摘要
表位肽疫苗作为一种新型疫苗,已成为口蹄疫(Foot-and-Mouth Disease,FMD)疫苗研究的热点之一。由于细胞免疫在口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)的免疫应答中发挥着重要作用,而结构蛋白VP1内又包含着FMDV的主要抗原位点。因此,筛选和鉴定VP1蛋白有效的H-2Ld限制性T细胞抗原表位,不仅可为进一步研究针对FMDV自然宿主的T细胞抗原表位提供方法和依据,也可为进一步研制FMDV表位肽疫苗奠定基础。
本研究通过扩增并测序,获得了完整的FMDV AF/72株VP1序列。采用密码子优化表达方法,在大肠杆菌中高效表达了该VP1蛋白。经过SDS-PAGE电泳及Western Blotting检测,其分子量与预期大小相符合,并且保持了良好的免疫原性。与此同时,利用相关生物信息学软件对VP1中潜在的H-2Dd、H-2Kd、H-2Ld限制性T细胞表位进行了预测分析,初步筛选并合成了30个10肽片段作为候选表位肽段。将VP1蛋白免疫BALB/c小鼠,制备经VP1蛋白致敏的脾淋巴细胞,与30个候选表位肽段分别进行体外共培养刺激,经淋巴细胞增殖实验和γ干扰素ELISPOT实验,鉴定出2个H-2d限制性T细胞表位,即H-2Kd限制性T细胞表位pK1(AYHKGPFTRL)和H-2Dd限制性T细胞表位pD7(TGESADPVTT)。本研究筛选并鉴定了FMDV AF/72株结构蛋白VP1中的H-2d限制性T细胞表位,为FMDV T细胞表位的研究提供方法和依据,为进一步研制FMDV表位肽疫苗奠定了基础。此外,通过鉴定小鼠这一FMDV研究中常用动物模型的CTL T细胞表位,也有利于了解细胞毒性淋巴细胞在抗FMDV方面的作用。
Epitope vaccine, as a new kind of vaccine, has become one of research hot spots of FMD vaccine. Screening and identification of T cell epitope of FMDV is significant to developing high-efficiency vaccines and further research due to cellular immunity play an important role in immune response of FMDV infection and structural protein VP1 include main antigen sites of FMDV.
In this study, The Complete sequence of VP1 of FMDV AF / 72 strains was obtain through cloning and sequencing, and then VP1 protein have high-level expressed in E. coli using the method of codon optimization.The detection of SDS-PAGE and Western Blotting suggest that the molecular weight of expressed VP1 proteins correspond to expected and keep a good immunogenicity. Meanwhile,30 the H-2d Restricted T Cell Epitope of VP1 have been forecast and synthesis using the relative bioinformatics software.The VP1 protein was used for immunizing BALB/c mice and spleen lymphocytes were isolated, and vitro common training stimulus was carried out to potential H-2Dd, H-2Kd and H-2Ld restricted T cell epitope.The H-2Kd restricted T cell epitope pK1 (AYHKGPFTRL) and H-2Dd restricted T cell epitope pD7 (GFIMDRFVKI) were identified through lymphocyte proliferation assay and IFN-γELISPOT experiments.This study provides method and basis through screen and identifies H-2d restricted T cell epitope of structural protein VP1 of foot-and-mouth disease virus type A. in addition, screening and identification of mouse's CTL epitope contribute to understanding the effect of cytotoxic lymphocytes against FMDV.
引文
1.范公忍,汪毅,张桂霞.有限稀释定量PCR检测血清HBV DNA的临床应用及意义[J].肝脏,2000,1(5):45-46
2.高蓉,舒翠莉,李靖等.丙肝病毒高变区1相关基因免疫小鼠诱导细胞免疫反应的研究[J].第二军医大学学报,2005,26(4):425-428
3.江勤,谢毅,谢弘.有限稀释法定量分析脐血中异基因特异的辅助性T细胞[J].中华血液学杂志,1997,11(18):588-591
4.刘光亮,王群,童铁钢等.H5亚型禽流感病毒核蛋白基因重组质粒的构建及其在Hela细胞的瞬时表达[J].中国兽医科学,2006,36(3):189-192
5.李梅,李宝全,宋文刚等.检测NDV诱导特异性细胞免疫的ELISPOT试验方法建立[J].中国免疫学杂志,2008,10(24):935-938
6.李庆章,刘忠贵.MTT比色分析法检测鸡T淋巴细胞体外增殖反应的研究[J].中国兽医杂志,1994,9:18-20
7.李祥瑞,金红,王秀丽等.以MTT比色法检测鸡脾淋巴细胞转化效果[J].畜牧与兽医,1996,28(1):3-5.
8.裘毅刚,陈月,张丽芸等.C3在迟发型超敏反应中的作用[J].第一军医大学学报,2005,25(11)18-21
9.张春涛,刘春雨,吴瑜等.ELISPOT,MHC肽五聚体和ICCD三种检测特异性细胞免疫应答方法的比较研究[J].中华实验和临床病毒学杂志,2006,20(4):416-419
10.司兴奎,郭鑫,杨汉春.猪圆环病毒2型感染后猪外周血淋巴细胞IL-2、IL-4、IL-10、IL-12p40、IFN-γ和TNF-αmRNA转录的变化[J].畜牧兽医学报,2009,40( 1) :78-82
11.周光炎.免疫学原理[M].北京:上海科学技术出版社,2007
12.郑敏,金宁一,尹革芬等.表达口蹄疫病毒复合多表位基因的重组鸡痘病毒以及DNA疫苗在豚鼠中的免疫原性研究[J].中国人兽共患病杂志,2005,21(11):931-936.
13.张宁媛,胡娅莉,孙海翔等.小鼠单个卵母细胞和早期胚胎中结节性硬化综合征2型基因mRNA定量检测方法的建立[J].中华男科学杂志,2009, 15 (5) : 417 - 421
14.郑永平,吴昌辉,廖家.尖锐湿疣组织中IL- 18 mRNA定量表达及意义[J].中国热带医学,2009 ,12(9):2221-2251
15.张中旺,张永光.抗原表位的研究方法及口蹄疫病毒抗原表位的研究进展[J].微生物学通报,2008,35(8):1302-1310
16.吴建设,张日俊,周毓平.全血法鸡淋巴细胞转化试验最佳试验条件的研究[J].畜牧兽医学报,1997,28(3):212-216
17.徐波,张建超,付作明等.地锦草总黄酮对小鼠免疫功能及细胞因子mRNA表达影响[J].中兽医医药杂志2010, 5:22-26
18.谢庆阁.口蹄疫[M].北京:中国农业出版社,2004:29-31
19.许文彬,徐丽慧,卢宏松等.棉酚对Jurkat T细胞增殖与凋亡的影响[J].细胞生物学杂志,2008,30: 742-746
20.赵晓甦,黄晓军.定量PCR检测细胞因子mRNA表达用于急性移植物抗宿主病诊断的研究[J].中华血液学杂志,2010,31(8) :12
21. Ana M. Espino, Francheska Rivera.Quantitation of cytokine mRNA by real-time RT-PCR during a vaccination trial in a rabbit model of fascioliasis[J].Veterinary Parasitology, 2010,169:82-92
22. Appay V, Dunbar PR, Callan M, et al. Memory CD8 T cells vary in differentiation phenotype in different persistent virus infections. Nat Med 2002, 8: 79 -85.
23. Arakaki R,Ishimaru N,Saito I,et a1.Development of autoimmune exocrinopathy resembling Sj- grens syndrome in adoptively transferred mice with autoreactive CD4+ T cells.Arthfitis Rheum, 2003,48:23-28
24. Bansal A,Carlson J,Matthews P,et al. Cryptic CTL epitopes derived from antisense transcription are frequently recognized in HIV-1 infection[J]. Retrovirology ,2009, 6(3): 23
25. Barey S,Huang ML,Corey L,et a1.Longitudinal analysis of herpes simplex virus-specific CD4+ cell clonotypes in infected tissues and blood[J].J Infect Dis,2005,191(12):2012-2021.
26. Barta O,Barta V.Optimum conditions for the chicken lymphocyte transformation test[J].Avian Diseases,1992,36(2):945-955.
27. Basto s R G, Dellago stin O A, Barletta R G, et al. Immune response of pigs inoculated with M ycobacterium bovis BCG expressing a truncated form of GP5 and M protein of porcine reproductive and respiratory syndrome virus [J]. Vaccine, 2004, 22: 467-474.
28. Benito JM, Lopez M, Lozano S, Martinez P, Kuroda M, Gonzalez-Lahoz J, Soriano V. Phenotype and functional characteristics of HIV-specific cytotoxic CD8+ T cells in chronically infected patients:dual effects of highly active antiretroviral therapy[J].J Acquir Immune Defic Syndr, 2003; 34:255-266
29. Betts MR,Casazza JP,Patterson BA,et al.Putative immunodominant human immunodeficiency virus-specific CD8+ T-cell responses cannot be predicted by major histocompatibility complex class I haplotype[J].J Virol,2000,74(19):9144-9151
30. Bhasin M, Raghava GPS. Prediction of CTL epitopes using QM, SVM and ANN techniques [J]. Vaccine, 2004, 22: 3195-3204
31. Blanco E, McCullough K,Summerfield A.Interspecies major histocompatibility complex-restricted Th cell epitope on foot-and-mouth disease virus capsid protein VP4[J].Virol, 2000, 74(10): 4902 -4907.
32. Blanco E,Garcia-Briones M,Sanz-Parra A,et al.Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus[J].Virol, 2001, 75(7):3164-3174.
33. Bounous V.Immunological effects of reticuloen-dotheliosis virus as potential contaminant of Marek's disease vaccines[J].Avian Pathol.1977.6:383-393.
34. Catherine Mazza, Bernard Malissen. What guides MHC-restricted TCR recognition [J].Seminars in Immunology. 2007, 19:225–235
35. Bras-Goncalves R,Le Gal FA,Avril MF,et al.In situ detection of antigen-specific tumor-infilt ratinglymphocytes using newly designed tetramers[J].J Immunol Methods,2003,280(122):103-111.
36. Chang Yi Wang, Tseng Yuan Chang, Alan M. Walfiel,et al.Effective synthetic peptide vaccine for foot-and-mouth disease in swine [J], Vaccine,2002, 20(19-20):2603-2610.
37. Collen T,Dimarchi R,Doel TR.AT cell epitope in VP1 of foot-and-mouth disease virus is immunodominant for vaccinated cattle [J].Immunol,1991,146(2): 749 -755.
38. Davis MM,Bjorkman PJ.T cell antigen receptor genes and T cell recognition[J].Nature,1988, 334(6181) :395-402
39. Dekun Chen, Allan Gould, Huayang Guo et al.HBsAg-vectored vaccines simultaneously deliver CTL responses to protective epitopes from multiple viral pathogens[J]. Virology, 2010,398:68-78
40. Deng Y,Yewdell JW,Eisenlohr LC,et al.MHC affinity,peptide liberation,T cell repertoire and immunodominance all contribute to the paucity of MHC class I-restricted peptides recognized by antiviral CTL [J].J Immunol,1997,158(4) :1507
41. Dhabar, F.S., McEwen, B.S.Acute stress enhances while chronic stress suppresses cell-mediated immunity in vivo: a potential role for leukocyte trafficking [J].Brain. Beh. Immun. 1997, 11, 286- 306.
42. Esteban Domingo, Cristina Escarm?′s, Eric Baranowski, Carmen M. Ruiz-Jarabo, Elisa Carrillo, Juan Ignacio Nu′n?ez, Francisco Sobrino.Evolution of foot-and-mouth disease virus[J].Virus Research,2003, 91:47-63
43. Filgueira MP,Wigdorovitz A,Romera A,et al. Detectionand characterization of functional T-Cell epitopes on the structural proteins V2,VP3, and VP4 of foot and mouth disease virus O1Campos[J]. Virology, 2000, 271: 234?239.
44. Francisco Co′rdoba, Grazyna Wieczorek, Ellen Preussing,et al. Modeling of delayed type hypersensitivity (DTH) in the non-human primate (NHP) [J].Drug Discovery Today:Disease Models,2008,5(2):63-71
45. Foster M, Cook A, Cedillo L, Parkhouse RM. Serological and cellular immune responses to non-structural proteins in animals infected with FMDV [J]. Vet. Q, 1998, 20 (2):S28–S30.
46. Garc?′a-Briones, Esther Blanco, Cristina Chiva, et al. Immunogenicity and T cell recognition in swine of foot-and-mouth disease virus polymerase 3D[J]. Virology, 2004, 322:264–275.
47. Gea-Banacloche JC, Migueles SA, Martino L,et al. Maintenance of large numbers of virus-specific CD8+ T cells in HIV-infected progressors and long-term nonprogressors[J].J Immunol.2000,165 (2):1082-92.
48. Geraldine Blanchard-Rohner, Grazia Galli, Elizabeth A. Clutterbuck et al.Comparison of a limiting dilution assay and ELISpot for detection of memory B-cells before and after immunisation with a protein-polysaccharide conjugate vaccine in children [J]. 2010,358( 1-2):46-55
49. Gouy M.Codon contexts in enterobacterial and coliphage genes[J].Mol Bio Evol,1987,4(4): 26-29
50. Haruo Hanawa,Tadashi Koike,Minori Sakaue.et al.Novel analysis of minimal residual disease in leukemia with TCRβrearrangement-Detection of monoclonality by single strand conformation polymorphism and PCR using a clonotype primer of leukemic T cell receptorβ-chainRNA[J].Leukemia Research,1997,21(3):201-210
51. Hel Z, Nacsa J, Kelsall B,et al.Impairment of Gag-specific CD8(+) T-cell function in mucosal and systemic compartments of simian immunodeficiency virus[J].J Virol,2001,75:11483-11495
52. Janeway CA,Trabers P,Walport M,etal.Immunobiology:the immune system in health and disease[M].lsevier:garland,1999,48:1-3
53. John D. Altman.et al.Phenotypic Analysis of Antigen-Specific T Lymphocytes [J]. Science,1996, 5284(274):94-96
54. Jorge Carneiro, Lurdes Duarte,Elisabetta Padovan.Limiting Dilution Analysis of Antigen-Specific T Cells[J]. Methods in molecular biology ,2009, 514:95-105
55. Kamm C, koberne M, Geginat G, et al. CD8 T cell immunomeanalysis of Listeria monocytogenes [J]. FEM S Immunology and Medical Microbiology, 2003, 35: 235-242.
56. Knowles N.J, Samuel A.R.Molecular epidemiology of foot-and-mouth disease virus[J].Virus Research ,2003,91:65-80
57. Kunisawa T,Kanaya S,Kutter E.Comparison of synonymous codon distribution patterns of bacteriophage and host genomes[J].DNA Res,1998.5.(6):319
58. Laplaud,Ruiz,Wierdewski,et a1.Blood T-cell receptor beta chain transcriptome in multiple sclerosis Characterization of the T cells witll altered CDR3 length distribution [J] .Brain, 2004 ,127 (5):981 -995.
59. Leisner C, Loeth N, Lamberth K, et al.One-Pot, Mix-and-Read Peptide-MHC Tetrmers [J]. PLoS ONE, 2008, 3(2): 1678-1682.
60. Li W, W atarai S, Iwasak i T, et al. Supp ression of Salmonella enterica serovar Enteritidis excretion by intraocular vaccination with fimbriae proteins incorporated in liposomes [J]. Developmental and Comparative Immunology, 2004, 28: 29-38.
61. Lyons AB,Parish CR.Determination of lymphocyte division by follow cytometry[J].J Immunol Methods,1994,171 (1) :131-137
62. Marchi R, Brooke G, Gale C, et al. Protection of cattle against foot-and-mouth disease by a synthetic peptide [J]. Science, 1986, 232(4750): 639-641.
63. Margarida Lima,et al. Immunophenotype and TCR-V-repertoire of peripheral blood T-cells in acute infectious mononucleosis[J].Blood Cells, Molecules and Diseases,2003, 30: 1-12
64. Matsumoto Y, Jee Y, Sugisaki M, et a l. Successful TCR-based immunotherapy for autoimmune myocarditis with DNA vaccines after rap id identification of pathogenic TCR[J].J Immunol, 2000,; 164:248-225
65. M Bhasin, GPS Raghava.A hybrid approach for predicting promiscuous MHC class I restricted T cell epitopes[J].Biosci, 2007, 32(1): 31-42
66. McCulloughKC,SobrinoF.Immunology of foot-and-mouth disease Current Perspectives[J].Horizon Bioscience Wymondham Norfolk England, 2004,19(56):173–222
67. MJ van Lierop,JP Wagenaar,JM van Noort,et al.Sequences derived from the highly antigenic VP1 region 140 to 160 of foot-and-mouth disease virus do not prime for a bovine T-cell responseagainst intact virus [J].J Virol,1995.69, 4511-4514.
68. Nishida K,Kawasaki T,Fujie M,et al Aminoacylation of tRNAs encoded by Chlorella virus CVK2[J].Virology,1999,263(1):220-225.
69. Nuntaprasert, Y. Mo ri, K. Fuk ita, et al. Characterization of specific antibodies and the establishment of sandwich EL ISA and EL ISPOT system s for swine IL 24 [J]. Comparative Immuno logy, Microbiology& Infectious Diseases, 2004, 27: 457-470.
70. Okamoto Y,Gotoh Y,Shlraishi H,et a1.A human dual-color enzyme-linked immunospot assay for simultaneous detection of interlenkin 2-and interleukin 4-secreting cells [J]. International lmmunopharmacology,2004,(4):149-156.
71. Parish CR. Fluorescent dyes for lymphocyte migration and proliferation studies [J].Immunol Cell Biol, 1999, 77 (6):499-508.
72. Parker, K. C., M. A. Bednarek,J. E. Coligan. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains [J]. J Immunol. 1994, 56(5):152-163.
73. Petersen, Sidorov, Charlotte.Limiting dilution analysis of interleukin-2 producing helper T-cell frequencies as a tool in allogeneic hematopoietic cell transplantation [J]. 2005, 80(5):573-581
74. Peter W,Mason,Grubman,et al.Molecular basis of pathogenesis of FMDV[J].Virus Research 2003,91:9 -32
75. Pietro PL, Tracy HL, Peter JM. Flow cytometrick measurement of intracellular cytokines [J]. Imm unolM ethods, 2000, 243: 107-124.
76. Pitcher CJ,Quittner C,Peterson DMet al.HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression[J].Nat Med.1999,5(5):518-525.
77. P. I. Zamorano, A. Wigdorovitz, D. M. Pérez Filgueira, et al.Induction of anti foot and mouth disease virus T and B cell responses in cattle immunized with a peptide representing ten amino acids of VP1[J].vaccine,1998, 16(6):558-563.
78. Precopio ML, Sullivan JL, Willard C, et al.Differential kinetics and specificity of EBV-specific CD4+ and CD8+ T cells during primary infection [J]. J Immunol.2003, 170(5):25-28.
79. Quiding,Granstrom,Nordstrom et al. High frequency of spontaneous interferon-gamma-producing cells in human tonsils:role of local accessory cells and soluble factors[J].Clin Exp Immunol, 1993,91(1):157-163
80. Radaelli A, Nacsa J, Tsai WP,et al.Prior DNA immunization enhances immune response to dominant and subdominant viral epitopes induced by a fowlpox-based SIVmac vaccine in long-term slow-progressor macaques infected with SIVmac251[J].Virology,2003;312:181-195
81. Rodriguez A, Saiz JC, Novella ZS, et al. Antigenic specificity of porcine T cell response against foot-and- mouth disease virus structural proteins:identification of T helper epitopes in VP1[J]. Virology, 1994, 205: 24-33.
82. Rowen L, Koop BF, Hood L. The complete 6852kilobase DNA sequence of the human beta T cellreceptor locus [J].Science ,1996 ,272(5269) :1755-1762
83. Schmittel A,Keilholz U,Bauer S,et al.Application of the IFN-gamma ELISPOT assay to quantify T cell responses against proteins[J].J Immunol Methods.2001,247(1-2):17-24.
84. Selim Ku?i, Johannes T. Wessels, Hans-J?rg Bühring,et al.Identification of a novel class of human adherent CD34 stem cells that give rise to SCID-repopulating cells [J]. Blood, 2003, 101, (3): 869 -876
85. Sette A, Vitiello A, Reherman B,et al.The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes [J].J Immunol.1994,153(12):5586-5592.
86. Siew-Cheng Wong, Andy Hee-Meng Tan, Kong-Peng Lam. Functional hierarchy and relative contribution of the CD28/B7 and ICOS/B7-H2 costimulatory pathways to T cell-mediated delayed-type hypersensitivity[J].Cellular Immunology, 2009,256 :64 -71
87. Taboga O, Tami C, Carrillo E. A large-scale evaluation of peptide vaccines against foot-and-mouth disease: Lack of solid protection in cattle and isolation of escape mutants [J]. J Virol, 1997, 71(4): 2606?2610.
88. Toja M, Escarmís C, Domingo E. Genomic nucleotide sequence of a foot-and-mouth disease virus clone and its persistent derivatives. Implications for the evolution of viral quasispecies during a persistent infection [J]. Virus Res, 1999, 64(2): 161?171.
89. Vonderheide RH, Domchek SM, Schultze JL, George DJ, Hoar KM, Chen DY, Stephans KF, Masutomi K, Loda M, Xia Z,Anderson KS, Hahn WC, Nadler LM. Vaccination of cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes[J].Clin Cancer Res,2004, 10: 828-839
90. Wang B, Chen H, Jiang X,et al.Identification of an HLA-A*0201-restricted CD8+ T-cell epitope SSp-1 of SARS-CoV spike protein[J].Blood,2004,1(104):200-206
91. Wang J L, Liu M Q, Han J, et al. A peptide of foot-and-mouth disease virus serotype Asia1 generating a neutralizing antibody response , and an immunostimulatory peptide[J].Vet Microbiol , 2007,125 :224-231
92. Waldrop SL,Pitcher CJ,Peterson DM,et al.Determination of antigen-specific memory,dffector CD4+ T cell frequencies by flow cytometry:evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency[J].J Clin Invest,1997,99(7):1739-1750
93. Wang P, Morgan AA, Zhang Q et al. Automating document classification for the Immune Epitope Database[J].BMC Bioinformatics, 2007, 8: 269-273
94. Wilhelm Gerner,Michael S. Denyer,Haru-Hisa,et al.Identification of novel foot-and-mouth disease virus specific T-cell epitopes in c/c and d/d haplotype miniature swine[J].Virus Research, 2006, 121:223-228
95. W?lfl M, Schalk S, Hellmich M,et al.Quantitation of MHC tetramer-positive cells from whole blood: Evaluation of a single-platform, six-parameter flow cytometric method [J].Cytometry, 2004, 57A:120-130
96. Wong FS, Karttunen J, Dumont C, et al. Identification of an MHC class I-restricted autoantigen intype 1 diabetes by screening an organ-specific cDNA library[J].Nat Med 1999;5:1026-1031.
97. YaoXS,Zhang GW,Ma L ,et a1.Analysis ofthe CDR3 letlgth of TCR alphabeta T cells in the peripheral blood of patients with chronic hepatitis B[J].Hepatol Res,2006,35(1):10-18
98. Zamorano P, Wigdorovitz A, Chaher MT, et al. Recognition of B and T cell epitopes by cattle immunized with a synthetic peptide containing the major immunogenic site of VP1 FMDV O1 Campos[J]. Virology, 1994, 201(2):383?387.