禽传染性支气管炎病毒N蛋白保守B细胞抗原表位的鉴定  

Identification of a conserved B-cell epitope located in the avian infectious bronchitis virus N protein

作　　者：张冰莎 张桃妮 张愉 刘玲 范文胜[1] 任丽娜 卢彦澎 磨美兰[1,2,3,4] ZHANG Bing-sha;ZHANG Tao-ni;ZHANG Yu;LIU Ling;FAN Wen-sheng;REN Li-na;LU Yan-peng;MO Mei-lan(College of Animal Science and Technology,Guangxi University,Nanning 530004,China;Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics,Nanning 530004,China;Guangxi Key Laboratory of Animal Breeding,Disease Control and Prevention,Nanning 530004,China;Guangxi Colleges and Universityies Key Laboratory of Prevention and Control for Animal Disease,Nanning 530004,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]广西壮族自治区兽用生物制品工程研究中心,广西南宁530004 [3]广西畜禽繁育与疾病防控重点实验室,广西南宁530004 [4]广西高校动物疫病预防与控制重点实验室,广西南宁530004

出　　处：《中国兽医科学》2023年第11期1406-1414,共9页Chinese Veterinary Science

基　　金：广西重点研发计划项目(桂科AB21238003);广西自然科学基金项目(2020GXNSFDA297004,2019GXNSFAA245009)。

摘　　要：应用噬菌体随机十二肽库技术对针对IBV GX-YL5株N蛋白的单抗N2D5进行抗原表位的鉴定。将淘选得到的噬菌体随机十二肽库第3轮洗脱液进行二代测序,将测序结果中出现频率最高的3个十二肽序列合成肽后验证是否为模拟表位;根据获得的模拟表位预测相应的天然表位序列并合成多肽和原核表达进行验证,同时对获得的天然表位进行保守性和交叉反应性分析。结果显示,第3轮筛选洗脱液测序出现频率最高的3个序列中有2个为模拟表位,即VVGRAMAYSTIP和DGVLLGTSGEST;预测天然表位序列为158IPLNRGRGGRST169;预测天然表位的合成肽的ELISA和Dot-blotting分析以及其原核表达蛋白的Western-blotting分析表明,158IPLNRGRGGRST169是IBV的一个天然表位序列;保守性和交叉反应性分析显示该天然表位具有高度保守性。结果表明,本研究成功鉴定出IBV GX-YL5 N蛋白上的一个高度保守的表位158IPLNRGRGGRST169,为IBV N蛋白的抗原结构和功能的研究及诊断试剂和表位疫苗的研制奠定了基础。The epitope targeted by monoclonal antibody(mAb)N2D5 which was against N protein of IBV GX-YL5 was identified by phage display random 12-peptide library.The third round of elution was sequenced using next-generation sequencing and the three dodecapeptides with the highest frequency in the sequencing results were synthesized to verify whether they were mimotopes.According to the obtained mimotopes,the corresponding natural epitope was predicted and verified by synthetic peptide and prokaryotic expression.At the same time,the conservation and cross-reactivity of the obtained natural epitope were analyzed.The results showed that VVGRAMAYSTIP and DGVLLGTSGEST were confirmed to be mimotopes,and the predicted epitope was 158IPLNRGRGGRST169.The ELISA and Dot-blotting analysis of the synthetic peptide and Westernblotting analysis of the prokaryotic expression protein of the predicted natural epitope showed that 158IPLNRGRGGRST169 was a natural epitope of IBV.The conservation and cross-reactivity analysis showed that the natural epitope was highly conserved.The results indicate that a highly conserved epitope 158IPLNRGRGGRST169 on the IBV GX-YL5 N protein was successfully identified in this study,which laying an important foundation for the study of the antigen structure and function of IBV N protein and the development of diagnostic reagents and epitope vaccines.

关 键 词：传染性支气管炎病毒 N蛋白 噬菌体随机十二肽库 抗原表位 交叉反应性 

分 类 号：S852.659.6[农业科学—基础兽医学]