一株H5亚型禽流感病毒NP蛋白两个鸡MHC分子限制性T细胞表位的鉴定  

Identification of two T cell epitopes of nucleoprotein from an H5 avian influenza virus in chickens

作　　者：侯艳霞[1] 郭莹莹[1] 沈楠[1] 吴春艳[1] 姜永萍 王靖飞[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室／疫病诊断中心,黑龙江哈尔滨150001 [2]国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室／农业部动物流感重点开放实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2011年第6期457-460,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：中国农业科学院哈尔滨兽医研究所院所长基金(SKLVBP201016)

摘　　要：为验证一株禽流感病毒(AIV)核蛋白(NP)中的3条T细胞表位候选肽NP89-97(PKKTGGPIY)、NP198-206(KRGINDRNF)和NP64-71(ERMVLSAF)的免疫原性,本研究构建了含有NP基因的重组质粒pCAGGS-NP,将其转染293T细胞,间接免疫荧光和western blot检测表明NP蛋白在293T细胞中获得表达。重组质粒免疫鸡后,用间接酶联免疫实验检测发现重组质粒在鸡体内能诱导产生相应的抗体。将多肽NP89-97、NP198-206、NP64-71和不相关肽N71-78(WRRQARYK)在体外刺激免疫后鸡的脾淋巴细胞,流式细胞术检测发现CD8+T淋巴细胞增殖分别为13.7%、11.9%、0.6%和0.4%;ELISA检测结果显示多肽NP89-97、NP198-206刺激后的淋巴细胞中IFN-γ的分泌量明显增加。本实验表明多肽NP89-97和NP198-206能在体外诱导活化的鸡淋巴细胞产生细胞毒性T淋巴细胞反应,为AIV NP的T细胞表位。该研究结果对研究禽类抗流感病毒的免疫机制及抗流感疫苗具有重要的意义。To verify 3 potential T cell epitope peptides of NP89-97（PKKTGGPIY）,NP198-206（KRGINDRNF） and NP64-71（ERMVLSAF） in nucleoprotein（NP） of avian influenza virus（AIV）,a recombinant plasmid pCAGGS-NP was constructed and the expression of NP was confirmed by indirect immunofluorescence assay and western blot in transfected 293T cell.The antibodies against NP were also detected by ELISA in pCAGGS-NP inoculated SPF chickens.The spleen cells were collected from inoculated chickens and stimulated with the peptides NP89-97,NP198-206,NP64-71 and irrelevant peptides N71-78（WRRQARYK）.Flow cytometry assay results showed that the CD8＋ T lymphocytes were proliferated by 13.8%,11.9%,0.6%,and 0.4%,respectively.Chicken IFN-γ secretions were also detected in cells stimulated with peptides NP89-97 and NP198-206 by ELISA.The results indicated that peptide NP89-97 and NP198-206 were T cell epitopes which could induce specific cytotoxic T lymphocyte reaction.

关 键 词：禽流感病毒 NP蛋白 T细胞表位 鉴定 

分 类 号：S852.4[农业科学—基础兽医学]