using DNAStar software,which determined the hydrophilicity,flexible regions,surface probability,and antigenic index of the protein.T-cell epitopes were analyzed using SYFPEITHI. Results The H9N2 virus infecting humans had 90 mutations while the H9N2 virus infecting birds had 75 mutations,including 38 mutations causing the same amino acid substitution,and 14 mutations causing different amino acid substitutions.Thirty-eight mutations were specific to the H9N2 virus infecting humans and 23 were specific to the H9N2 virus infecting birds.In addition,the HA protein was found to have multiple post-translational modification sites.With the exception of 13 protein kinase C phosphorylation sites,other post-translational modification sites differed between the two types of H9N2 virus,such as 9 N-glycosylation sites in the H9N2 virus infecting humans and 7 N-glycosylation sites in the H9N2 virus infecting birds,6 casein kinase II phosphorylation sites in the H9N2 virus infecting humans and 7 casein kinase II phosphorylation sites in the H9N2 virus infecting birds,1 kinase phosphorylation site in the H9N2 virus infecting humans and 2 kinase phosphorylation sites in the H9N2 virus infecting birds,and 10 N-myristoylation sites in both the H9N2 virus infecting humans and the H9N2 virus infecting birds.However,mutations at these sites differed.In addition,4 potential B-cell epitopes,8 potential CTL cell epitopes,and 6 Thcell epitopes of the HA protein were predicted for the H9N2virus infecting humans and the H9N2virus infecting birds.The predicted potential B-cell epitopes and potential CTL cell epitopes of the HA protein differed widely between the two types of H9N2virus,though the Th cell epitopes of the HA protein were similar.Conclusion The amino acid sequences of the H9N2virus infecting humans and the H9N2virus infecting differ somewhat.Specific prediction of the characteristics and B-and T-cell epitopes of the HA protein might help to develop vaccines against H9N2based on peptides of human and avian strains of the H9N2virus.
引文
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[22]Huang Y,Zhang H,Li X,et al.Detection and GENETIC Characteristics of H9N2avian influenza viruses from live poultry markets in Hunan Province,China[J].PloS One,2015,10(11):e0142584.
[23]陈陆,刘守川,赵军,等.不同H9N2亚型禽流感病毒分离株致病力研究及HA抗原性变异分析[J].中国农业科学,2011,44(24):5100-7.
[24]孟令楠,任志广,冀显亮,等.H5N1亚型流感病毒血凝素(HA)蛋白的表达鉴定及免疫原性研究[J].中国病原生物学杂志,2016,11(3):209-14.
[25]刘军.山东省首起人感染H7N9禽流感聚集性疫情及血凝素基因分析[J].中国病原生物学杂志,2015,10(10):888-91.
[26]杨依丽,吴军.病毒抗原糖基化与免疫关系的研究进展[J].生物技术通讯,2008,19(5):728-30.
[2]Gu M,Chen H,Li Q,et al.Enzootic genotype S of H9N2avian influenza viruses donates internal genes to emerging zoonotic influenza viruses in China[J].Vet Microbiol,2014,174(3-4):309-15.
[3]Cheung CL,Vijaykrishna D,Smith GJ,et al.Establishment of influenza A virus(H6N1)in minor poultry species in southern China[J].J Virol,2007,81(19):10402-12.
[4]Liu D,Shi W,Shi Y,et al.Origin and diversity of novel avian influenza A H7N9viruses causing human infection:phylogenetic,structural,and coalescent analyses[J].Lancet,2013,381(9881):1926-32.
[5]Chen H,Yuan H,Gao R,et al.Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8virus infection:a descriptive study[J].Lancet,2014,383(9918):714-21.
[6]Thompson JD,Higgins DG,Gibson TJ.CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice[J].Nucleic Acids Res,1994,22(22):4673-80.
[7]Schwede T,Kopp J,Guex N,et al.SWISS-MODEL:An automated protein homology-modeling server[J].Nucleic Acids Res,2003,31(13):3381-5.
[8]Wilkins MR,Gasteiger E,Bairoch A,et al.Protein identification and analysis tools in the ExPASy server[J].Methods Mol Biol,1998(112):531-52.
[9]Combet C,Blanchet C,Geourjon C,et al.NPS@:network protein sequence analysis[J].Trends Biochem Sci,2000,25(3):147-50.
[10]Nikitina TV,Tishchenko LI.Computational search for potential post-translational modification sites in human RNA polymerase III subunits[J].Mol Biol(Mosk),2005,39(3):437-44.
[11]Kyte J,Doolittle RF.A simple method for displaying the hydropathic character of a protein[J].J Mol Biol,1982,157(1):105-32.
[12]Vihinen M,Torkkila E,Riikonen P.Accuracy of protein flexibility predictions[J].Proteins,1994,19(2):141-9.
[13]Emini EA,Hughes JV,Perlow DS,et al.Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide[J].J Virol,1985,55(3):836-9.
[14]Jameson BA,Wolf H.The antigenic index:a novel algorithm for predicting antigenic determinants[J].Comput Appl Biosci,4(1):181-6.
[15]Wang F,Ye B.In silico cloning and B/T cell epitope prediction of triosephosphate isomerase from Echinococcus granulosus[J].Parasitol Res,2016,115(10):3991-8.
[16]赵奇,王远强,林勇,等.H5N1型高致病性禽流感病毒血凝素蛋白及神经氨酸酶的B细胞抗原表位预测[J].免疫学杂志,2010,26(5):408-11.
[17]Chen H,Deng G,Li Z,et al.The evolution of H5N1influenza viruses in ducks in southern China[J].Proc National Acad Sci USA,2004,101(28):10452-7.
[18]Wang X,Sun Q,Ye Z,et al.Computational approach for predicting the conserved B-cell epitopes of hemagglutinin H7subtype influenza virus[J].Exp Ther Med,2016,12(4):2439-46.
[19]El-Manzalawy Y,Honavar V.Recent advances in B-cell epitope prediction methods[J].Immunome Res,2010,6(Suppl 2):S2.
[20]蓝佳明,卢帅,邓瑶,等.MERS-CoV棘突蛋白表位多肽疫苗设计及在小鼠体内免疫效果分析[J].病毒学报,2016,32(1):77-81.
[21]柴丹,崔卓栋,郭焱,等.2013年广西分离株H9N2亚型禽流感病毒全基因组特性分析[J].中国病原生物学杂志,2017,12(1):10-4,19.
[22]Huang Y,Zhang H,Li X,et al.Detection and GENETIC Characteristics of H9N2avian influenza viruses from live poultry markets in Hunan Province,China[J].PloS One,2015,10(11):e0142584.
[23]陈陆,刘守川,赵军,等.不同H9N2亚型禽流感病毒分离株致病力研究及HA抗原性变异分析[J].中国农业科学,2011,44(24):5100-7.
[24]孟令楠,任志广,冀显亮,等.H5N1亚型流感病毒血凝素(HA)蛋白的表达鉴定及免疫原性研究[J].中国病原生物学杂志,2016,11(3):209-14.
[25]刘军.山东省首起人感染H7N9禽流感聚集性疫情及血凝素基因分析[J].中国病原生物学杂志,2015,10(10):888-91.
[26]杨依丽,吴军.病毒抗原糖基化与免疫关系的研究进展[J].生物技术通讯,2008,19(5):728-30.