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.
引文
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