H5亚型禽流感病毒NP中鸡MHC限制性CTL表位的预测及鉴定
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摘要
禽流感是由禽流感病毒(Avian influenza virus, AIV)引起的禽的一种高度传染病。AIV中节段5编码的核蛋白(Nucleoprotein, NP)上含有大量的细胞毒性淋巴细胞识别的抗原表位,其诱导的特异性的细胞毒性T淋巴细胞(Cytotoxic T lymphocyte, CTL)反应在不同亚型的AIV之间具有一定的交叉保护力。目前许多关于流感病毒的T细胞表位在人和鼠中已获得鉴定。然而,由于禽主要组织相容性复合体(Major histocompatibility complex, MHC)的结构和功能方面研究很少,已鉴定的禽流感病毒结构蛋白中鸡MHC限制性T细胞表位非常有限,限制了禽类抗流感病毒细胞免疫研究的开展。
为鉴定H5亚型禽流感病毒NP中能被鸡MHC分子特异性识别的CTL表位,本研究首先模建了B4、B12、B15和B19单倍型鸡的MHC I类分子的α1和α2结构域的三维结构,并分析了各个单倍型结构的肽结合槽的表面性质。结果显示:结合槽的表面都高度疏水;结合槽内B口袋比F口袋的柔性大;B4单倍型的结合槽表面大部分区域带正电,B15和B19的结合槽表面以负电荷聚集为主,B12的表面相对呈中性。将AIV代表毒株A/Goose/Guangdong/1/1996 (H5N1)的NP蛋白截取重叠肽,选出符合B4、B12、B15和B19单倍型MHC I类分子结合基序的多肽,利用分子对接方法将多肽与相应的MHC I类分子结构对接,预测可以和各单倍型具有高亲和力的多肽。经以构象和打分函数相结合的方法筛选,最终获得25条(其中结合B4、B12、B15和B19的多肽数分别为10、6、2和7)与各个单倍型的MHC I类分子结合较好的潜在CTL表位肽,并选出结合能最高的9条短肽用于实验室免疫原性检测。
为了诱导SPF鸡产生针对AIV NP的记忆性T细胞,本研究将优化成鸡偏嗜性的NP基因双酶切后亚克隆到含禽源启动子的真核表达载体pCAGGS中,构建重组质粒pCAGGS-NP,经筛选鉴定正确的阳性重组质粒转染293T细胞,间接免疫荧光和Western blot检测表明NP蛋白在真核细胞中能正确表达并具有良好的免疫原性。重组质粒pCAGGS-NP以100 ug/羽的剂量腿部肌肉免疫3周龄SPF鸡,3周后以同样的剂量加强免疫,间接酶联免疫实验检测结果显示pCAGGS-NP能诱导SPF鸡产生针对AIV NP的抗体,抗体水平在加强免疫后迅速升高。取加强免疫三周后的鸡脾脏制成淋巴单细胞悬液,调整细胞浓度为1×106 /mL,一部分细胞加入上述9条短肽和1条不相关肽进行体外刺激培养,24 h后收集细胞,采用ELISA试剂盒检测培养上清中IFN-γ分泌量;一部分细胞加入CFSE贮存液和10条合成肽体外培养,5 d后用流式细胞术对培养后细胞中CD8~+T淋巴细胞增殖情况进行检测。检测结果一致显示肽NP89-97(PKKTGGPIY)和NP198-206(KRGINDRNF)能刺激活化的鸡脾淋巴细胞IFN-γ分泌量明显增加,CD8~+T淋巴细胞分别有13.7 %、11.9 %的增值,表明NP89-97和NP198-206能诱导较强的细胞免疫反应,为AIV NP的T细胞表位。本研究所构建的鸡MHC I类分子的结构及鉴定的表位为进一步探讨AIV所引起的细胞免疫及T细胞表位的鉴定奠定了基础。
Avian influenza virus(AIV) is a causative agent of highly contagious viral disease in chickens. Previous studies showed that T cell-mediated immune responses play a critical role in defense against influenza virus infection. The nucleoprotein (NP) encoded by the fifth fragment of AIV is a conserved inner protein, and it contains many T cell epitopes. Those epitopes can induce Cytotoxic T lymphocyte(CTL) response against the infection of different subtypes of influenza viruses. Some T cell epitopes from various proteins of influenza virus have been identified in human and mouse. However, little is known about which in chicken partially for lacking of the structure of the chicken major histocompatibility complex (MHC).
In this study, we have developed three-dimensional structures ofα1 andα2 domains of chicken MHC classΙmolecules from B4, B12, B15 and B19 haplotypes, those models provide clear insight into the structural characters of their peptide binding grooves. The surface of peptide binding grooves in those four structures are high lipothilic; The B pockets of the four structures are more flexible than the F pockets; The electrostatic potential of the peptide binding groove of B4 haplotype is highly positive charged,B15 and B19 haplotypes are negatively charged, while that of B12 is neutral. Based on structural properties, we screened antigen peptides derived from an AIV NP according to peptide-binding motifs and molecular docking, all together 25 peptides (10 for B4, 6 for B12, 2 for B15 and 7 for B19 haplotype) were predicted with potentials to bind to those MHC class I molecules. The top 9 peptides with high binding energy were selected to verify their activity.
In order to induce CD8~+ CTL specific to AIV NP in SPF chicken, NP gene, which was optimized with chicken biased codons, was sub-cloned into the eukaryotic expression vector pCAGGS. The recombinant plasmid pCAGGS-NP was constructed and the expression of NP was confirmed by IFA and western blot in transfected 293T cells. SPF chickens were immunized with 100μg pCAGG-NP and boosted with the same dose three weeks later. A dramatically increase NP antibody was detected by ELISA after being boosted. The spleens were isolated from vaccinated chickens for the preparation of lymphocytes. The peptide-stimulated lymphocytes were used to detect the secretions of chicken IFN-γand proliferation of CD8~+ T cell by ELISA kit and flow cytometry analysis respectively. The results showed that the increase of chicken IFN-γsecretions and proliferation of CD8~+ T lymphocytes by 13.8%, 11.9 % were detected in cells stimulated with peptides NP89-97 and NP198-206. The results indicate that peptides NP89-97 and NP198-206 are NP T cell epitopes in chicken. The homology modeling structures of chicken MHC class I molecules and the identified epitopes will extend our understanding of the mechanisms of immune response to AIV in chickens.
为鉴定H5亚型禽流感病毒NP中能被鸡MHC分子特异性识别的CTL表位,本研究首先模建了B4、B12、B15和B19单倍型鸡的MHC I类分子的α1和α2结构域的三维结构,并分析了各个单倍型结构的肽结合槽的表面性质。结果显示:结合槽的表面都高度疏水;结合槽内B口袋比F口袋的柔性大;B4单倍型的结合槽表面大部分区域带正电,B15和B19的结合槽表面以负电荷聚集为主,B12的表面相对呈中性。将AIV代表毒株A/Goose/Guangdong/1/1996 (H5N1)的NP蛋白截取重叠肽,选出符合B4、B12、B15和B19单倍型MHC I类分子结合基序的多肽,利用分子对接方法将多肽与相应的MHC I类分子结构对接,预测可以和各单倍型具有高亲和力的多肽。经以构象和打分函数相结合的方法筛选,最终获得25条(其中结合B4、B12、B15和B19的多肽数分别为10、6、2和7)与各个单倍型的MHC I类分子结合较好的潜在CTL表位肽,并选出结合能最高的9条短肽用于实验室免疫原性检测。
为了诱导SPF鸡产生针对AIV NP的记忆性T细胞,本研究将优化成鸡偏嗜性的NP基因双酶切后亚克隆到含禽源启动子的真核表达载体pCAGGS中,构建重组质粒pCAGGS-NP,经筛选鉴定正确的阳性重组质粒转染293T细胞,间接免疫荧光和Western blot检测表明NP蛋白在真核细胞中能正确表达并具有良好的免疫原性。重组质粒pCAGGS-NP以100 ug/羽的剂量腿部肌肉免疫3周龄SPF鸡,3周后以同样的剂量加强免疫,间接酶联免疫实验检测结果显示pCAGGS-NP能诱导SPF鸡产生针对AIV NP的抗体,抗体水平在加强免疫后迅速升高。取加强免疫三周后的鸡脾脏制成淋巴单细胞悬液,调整细胞浓度为1×106 /mL,一部分细胞加入上述9条短肽和1条不相关肽进行体外刺激培养,24 h后收集细胞,采用ELISA试剂盒检测培养上清中IFN-γ分泌量;一部分细胞加入CFSE贮存液和10条合成肽体外培养,5 d后用流式细胞术对培养后细胞中CD8~+T淋巴细胞增殖情况进行检测。检测结果一致显示肽NP89-97(PKKTGGPIY)和NP198-206(KRGINDRNF)能刺激活化的鸡脾淋巴细胞IFN-γ分泌量明显增加,CD8~+T淋巴细胞分别有13.7 %、11.9 %的增值,表明NP89-97和NP198-206能诱导较强的细胞免疫反应,为AIV NP的T细胞表位。本研究所构建的鸡MHC I类分子的结构及鉴定的表位为进一步探讨AIV所引起的细胞免疫及T细胞表位的鉴定奠定了基础。
Avian influenza virus(AIV) is a causative agent of highly contagious viral disease in chickens. Previous studies showed that T cell-mediated immune responses play a critical role in defense against influenza virus infection. The nucleoprotein (NP) encoded by the fifth fragment of AIV is a conserved inner protein, and it contains many T cell epitopes. Those epitopes can induce Cytotoxic T lymphocyte(CTL) response against the infection of different subtypes of influenza viruses. Some T cell epitopes from various proteins of influenza virus have been identified in human and mouse. However, little is known about which in chicken partially for lacking of the structure of the chicken major histocompatibility complex (MHC).
In this study, we have developed three-dimensional structures ofα1 andα2 domains of chicken MHC classΙmolecules from B4, B12, B15 and B19 haplotypes, those models provide clear insight into the structural characters of their peptide binding grooves. The surface of peptide binding grooves in those four structures are high lipothilic; The B pockets of the four structures are more flexible than the F pockets; The electrostatic potential of the peptide binding groove of B4 haplotype is highly positive charged,B15 and B19 haplotypes are negatively charged, while that of B12 is neutral. Based on structural properties, we screened antigen peptides derived from an AIV NP according to peptide-binding motifs and molecular docking, all together 25 peptides (10 for B4, 6 for B12, 2 for B15 and 7 for B19 haplotype) were predicted with potentials to bind to those MHC class I molecules. The top 9 peptides with high binding energy were selected to verify their activity.
In order to induce CD8~+ CTL specific to AIV NP in SPF chicken, NP gene, which was optimized with chicken biased codons, was sub-cloned into the eukaryotic expression vector pCAGGS. The recombinant plasmid pCAGGS-NP was constructed and the expression of NP was confirmed by IFA and western blot in transfected 293T cells. SPF chickens were immunized with 100μg pCAGG-NP and boosted with the same dose three weeks later. A dramatically increase NP antibody was detected by ELISA after being boosted. The spleens were isolated from vaccinated chickens for the preparation of lymphocytes. The peptide-stimulated lymphocytes were used to detect the secretions of chicken IFN-γand proliferation of CD8~+ T cell by ELISA kit and flow cytometry analysis respectively. The results showed that the increase of chicken IFN-γsecretions and proliferation of CD8~+ T lymphocytes by 13.8%, 11.9 % were detected in cells stimulated with peptides NP89-97 and NP198-206. The results indicate that peptides NP89-97 and NP198-206 are NP T cell epitopes in chicken. The homology modeling structures of chicken MHC class I molecules and the identified epitopes will extend our understanding of the mechanisms of immune response to AIV in chickens.
引文
1.甘孟侯.禽流感(第二版).北京:中国农业大学出版社,2002,1-14。
2.侯艳霞,王靖飞.鸡主要组织相容性复合体的结构和功能研究进展.动物医学进展,2010,31(12):112-115。
3.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.中国兽医科学,2006,36(3):189-192。
4.刘凯军,李晋军.细胞毒性T淋巴细胞表位预测.生命的化学,2008,28(3):331-334。
5.王美亮,朱勇,王九平等.汉滩病毒核蛋白T细胞表位的鉴定及其特异性T细胞应答规律的初步探讨.细胞与分子免疫学杂志2005,21(6):704-706。
6.王群. H5N1亚型禽流感病毒核蛋白T细胞表位区的研究. [中国农业科学院博士学位论文].哈尔滨:中国农业科学院,2009。
7.杨汉春.动物免疫学(第二版).北京:中国农业大学出版社,2003,378。
8.支轶.生物信息学方法在CTL表位预测中的应用.免疫学杂志,2005,21(2):155-159。
9.周光炎.免疫学原理(第二版).上海:上海科学技术出版社,2007,8。
10. Adams S C, Xing Z, Li J et al. Immune-related gene expression in response to H11N9 low pathogenic avian influenza virus infection in chicken and Pekin duck peripheral blood mononuclear cells. Molecular immunology. 2009,46(8-9): 1744-1749.
11. Allen H. Glycoconyugate,composition,Structure and Function. NewYork, Basel, Hongkong: Marcel Deker Inc, 1992,
12. Altman J D, Moss P A, Goulder P J et al. Phenotypic analysis of antigen-specific T lymphocytes. Science. 1996,274(5284): 94-96.
13. Altuvia Y, Sette A, Sidney J et al. A structure-based algorithm to predict potential binding peptides to MHC molecules with hydrophobic binding pockets. Human immunology. 1997,58(1): 1-11.
14. Appay V, Nixon D F, Donahoe S M et al. HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function. The Journal of experimental medicine. 2000,192(1): 63-75.
15. Barlow D J, Edwards M S, Thornton J M. Continuous and discontinuous protein antigenic determinants. Nature. 1986,322(6081): 747-748.
16. Bastin J, Rothbard J, Davey J et al. Use of synthetic peptides of influenza nucleoprotein to define epitopes recognized by class I-restricted cytotoxic T lymphocytes. The Journal of experimental medicine. 1987,165(6): 1508-1523.
17. Berkhoff E G, Boon A C, Nieuwkoop N J et al. A mutation in the HLA-B*2705-restricted NP383-391 epitope affects the human influenza A virus-specific cytotoxic T-lymphocyte response in vitro. Journal of virology. 2004,78(10): 5216-5222.
18. Bhasin M, Raghava G P. Prediction of CTL epitopes using QM, SVM and ANN techniques. Vaccine. 2004,22(23-24): 3195-3204.
19. Bodewes R, Geelhoed-Mieras M M, Nieuwkoop N J et al. Redundancy of the influenza Avirus-specific cytotoxic T lymphocyte response in HLA-B*2705 transgenic mice limits the impact of a mutation in the immunodominant NP(383-391) epitope on influenza pathogenesis. Virus research. 2011,155(1): 123-130.
20. Boon A C, de Mutsert G, Graus Y M et al. The magnitude and specificity of influenza A virus-specific cytotoxic T-lymphocyte responses in humans is related to HLA-A and -B phenotype. Journal of virology. 2002,76(2): 582-590.
21. Brunner K T, Mauel J, Cerottini J C et al. Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology. 1968,14(2): 181-196.
22. Brusic V, Rudy G, Honeyman G et al. Prediction of MHC class II-binding peptides using an evolutionary algorithm and artificial neural network. Bioinformatics. 1998,14(2): 121-130.
23. Bui H H, Peters B, Assarsson E et al. Ab and T cell epitopes of influenza A virus, knowledge and opportunities. Proceedings of the National Academy of Sciences of the United States of America. 2007,104(1): 246-251.
24. Cascio P, Hilton C, Kisselev A F et al. 26S proteasomes and immunoproteasomes produce mainly N-extended versions of an antigenic peptide. The EMBO journal. 2001,20(10): 2357-2366.
25. Chen W, Calvo P A, Malide D et al. A novel influenza A virus mitochondrial protein that induces cell death. Nature medicine. 2001,7(12): 1306-1312.
26. Cole G A, Katz J M, Hogg T L et al. Analysis of the primary T-cell response to Sendai virus infection in C57BL/6 mice: CD4+ T-cell recognition is directed predominantly to the hemagglutinin-neuraminidase glycoprotein. Journal of virology. 1994,68(11): 6863-6870.
27. Crowe S R, Miller S C, Brown D M et al. Uneven distribution of MHC class II epitopes within the influenza virus. Vaccine. 2006,24(4): 457-467.
28. Daniel S, Brusic V, Caillat-Zucman S et al. Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules. J Immunol. 1998,161(2): 617-624.
29. Davenport M P, Ho Shon I A, Hill A V. An empirical method for the prediction of T-cell epitopes. Immunogenetics. 1995,42(5): 392-397.
30. Deml L, Bojak A, Steck S et al. Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. Journal of virology. 2001,75(22): 10991-11001.
31. Desselberger U, Racaniello V R, Zazra J J et al. The 3' and 5'-terminal sequences of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity. Gene. 1980,8(3): 315-328.
32. Ewald S J, Livant E J. Distinctive polymorphism of chicken B-FI (major histocompatibility complex class I) molecules. Poultry science. 2004,83(4): 600-605.
33. Fiser A, Sali A. Modeller: generation and refinement of homology-based protein structure models. Methods in enzymology. 2003,374: 461-491.
34. Fodor E, Pritlove D C, Brownlee G G. Characterization of the RNA-fork model of virion RNA in the initiation of transcription in influenza A virus. Journal of virology. 1995,69(7): 4012-4019.
35. Fogg M H, Parsons K R, Thomas L H et al. Identification of CD4+ T cell epitopes on the fusion (F) and attachment (G) proteins of bovine respiratory syncytial virus (BRSV). Vaccine. 2001,19(23-24): 3226-3240.
36. Forsthuber T, Yip H C, Lehmann P V. Induction of TH1 and TH2 immunity in neonatal mice. Science. 1996,271(5256): 1728-1730.
37. Fujii K, Fujii Y, Noda T et al. Importance of both the coding and the segment-specific noncoding regions of the influenza A virus NS segment for its efficient incorporation into virions. Journal of virology. 2005,79(6): 3766-3774.
38. Fulcher D, Wong S. Carboxyfluorescein succinimidyl ester-based proliferative assays for assessment of T cell function in the diagnostic laboratory. Immunology and cell biology. 1999,77(6): 559-564.
39. Green K J. Improving understanding of exercise effects on in vitro T-lymphocyte function--the role of fluorescent cell division tracking. Exercise immunology review. 2002,8: 101-115.
40. Gruener N H, Lechner F, Jung M C et al. Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus. Journal of virology. 2001,75(12): 5550-5558.
41. Haghighi H R, Read L R, Haeryfar S M et al. Identification of a dual-specific T cell epitope of the hemagglutinin antigen of an h5 avian influenza virus in chickens. PloS one. 2009,4(11): e7772.
42. Hansson L, Rabbani H, Fagerberg J et al. T-cell epitopes within the complementarity-determining and framework regions of the tumor-derived immunoglobulin heavy chain in multiple myeloma. Blood. 2003,101(12): 4930-4936.
43. He J F, Fang S S, Cheng X W et al. Evaluation on construction and expression in vitro of nucleic acid vaccine of nucleoprotein of influenza virus A. Wei sheng yan jiu. 2005,34(4): 419-422.
44. Helms T, Boehm B O, Asaad R J et al. Direct visualization of cytokine-producing recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. J Immunol. 2000,164(7): 3723-3732.
45. Hofmann A, Plachy J, Hunt L et al. v-src oncogene-specific carboxy-terminal peptide is immunoprotective against Rous sarcoma growth in chickens with MHC class I allele B-F12. Vaccine. 2003,21(32): 4694-4699.
46. Holzhutter H G, Frommel C, Kloetzel P M. A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20 S proteasome. Journal of molecular biology. 1999,286(4): 1251-1265.
47. Holzhutter H G, Kloetzel P M. A kinetic model of vertebrate 20S proteasome accounting for the generation of major proteolytic fragments from oligomeric peptide substrates. Biophysical journal. 2000,79(3): 1196-1205.
48. Jiang Y, Yu K, Zhang H et al. Enhanced protective efficacy of H5 subtype avian influenza DNA vaccine with codon optimized HA gene in a pCAGGS plasmid vector. Antiviral research.2007,75(3): 234-241.
49. Kaufman J. The simple chicken major histocompatibility complex: life and death in the face of pathogens and vaccines. Philosophical transactions of the Royal Society of London. 2000,355(1400): 1077-1084.
50. Kaufman J, Volk H, Wallny H J. A "minimal essential Mhc" and an "unrecognized Mhc": two extremes in selection for polymorphism. Immunological reviews. 1995,143(63-88.
51. Kesmir C, Nussbaum A K, Schild H et al. Prediction of proteasome cleavage motifs by neural networks. Protein engineering. 2002,15(4): 287-296.
52. Ketha K M, Atreya C D. Application of bioinformatics-coupled experimental analysis reveals a new transport-competent nuclear localization signal in the nucleoprotein of influenza A virus strain. BMC cell biology. 2008,9: 22.
53. Kobayashi M, Toyoda T, Adyshev D M et al. Molecular dissection of influenza virus nucleoprotein: deletion mapping of the RNA binding domain. Journal of virology. 1994,68(12): 8433-8436.
54. Koch M, Camp S, Collen T et al. Structures of an MHC class I molecule from B21 chickens illustrate promiscuous peptide binding. Immunity. 2007,27(6): 885-899.
55. Kozak M. Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6. The EMBO journal. 1997,16(9): 2482-2492.
56. Kuttler C, Nussbaum A K, Dick T P et al. An algorithm for the prediction of proteasomal cleavages. Journal of molecular biology. 2000,298(3): 417-429.
57. Laskowski R A, Macarthur M W, Moss D S et al. PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst. 1993,26: 283-291.
58. Lechner F, Wong D K, Dunbar P R et al. Analysis of successful immune responses in persons infected with hepatitis C virus. The Journal of experimental medicine. 2000,191(9): 1499-1512.
59. Lee K Y, Chun E, Kim N Y et al. Characterization of HLA-A2.1-restricted epitopes, conserved in both Hantaan and Sin Nombre viruses, in Hantaan virus-infected patients. The Journal of general virology. 2002,83(5): 1131-1136.
60. Lee Y J, Shin J Y, Song M S et al. Continuing evolution of H9 influenza viruses in Korean poultry. Virology. 2007,359(2): 313-323.
61. Lim J S, Kim S, Lee H G et al. Selection of peptides that bind to the HLA-A2.1 molecule by molecular modelling. Molecular immunology. 1996,33(2): 221-230.
62. Liu G, Wang Q, Tong T et al. Construction and functional test of a chicken MHC-I (BF2*15)/peptide tetramer. Veterinary immunology and immunopathology. 2008,122(1-2): 1-7.
63. Liu N, Song W, Wang P et al. Identification of unusual truncated forms of nucleocapsid protein in MDCK cells infected by Avian influenza virus (H9N2). Proteomics. 2010,10(9): 1875-1879.
64. Logean A, Rognan D. Recovery of known T-cell epitopes by computational scanning of a viral genome. Journal of computer-aided molecular design. 2002,16(4): 229-243.
65. Luthy R, Bowie J U, Eisenberg D. Assessment of protein models with three-dimensional profiles.Nature. 1992,356(6364): 83-85.
66. Maeno K, Aoki H, Hamaguchi M et al. Analysis of nuclear accumulation of influenza NP antigen in von Magnus virus-infected cells. Microbiology and immunology. 1981,25(3): 283-294.
67. Makoff A J, Oxer M D, Romanos M A et al. Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons. Nucleic acids research. 1989,17(24): 10191-10202.
68. McMichael A J, Kelleher A. The arrival of HLA class II tetramers. The Journal of clinical investigation. 1999,104(12): 1669-1670.
69. Neumann G, Castrucci M R, Kawaoka Y. Nuclear import and export of influenza virus nucleoprotein. Journal of virology. 1997,71(12): 9690-9700.
70. Ng A K, Zhang H, Tan K et al. Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design. Faseb J. 2008,22(10): 3638-3647.
71. Ng S S, Li O T, Cheung T K et al. Heterologous influenza vRNA segments with identical non-coding sequences stimulate viral RNA replication in trans. Virology journal. 2008,5(2.
72. Nielsen M, Lundegaard C, Lund O et al. The role of the proteasome in generating cytotoxic T-cell epitopes: insights obtained from improved predictions of proteasomal cleavage. Immunogenetics. 2005,57(1-2): 33-41.
73. Nussbaum A K, Kuttler C, Hadeler K P et al. PAProC: a prediction algorithm for proteasomal cleavages available on the WWW. Immunogenetics. 2001,53(2): 87-94.
74. O'Neill A M, Livant E J, Ewald S J. The chicken BF1 (classical MHC class I) gene shows evidence of selection for diversity in expression and in promoter and signal peptide regions. Immunogenetics. 2009,61(4): 289-302.
75. Ozawa M, Fujii K, Muramoto Y et al. Contributions of two nuclear localization signals of influenza A virus nucleoprotein to viral replication. Journal of virology. 2007,81(1): 30-41.
76. Peiris J S, Yu W C, Leung C W et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004,363(9409): 617-619.
77. Peters B, Sette A. Integrating epitope data into the emerging web of biomedical knowledge resources. Nature reviews. 2007,7(6): 485-490.
78. Popma S H, Krasinskas A M, McLean A D et al. Immune monitoring in xenotransplantation: the multiparameter flow cytometric mixed lymphocyte culture assay. Cytometry. 2000,42(5): 277-283.
79. Privalsky M L, Penhoet E E. Influenza virus proteins: identity, synthesis, and modification analyzed by two-dimensional gel electrophoresis. Proceedings of the National Academy of Sciences of the United States of America. 1978,75(8): 3625-3629.
80. Reid A H, Fanning T G, Janczewski T A et al. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene. Journal of virology. 2004,78(22): 12462-12470.
81. Reperant L A, Rimmelzwaan G F, Kuiken T. Avian influenza viruses in mammals. Revue scientifique et technique (International Office of Epizootics). 2009,28(1): 137-159.
82. Rimmelzwaan G F, Boon A C, Voeten J T et al. Sequence variation in the influenza A virus nucleoprotein associated with escape from cytotoxic T lymphocytes. Virus research. 2004,103(1-2):97-100.
83. Robertson J S. 5' and 3' terminal nucleotide sequences of the RNA genome segments of influenza virus. Nucleic acids research. 1979,6(12): 3745-3757.
84. Robinson H L, Pertmer T M. DNA vaccines for viral infections: basic studies and applications. Advances in virus research. 2000,55: 1-74.
85. Rotzschke O, Falk K, Stevanovic S et al. Exact prediction of a natural T cell epitope. European journal of immunology. 1991,21(11): 2891-2894.
86. Schueler-Furman O, Altuvia Y, Sette A et al. Structure-based prediction of binding peptides to MHC class I molecules: application to a broad range of MHC alleles. Protein Sci. 2000,9(9): 1838-1846.
87. Sedgwick J D. ELISPOT assay: a personal retrospective. Methods in molecular biology (Clifton, NJ. 2005,302: 3-14.
88. Sette A, Sidney J, Oseroff C et al. HLA DR4w4-binding motifs illustrate the biochemical basis of degeneracy and specificity in peptide-DR interactions. J Immunol. 1993,151(6): 3163-3170.
89. Shaw I, Powell T J, Marston D A et al. Different evolutionary histories of the two classical class I genes BF1 and BF2 illustrate drift and selection within the stable MHC haplotypes of chickens. J Immunol. 2007,178(9): 5744-5752.
90. Shu L L, Bean W J, Webster R G. Analysis of the evolution and variation of the human influenza A virus nucleoprotein gene from 1933 to 1990. Journal of virology. 1993,67(5): 2723-2729.
91. Skorko R, Summers D F, Galarza J M. Influenza A virus in vitro transcription: roles of NS1 and NP proteins in regulating RNA synthesis. Virology. 1991,180(2): 668-677.
92. Stenger S, Mazzaccaro R J, Uyemura K et al. Differential effects of cytolytic T cell subsets on intracellular infection. Science. 1997,276(5319): 1684-1687.
93. Stitz L, Schmitz C, Binder D et al. Characterization and immunological properties of influenza A virus nucleoprotein (NP): cell-associated NP isolated from infected cells or viral NP expressed by vaccinia recombinant virus do not confer protection. The Journal of general virology. 1990,71 ( Pt 5)(1169-1179).
94. Sugawara K, Muraki Y, Takashita E et al. Conformational maturation of the nucleoprotein synthesized in influenza C virus-infected cells. Virus research. 2006,122(1-2): 45-52.
95. Tan L C, Gudgeon N, Annels N E et al. A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers. J Immunol. 1999,162(3): 1827-1835.
96. Trojan A, Schultze J L, Witzens M et al. Immunoglobulin framework-derived peptides function as cytotoxic T-cell epitopes commonly expressed in B-cell malignancies. Nature medicine. 2000,6(6): 667-672.
97. Van Epps H L, Schmaljohn C S, Ennis F A. Human memory cytotoxic T-lymphocyte (CTL) responses to Hantaan virus infection: identification of virus-specific and cross-reactive CD8(+) CTL epitopes on nucleocapsid protein. Journal of virology. 1999,73(7): 5301-5308.
98. Voeten J T, Bestebroer T M, Nieuwkoop N J et al. Antigenic drift in the influenza A virus (H3N2)nucleoprotein and escape from recognition by cytotoxic T lymphocytes. Journal of virology. 2000,74(15): 6800-6807.
99. Voeten J T, Rimmelzwaan G F, Nieuwkoop N J et al. Antigen processing for MHC class I restricted presentation of exogenous influenza A virus nucleoprotein by B-lymphoblastoid cells. Clinical and experimental immunology. 2001,125(3): 423-431.
100. Wallny H J, Avila D, Hunt L G et al. Peptide motifs of the single dominantly expressed class I molecule explain the striking MHC-determined response to Rous sarcoma virus in chickens. Proceedings of the National Academy of Sciences of the United States of America. 2006,103(5): 1434-1439.
101. Wong D K, Dudley D D, Dohrenwend P B et al. Detection of diverse hepatitis C virus (HCV)-specific cytotoxic T lymphocytes in peripheral blood of infected persons by screening for responses to all translated proteins of HCV. Journal of virology. 2001,75(3): 1229-1235.
102. Ye Q, Krug R M, Tao Y J. The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature. 2006,444(7122): 1078-1082.
103. Zhang C, Anderson A, DeLisi C. Structural principles that govern the peptide-binding motifs of class I MHC molecules. Journal of molecular biology. 1998,281(5): 929-947.
2.侯艳霞,王靖飞.鸡主要组织相容性复合体的结构和功能研究进展.动物医学进展,2010,31(12):112-115。
3.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.中国兽医科学,2006,36(3):189-192。
4.刘凯军,李晋军.细胞毒性T淋巴细胞表位预测.生命的化学,2008,28(3):331-334。
5.王美亮,朱勇,王九平等.汉滩病毒核蛋白T细胞表位的鉴定及其特异性T细胞应答规律的初步探讨.细胞与分子免疫学杂志2005,21(6):704-706。
6.王群. H5N1亚型禽流感病毒核蛋白T细胞表位区的研究. [中国农业科学院博士学位论文].哈尔滨:中国农业科学院,2009。
7.杨汉春.动物免疫学(第二版).北京:中国农业大学出版社,2003,378。
8.支轶.生物信息学方法在CTL表位预测中的应用.免疫学杂志,2005,21(2):155-159。
9.周光炎.免疫学原理(第二版).上海:上海科学技术出版社,2007,8。
10. Adams S C, Xing Z, Li J et al. Immune-related gene expression in response to H11N9 low pathogenic avian influenza virus infection in chicken and Pekin duck peripheral blood mononuclear cells. Molecular immunology. 2009,46(8-9): 1744-1749.
11. Allen H. Glycoconyugate,composition,Structure and Function. NewYork, Basel, Hongkong: Marcel Deker Inc, 1992,
12. Altman J D, Moss P A, Goulder P J et al. Phenotypic analysis of antigen-specific T lymphocytes. Science. 1996,274(5284): 94-96.
13. Altuvia Y, Sette A, Sidney J et al. A structure-based algorithm to predict potential binding peptides to MHC molecules with hydrophobic binding pockets. Human immunology. 1997,58(1): 1-11.
14. Appay V, Nixon D F, Donahoe S M et al. HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function. The Journal of experimental medicine. 2000,192(1): 63-75.
15. Barlow D J, Edwards M S, Thornton J M. Continuous and discontinuous protein antigenic determinants. Nature. 1986,322(6081): 747-748.
16. Bastin J, Rothbard J, Davey J et al. Use of synthetic peptides of influenza nucleoprotein to define epitopes recognized by class I-restricted cytotoxic T lymphocytes. The Journal of experimental medicine. 1987,165(6): 1508-1523.
17. Berkhoff E G, Boon A C, Nieuwkoop N J et al. A mutation in the HLA-B*2705-restricted NP383-391 epitope affects the human influenza A virus-specific cytotoxic T-lymphocyte response in vitro. Journal of virology. 2004,78(10): 5216-5222.
18. Bhasin M, Raghava G P. Prediction of CTL epitopes using QM, SVM and ANN techniques. Vaccine. 2004,22(23-24): 3195-3204.
19. Bodewes R, Geelhoed-Mieras M M, Nieuwkoop N J et al. Redundancy of the influenza Avirus-specific cytotoxic T lymphocyte response in HLA-B*2705 transgenic mice limits the impact of a mutation in the immunodominant NP(383-391) epitope on influenza pathogenesis. Virus research. 2011,155(1): 123-130.
20. Boon A C, de Mutsert G, Graus Y M et al. The magnitude and specificity of influenza A virus-specific cytotoxic T-lymphocyte responses in humans is related to HLA-A and -B phenotype. Journal of virology. 2002,76(2): 582-590.
21. Brunner K T, Mauel J, Cerottini J C et al. Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology. 1968,14(2): 181-196.
22. Brusic V, Rudy G, Honeyman G et al. Prediction of MHC class II-binding peptides using an evolutionary algorithm and artificial neural network. Bioinformatics. 1998,14(2): 121-130.
23. Bui H H, Peters B, Assarsson E et al. Ab and T cell epitopes of influenza A virus, knowledge and opportunities. Proceedings of the National Academy of Sciences of the United States of America. 2007,104(1): 246-251.
24. Cascio P, Hilton C, Kisselev A F et al. 26S proteasomes and immunoproteasomes produce mainly N-extended versions of an antigenic peptide. The EMBO journal. 2001,20(10): 2357-2366.
25. Chen W, Calvo P A, Malide D et al. A novel influenza A virus mitochondrial protein that induces cell death. Nature medicine. 2001,7(12): 1306-1312.
26. Cole G A, Katz J M, Hogg T L et al. Analysis of the primary T-cell response to Sendai virus infection in C57BL/6 mice: CD4+ T-cell recognition is directed predominantly to the hemagglutinin-neuraminidase glycoprotein. Journal of virology. 1994,68(11): 6863-6870.
27. Crowe S R, Miller S C, Brown D M et al. Uneven distribution of MHC class II epitopes within the influenza virus. Vaccine. 2006,24(4): 457-467.
28. Daniel S, Brusic V, Caillat-Zucman S et al. Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules. J Immunol. 1998,161(2): 617-624.
29. Davenport M P, Ho Shon I A, Hill A V. An empirical method for the prediction of T-cell epitopes. Immunogenetics. 1995,42(5): 392-397.
30. Deml L, Bojak A, Steck S et al. Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. Journal of virology. 2001,75(22): 10991-11001.
31. Desselberger U, Racaniello V R, Zazra J J et al. The 3' and 5'-terminal sequences of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity. Gene. 1980,8(3): 315-328.
32. Ewald S J, Livant E J. Distinctive polymorphism of chicken B-FI (major histocompatibility complex class I) molecules. Poultry science. 2004,83(4): 600-605.
33. Fiser A, Sali A. Modeller: generation and refinement of homology-based protein structure models. Methods in enzymology. 2003,374: 461-491.
34. Fodor E, Pritlove D C, Brownlee G G. Characterization of the RNA-fork model of virion RNA in the initiation of transcription in influenza A virus. Journal of virology. 1995,69(7): 4012-4019.
35. Fogg M H, Parsons K R, Thomas L H et al. Identification of CD4+ T cell epitopes on the fusion (F) and attachment (G) proteins of bovine respiratory syncytial virus (BRSV). Vaccine. 2001,19(23-24): 3226-3240.
36. Forsthuber T, Yip H C, Lehmann P V. Induction of TH1 and TH2 immunity in neonatal mice. Science. 1996,271(5256): 1728-1730.
37. Fujii K, Fujii Y, Noda T et al. Importance of both the coding and the segment-specific noncoding regions of the influenza A virus NS segment for its efficient incorporation into virions. Journal of virology. 2005,79(6): 3766-3774.
38. Fulcher D, Wong S. Carboxyfluorescein succinimidyl ester-based proliferative assays for assessment of T cell function in the diagnostic laboratory. Immunology and cell biology. 1999,77(6): 559-564.
39. Green K J. Improving understanding of exercise effects on in vitro T-lymphocyte function--the role of fluorescent cell division tracking. Exercise immunology review. 2002,8: 101-115.
40. Gruener N H, Lechner F, Jung M C et al. Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus. Journal of virology. 2001,75(12): 5550-5558.
41. Haghighi H R, Read L R, Haeryfar S M et al. Identification of a dual-specific T cell epitope of the hemagglutinin antigen of an h5 avian influenza virus in chickens. PloS one. 2009,4(11): e7772.
42. Hansson L, Rabbani H, Fagerberg J et al. T-cell epitopes within the complementarity-determining and framework regions of the tumor-derived immunoglobulin heavy chain in multiple myeloma. Blood. 2003,101(12): 4930-4936.
43. He J F, Fang S S, Cheng X W et al. Evaluation on construction and expression in vitro of nucleic acid vaccine of nucleoprotein of influenza virus A. Wei sheng yan jiu. 2005,34(4): 419-422.
44. Helms T, Boehm B O, Asaad R J et al. Direct visualization of cytokine-producing recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. J Immunol. 2000,164(7): 3723-3732.
45. Hofmann A, Plachy J, Hunt L et al. v-src oncogene-specific carboxy-terminal peptide is immunoprotective against Rous sarcoma growth in chickens with MHC class I allele B-F12. Vaccine. 2003,21(32): 4694-4699.
46. Holzhutter H G, Frommel C, Kloetzel P M. A theoretical approach towards the identification of cleavage-determining amino acid motifs of the 20 S proteasome. Journal of molecular biology. 1999,286(4): 1251-1265.
47. Holzhutter H G, Kloetzel P M. A kinetic model of vertebrate 20S proteasome accounting for the generation of major proteolytic fragments from oligomeric peptide substrates. Biophysical journal. 2000,79(3): 1196-1205.
48. Jiang Y, Yu K, Zhang H et al. Enhanced protective efficacy of H5 subtype avian influenza DNA vaccine with codon optimized HA gene in a pCAGGS plasmid vector. Antiviral research.2007,75(3): 234-241.
49. Kaufman J. The simple chicken major histocompatibility complex: life and death in the face of pathogens and vaccines. Philosophical transactions of the Royal Society of London. 2000,355(1400): 1077-1084.
50. Kaufman J, Volk H, Wallny H J. A "minimal essential Mhc" and an "unrecognized Mhc": two extremes in selection for polymorphism. Immunological reviews. 1995,143(63-88.
51. Kesmir C, Nussbaum A K, Schild H et al. Prediction of proteasome cleavage motifs by neural networks. Protein engineering. 2002,15(4): 287-296.
52. Ketha K M, Atreya C D. Application of bioinformatics-coupled experimental analysis reveals a new transport-competent nuclear localization signal in the nucleoprotein of influenza A virus strain. BMC cell biology. 2008,9: 22.
53. Kobayashi M, Toyoda T, Adyshev D M et al. Molecular dissection of influenza virus nucleoprotein: deletion mapping of the RNA binding domain. Journal of virology. 1994,68(12): 8433-8436.
54. Koch M, Camp S, Collen T et al. Structures of an MHC class I molecule from B21 chickens illustrate promiscuous peptide binding. Immunity. 2007,27(6): 885-899.
55. Kozak M. Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6. The EMBO journal. 1997,16(9): 2482-2492.
56. Kuttler C, Nussbaum A K, Dick T P et al. An algorithm for the prediction of proteasomal cleavages. Journal of molecular biology. 2000,298(3): 417-429.
57. Laskowski R A, Macarthur M W, Moss D S et al. PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst. 1993,26: 283-291.
58. Lechner F, Wong D K, Dunbar P R et al. Analysis of successful immune responses in persons infected with hepatitis C virus. The Journal of experimental medicine. 2000,191(9): 1499-1512.
59. Lee K Y, Chun E, Kim N Y et al. Characterization of HLA-A2.1-restricted epitopes, conserved in both Hantaan and Sin Nombre viruses, in Hantaan virus-infected patients. The Journal of general virology. 2002,83(5): 1131-1136.
60. Lee Y J, Shin J Y, Song M S et al. Continuing evolution of H9 influenza viruses in Korean poultry. Virology. 2007,359(2): 313-323.
61. Lim J S, Kim S, Lee H G et al. Selection of peptides that bind to the HLA-A2.1 molecule by molecular modelling. Molecular immunology. 1996,33(2): 221-230.
62. Liu G, Wang Q, Tong T et al. Construction and functional test of a chicken MHC-I (BF2*15)/peptide tetramer. Veterinary immunology and immunopathology. 2008,122(1-2): 1-7.
63. Liu N, Song W, Wang P et al. Identification of unusual truncated forms of nucleocapsid protein in MDCK cells infected by Avian influenza virus (H9N2). Proteomics. 2010,10(9): 1875-1879.
64. Logean A, Rognan D. Recovery of known T-cell epitopes by computational scanning of a viral genome. Journal of computer-aided molecular design. 2002,16(4): 229-243.
65. Luthy R, Bowie J U, Eisenberg D. Assessment of protein models with three-dimensional profiles.Nature. 1992,356(6364): 83-85.
66. Maeno K, Aoki H, Hamaguchi M et al. Analysis of nuclear accumulation of influenza NP antigen in von Magnus virus-infected cells. Microbiology and immunology. 1981,25(3): 283-294.
67. Makoff A J, Oxer M D, Romanos M A et al. Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons. Nucleic acids research. 1989,17(24): 10191-10202.
68. McMichael A J, Kelleher A. The arrival of HLA class II tetramers. The Journal of clinical investigation. 1999,104(12): 1669-1670.
69. Neumann G, Castrucci M R, Kawaoka Y. Nuclear import and export of influenza virus nucleoprotein. Journal of virology. 1997,71(12): 9690-9700.
70. Ng A K, Zhang H, Tan K et al. Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design. Faseb J. 2008,22(10): 3638-3647.
71. Ng S S, Li O T, Cheung T K et al. Heterologous influenza vRNA segments with identical non-coding sequences stimulate viral RNA replication in trans. Virology journal. 2008,5(2.
72. Nielsen M, Lundegaard C, Lund O et al. The role of the proteasome in generating cytotoxic T-cell epitopes: insights obtained from improved predictions of proteasomal cleavage. Immunogenetics. 2005,57(1-2): 33-41.
73. Nussbaum A K, Kuttler C, Hadeler K P et al. PAProC: a prediction algorithm for proteasomal cleavages available on the WWW. Immunogenetics. 2001,53(2): 87-94.
74. O'Neill A M, Livant E J, Ewald S J. The chicken BF1 (classical MHC class I) gene shows evidence of selection for diversity in expression and in promoter and signal peptide regions. Immunogenetics. 2009,61(4): 289-302.
75. Ozawa M, Fujii K, Muramoto Y et al. Contributions of two nuclear localization signals of influenza A virus nucleoprotein to viral replication. Journal of virology. 2007,81(1): 30-41.
76. Peiris J S, Yu W C, Leung C W et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004,363(9409): 617-619.
77. Peters B, Sette A. Integrating epitope data into the emerging web of biomedical knowledge resources. Nature reviews. 2007,7(6): 485-490.
78. Popma S H, Krasinskas A M, McLean A D et al. Immune monitoring in xenotransplantation: the multiparameter flow cytometric mixed lymphocyte culture assay. Cytometry. 2000,42(5): 277-283.
79. Privalsky M L, Penhoet E E. Influenza virus proteins: identity, synthesis, and modification analyzed by two-dimensional gel electrophoresis. Proceedings of the National Academy of Sciences of the United States of America. 1978,75(8): 3625-3629.
80. Reid A H, Fanning T G, Janczewski T A et al. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene. Journal of virology. 2004,78(22): 12462-12470.
81. Reperant L A, Rimmelzwaan G F, Kuiken T. Avian influenza viruses in mammals. Revue scientifique et technique (International Office of Epizootics). 2009,28(1): 137-159.
82. Rimmelzwaan G F, Boon A C, Voeten J T et al. Sequence variation in the influenza A virus nucleoprotein associated with escape from cytotoxic T lymphocytes. Virus research. 2004,103(1-2):97-100.
83. Robertson J S. 5' and 3' terminal nucleotide sequences of the RNA genome segments of influenza virus. Nucleic acids research. 1979,6(12): 3745-3757.
84. Robinson H L, Pertmer T M. DNA vaccines for viral infections: basic studies and applications. Advances in virus research. 2000,55: 1-74.
85. Rotzschke O, Falk K, Stevanovic S et al. Exact prediction of a natural T cell epitope. European journal of immunology. 1991,21(11): 2891-2894.
86. Schueler-Furman O, Altuvia Y, Sette A et al. Structure-based prediction of binding peptides to MHC class I molecules: application to a broad range of MHC alleles. Protein Sci. 2000,9(9): 1838-1846.
87. Sedgwick J D. ELISPOT assay: a personal retrospective. Methods in molecular biology (Clifton, NJ. 2005,302: 3-14.
88. Sette A, Sidney J, Oseroff C et al. HLA DR4w4-binding motifs illustrate the biochemical basis of degeneracy and specificity in peptide-DR interactions. J Immunol. 1993,151(6): 3163-3170.
89. Shaw I, Powell T J, Marston D A et al. Different evolutionary histories of the two classical class I genes BF1 and BF2 illustrate drift and selection within the stable MHC haplotypes of chickens. J Immunol. 2007,178(9): 5744-5752.
90. Shu L L, Bean W J, Webster R G. Analysis of the evolution and variation of the human influenza A virus nucleoprotein gene from 1933 to 1990. Journal of virology. 1993,67(5): 2723-2729.
91. Skorko R, Summers D F, Galarza J M. Influenza A virus in vitro transcription: roles of NS1 and NP proteins in regulating RNA synthesis. Virology. 1991,180(2): 668-677.
92. Stenger S, Mazzaccaro R J, Uyemura K et al. Differential effects of cytolytic T cell subsets on intracellular infection. Science. 1997,276(5319): 1684-1687.
93. Stitz L, Schmitz C, Binder D et al. Characterization and immunological properties of influenza A virus nucleoprotein (NP): cell-associated NP isolated from infected cells or viral NP expressed by vaccinia recombinant virus do not confer protection. The Journal of general virology. 1990,71 ( Pt 5)(1169-1179).
94. Sugawara K, Muraki Y, Takashita E et al. Conformational maturation of the nucleoprotein synthesized in influenza C virus-infected cells. Virus research. 2006,122(1-2): 45-52.
95. Tan L C, Gudgeon N, Annels N E et al. A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers. J Immunol. 1999,162(3): 1827-1835.
96. Trojan A, Schultze J L, Witzens M et al. Immunoglobulin framework-derived peptides function as cytotoxic T-cell epitopes commonly expressed in B-cell malignancies. Nature medicine. 2000,6(6): 667-672.
97. Van Epps H L, Schmaljohn C S, Ennis F A. Human memory cytotoxic T-lymphocyte (CTL) responses to Hantaan virus infection: identification of virus-specific and cross-reactive CD8(+) CTL epitopes on nucleocapsid protein. Journal of virology. 1999,73(7): 5301-5308.
98. Voeten J T, Bestebroer T M, Nieuwkoop N J et al. Antigenic drift in the influenza A virus (H3N2)nucleoprotein and escape from recognition by cytotoxic T lymphocytes. Journal of virology. 2000,74(15): 6800-6807.
99. Voeten J T, Rimmelzwaan G F, Nieuwkoop N J et al. Antigen processing for MHC class I restricted presentation of exogenous influenza A virus nucleoprotein by B-lymphoblastoid cells. Clinical and experimental immunology. 2001,125(3): 423-431.
100. Wallny H J, Avila D, Hunt L G et al. Peptide motifs of the single dominantly expressed class I molecule explain the striking MHC-determined response to Rous sarcoma virus in chickens. Proceedings of the National Academy of Sciences of the United States of America. 2006,103(5): 1434-1439.
101. Wong D K, Dudley D D, Dohrenwend P B et al. Detection of diverse hepatitis C virus (HCV)-specific cytotoxic T lymphocytes in peripheral blood of infected persons by screening for responses to all translated proteins of HCV. Journal of virology. 2001,75(3): 1229-1235.
102. Ye Q, Krug R M, Tao Y J. The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature. 2006,444(7122): 1078-1082.
103. Zhang C, Anderson A, DeLisi C. Structural principles that govern the peptide-binding motifs of class I MHC molecules. Journal of molecular biology. 1998,281(5): 929-947.