蓝舌病病毒VP7抗原在重组杆状病毒表达系统中的表达与鉴定  

Expression and Identification of Recombinant Bluetongue Virus VP7 Antigen in Baculovirus Expression Vector System

作　　者：王乃福 刘志玲 吴冬雪 孙雨[3,4] 孙航 宋晓晖 张海滨[1] WANG Naifu;LIU Zhiling;WU Dongxue;SUN Yu;SUN Hang;SONG Xiaohui;ZHANG Haibin(Tianjin Customs Animal,Plant and Food Testing Center,Tianjin 300461,China;Guangzhou Customs Technology Center,Guangzhou 510623,China;China Animal Disease Control Center,Beijing 102600,China;Beijing University of Agriculture,Beijing 100096,China)

机构地区：[1]天津海关动植物与食品检测中心,天津300461 [2]广州海关技术中心,广州510623 [3]中国动物疫病预防控制中心,北京102600 [4]北京农学院,北京100096

出　　处：《中国草食动物科学》2024年第5期31-36,共6页China Herbivore Science

基　　金：国家重点研发计划(2022YFD1800500);国家肉羊产业技术体系(CARS39)。

摘　　要：本试验通过在重组杆状病毒表达系统中制备、纯化与鉴定蓝舌病病毒VP7抗原,为开发蓝舌病病毒免疫学抗体检测方法提供科学依据。利用SignalP与TMHMM在线软件分析VP7序列的信号肽与跨膜疏水区序列,对选择的目的基因片段进行稀有密码子优化,将化学合成后的核苷酸序列连接至pFastBacHTA载体上,并构建相应的重组杆粒Bacmid-VP7-DH10Bac,转染sf9昆虫细胞后获得重组杆状病毒Bacmid-BTV-VP7株,利用杆状病毒-昆虫细胞表达系统表达制备蓝舌病病毒VP7抗原。结果显示,重组杆粒Bacmid-VP7-DH10Bac转染sf9昆虫细胞并连续盲传5代后,成功获得重组杆状病毒Bacmid-BTV-VP7株,并成功表达纯化出VP7蛋白,大小约为43 kDa,蛋白纯度约为94%;VP7重组蛋白与其他疫病抗体无非特异性交叉。综上,重组VP7蛋白能够与BTV阳性血清呈现特异性反应,具有良好的反应原性,蛋白纯度高,并且具有病毒蛋白的生物学活性,将其作为诊断抗原,后期可用于开发系列免疫学抗体诊断检测技术。This experiment was conducted to prepare,purify,and identify the bluetongue virus VP7 antigen in a recombinant baculovirus expression system,and lay the foundation for the development of immunological antibody detection methods for bluetongue disease virus.The signal peptide and transmembrane hydrophobic region sequence of VP7 sequence was analyzed by SignalP and TMHMM online software,and rare codon optimization was performed on the selected target gene fragment.The chemically synthesized nucleotide sequence was connected to the pFastBacHTA vector,and the corresponding recombinant plasmid Bacmid-VP7-DH10Bac was constructed.After transfection into sf9 insect cells,a recombinant baculovirus Bacmid-BTV-VP7 strain was obtained,then,using the rod-shaped virus insect cell expression system to express and prepare the bluetongue disease virus VP7 antigen.The results showed that after transfection of recombinant Bacmid-VP7-DH10Bac into sf9 insect cells and continuous blind transmission for 5 generations,a recombinant Bacmid-BTV-VP7 strain was successfully obtained,and the VP7 protein was successfully expressed and purified,with a size of about 43 kDa and a protein purity of about 94%.The VP7 recombinant protein did not cross with other disease antibodies.In summary,the recombinant VP7 protein can exhibit specific reactions with BTV positive serum,with good reactivity,high protein purity,and biological activity as a viral protein.It can be used as a diagnostic antigen for the development of a series of immunological antibody diagnostic detection technologies in the later stage.

关 键 词：蓝舌病 重组VP7抗原 杆状病毒 真核表达 蛋白鉴定 

分 类 号：S852.65[农业科学—基础兽医学]