新城疫病毒NP蛋白MHCI类分子限制性T细胞表位研究  

LOCALIZATION OF RESTRICTED T CELL EPITOPES ON NP PROTEIN MHC CLASS I MOLECULES OF NEWCASTLE DISEASE VIRUS

作　　者：乔长涛[1,2] 谭磊[2] 于圣青[2] 仇旭升[2] 宋翠萍[2] 孙英杰[2] 胡桂学[1] 丁铲[2] 

机构地区：[1]吉林农业大学动物科学技术学院,长春130118 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2014年第1期9-15,共7页Chinese Journal of Animal Infectious Diseases

基　　金：上海市科技兴农重点攻关项目资助(沪农科攻字(2013)第3-7号);公益性行业(农业)科研专项资助(201303038)

摘　　要：为了对新城疫病毒(Newcastle disease virus,NDV)的细胞毒性T细胞(cytotoxic T cell,CTL)表位进行研究,本研究通过在线分析软件预测了NDV弱毒株La Sota和强毒株Herts/33 NP蛋白的MHC I类分子限制性表位,共获得9条可能的表位多肽序列,并人工合成了这些短肽。分别构建了含有La Sota和Herts/33 NP基因的DNA重组质粒并免疫4周龄SPF级C57BL/6小鼠,首免后21 d加强免疫1次,二免后10 d无菌采集脾脏制备脾淋巴细胞悬液,采用ELISpot法测定预测多肽诱导脾淋巴细胞分泌IFN-γ的能力,根据分泌IFN-γ形成的特异性斑点数进行生物统计学分析,发现2条多肽产生的斑点数显著高于无关肽刺激组(P<0.01)。确定了针对小鼠H-2 kb的限制性T细胞抗原表位的2条多肽。这些可用于合成特异性的MHC-肽四聚体,为后续研究NDV与树突状细胞(dendritic cells,DC)及T淋巴细胞之间的免疫抑制机制提供重要保障。The NP protein MHC I restricted epitopes of attenuated strain La Sota and virulent strain Herts/33 of Newcastle disease virus （NDV） were predicted using online analysis software for localization of cytotoxic T cell （CTL） epitopes. A total of 9 possible epitope sequences were identified and synthesized. The DNA plasmids were constructed to contain La Sota, Herts/33 strain NP genes and then used to immunize 4-week-old SPF C57BL/6 mice. A booster immunization was performed at day 21 post the first vaccination. Spleens of immunized mice were aseptically collected at day 10 post the second immunization and lymphocyte suspensions were prepared to detect IFN-γsecretions in ELISpot. Specific spots of lymphocyte IFN-γsecretions were analyzed. Two out of 9 peptides showed significantly higher levels of lymphocyte IFN-γsecretions than irrelevance peptide （p〈0.01）. As a result, two restricted T cell epitopes were determined on mouse H-2 kb. Information can be used for synthesis of specific MHC-peptide tetramer for further investigation into the mechanisms of immunosuppression between NDV and dendritic cells （DC） and T lymphocytes.

关 键 词：新城疫病毒 NP蛋白 CTL表位 筛选 

分 类 号：S852.659.5[农业科学—基础兽医学]