摘要
根据ClassI新城疫病毒分离株9a5b编码序列设计特异性引物扩增P蛋白基因，原核表达后利用纯化的蛋白免疫BALB／C小鼠制备单克隆抗体，经二次亚克隆后筛选到4株针对P蛋白的单克隆抗体，利用所得单抗进行P蛋白在细胞内定位分析，NDV9a5b株按5M．O．I感染量接种DFl单层细胞，当PI=4h时，P蛋白呈点状弥散分布于细胞浆内；PI=6～8h时，P蛋白聚集于细胞核周围；PI=12h时，P蛋白呈单极化聚集于细胞核一侧；而PI=16h后开始形成合胞体，P蛋白在单个细胞中仍呈现单极分布；当PI=48h时，细胞核已发生碎裂，荧光强度有所减弱。本研究为深入开展P蛋白在病毒增殖以及细胞周期中的作用机制研究奠定了基础。
According to the sequence of Class I Newcastle disease virus （NDV）9a5b strain, a pair of primes was designed to amplify P genes. After procaryotic expression and purification, P protein was immunized BALB/c mouse to prepare monoclonal antibodies （MAbs）. After subcloning for 2 times, 4 specific MAbs were obtained to analysis the subcellular localization of P protein. The DF-1 monolayer cells were infected by 5 M.O.I NDV 9a5b virus. PI=4 h, P protein diffusely distributed in cytoplasm in punctiform. PI=6-8 h, P protein accumulated mainly in the surrounding of nucleus. PI=12 h, P protein localized in the monopole of cells. After the formation of syncytium in 16 h, P protein still accumulated mainly in the monopole of single cells. PI=48 h, with the cell nucleus splitting up, fluorescence intensity attenuated. The research established the basis for further study on the effect P protein participated in virus generation and cell cycle.
出处
《中国动物传染病学报》
CAS
2013年第1期17-22,共6页
Chinese Journal of Animal Infectious Diseases
基金
国家自然科学基金(31101822)
公益性行业(农业)专项(201003012)
