H5N1亚型禽流感病毒NP蛋白一个鸡MHCⅠ分子限制性T细胞表位的鉴定
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摘要
为鉴定禽流感病毒核蛋白(NP)中的T细胞表位,本研究根据蛋白结构预测技术预测了4个潜在细胞毒性T淋巴细胞(CTL)表位(NP120-128、NP163-172、NP85-92、NP67-74),并合成相应的候选短肽。构建了含有NP基因的重组质粒p CAGGS-NP,将其转染293T细胞。间接免疫荧光和western blot结果表明NP蛋白能够在293T细胞中获得表达。重组质粒免疫SPF鸡后能够诱导鸡体产生特异的NP抗体。在合成的4条短肽中,NP67-74体外刺激免疫过p CAGGS-NP的鸡脾淋巴细胞可以显著提高脾淋巴细胞IFN-γ的分泌量,提示短肽NP67-74能够在体外诱导活化鸡淋巴细胞产生CTL,可能是AIV NP的CTL表位。该研究结果对流感病毒免疫机制和通用疫苗研究具有借鉴意义。
In order to identify the T cell epitope in avian influenza virus(AIV) nucleoprotein(NP),four T cell epitope candidate peptides(NP104-112,NP163-172,NP85-92 and NP67-74) of the AIV NP were predicted with the protein structure prediction technology and synthesized.To verify the immunogenicity of these peptides,the recombinant plasmid p CAGGS-NP was constructed and the expression of NP was confirmed by indirect immunofluorescence and western blot in transfected 293 T cell.The antibodies against NP were also detected by ELISA in p CAGGS-NP inoculated SPF chickens.The splenic cells were collected from inoculated chickens and stimulated with the peptides of NP120-128,NP163-172,NP85-92,NP67-74 or negative control,respectively.The results of real-time PCR show that spleen lymphocytes stimulated with the peptide NP67-74 significantly increased the IFN-γsecretion,which indicated that the peptide NP67-74 might be AIV NP CTL epitope.The results possessed significance to mechanism of protective immunity against influenza virus and the research towards universal flu vaccine.
In order to identify the T cell epitope in avian influenza virus(AIV) nucleoprotein(NP),four T cell epitope candidate peptides(NP104-112,NP163-172,NP85-92 and NP67-74) of the AIV NP were predicted with the protein structure prediction technology and synthesized.To verify the immunogenicity of these peptides,the recombinant plasmid p CAGGS-NP was constructed and the expression of NP was confirmed by indirect immunofluorescence and western blot in transfected 293 T cell.The antibodies against NP were also detected by ELISA in p CAGGS-NP inoculated SPF chickens.The splenic cells were collected from inoculated chickens and stimulated with the peptides of NP120-128,NP163-172,NP85-92,NP67-74 or negative control,respectively.The results of real-time PCR show that spleen lymphocytes stimulated with the peptide NP67-74 significantly increased the IFN-γsecretion,which indicated that the peptide NP67-74 might be AIV NP CTL epitope.The results possessed significance to mechanism of protective immunity against influenza virus and the research towards universal flu vaccine.
引文
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[14]Corr M,Slanetz A E,Boyd L F,et al.T cell receptor-MHC class I peptide interactions:affinity,kinetics,and specificity[J].Science,1994,265(5174):946-949.
[15]Adams S C,Kiefer M.Testing the attentional boundary conditions of subliminal semantic priming:the influence of semantic and phonological task sets[J].Front Hum Neurosci,2012,29(6):241.
[2]Li K S,Guan Y,Wang J,et al.Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia[J].Nature,2004,430(6996):209-213.
[3]焦同路,王靖飞,赵双成,等.表达H5亚型禽流感病毒HA蛋白的重组禽痘病毒的构建[J].中国预防兽医学报,2012,34(5):341-344.
[4]Lambert L C,Fauci A S.Influenza vaccines for the future[J].N Engl J Med,2010,363(21):2036-2044.
[5]Saha S,Yoshida S,Ohba K,et al.A fused gene of nucleoprotein(NP)and herpes simplex virus genes(VP22)induces highly protective immunity against different subtypes of influenza virus[J].Virology,2006,354(1):48-57.
[6]Ohba K,Yoshida S,Zahidunnabi D M,et al.Mutant influenza A virus nucleoprotein is preferentially localized in the cytoplasm and its immunization in mice shows higher immunogenicity and cross-reactivity[J].Vaccine,2007,25(21):4291-4300.
[7]Altstein A D,Gitelman A K,Smirnov Y A,et al.Immunization with influenza A NP-expressing vaccinia virus recombinant protects mice against experimental infection with human and avian influenza viruses[J].Arch Virol,2006,151(5):921-931.
[8]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[J].J Virol,2002,76(2):582-590.
[9]Rimmelzwaan G F,Berkhoff E G,Nieuwkoop N J,et al.Functional compensation of a detrimental amino acid substitution in a cytotoxic-T-lymphocyte epitope of influenza a viruses by comutations[J].J Virol,2004,78(16):8946-8949.
[10]Ng A K,Zhang Hong-min,Tan Ke-min,et al.Structure of the influenza virus A H5N1 nucleoprotein:implications for RNA binding,oligomerization,and vaccine design'[J].FASEB J,2008,22(10):3638-3647.
[11]Bui H H,Peters B,Assarsson E,et al.Ab and T cell epitopes of influenza A virus,knowledge and opportunities[J].PNAS USA,2007,104(1):246-251.
[12]McMurry J A,Johansson B E,De Groot A S,et al.A call to cellular&humoral arms:enlisting cognate T cell help to develop broad-spectrum vaccines against influenza A[J].Hum Vaccin,2008,4(2):148-157.
[13]Seo S H,Webster R G.Cross-reactive,cell-mediated immunity and protection of chickens from lethal H5N1 influenza virus infection in Hong Kong poultry markets[J].J Virol,2001,75(6):2516-2525.
[14]Corr M,Slanetz A E,Boyd L F,et al.T cell receptor-MHC class I peptide interactions:affinity,kinetics,and specificity[J].Science,1994,265(5174):946-949.
[15]Adams S C,Kiefer M.Testing the attentional boundary conditions of subliminal semantic priming:the influence of semantic and phonological task sets[J].Front Hum Neurosci,2012,29(6):241.