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中国明对虾(Fenneropenaeus chinensis) Tetraspanin-3与WSSV的体外相互作用
关广阔,刘庆慧,黄 倢
作者单位
关广阔 上海海洋大学 上海 201306;农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071; 
刘庆慧 农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071;青岛国家海洋科学重点实验室 青岛 266071 
黄 倢 农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071;青岛国家海洋科学重点实验室 青岛 266071 
摘要:
Tetraspanin-3是四跨膜蛋白超家族的一员,在信号转导和免疫等过程中起到重要作用。本研究将中国明对虾(Fenneropenaeus chinensis) Tetraspanin-3 (FcT3)的大胞外环区(Large extracellular loop, LEL)基因片段克隆,与原核表达载体pBAD/gⅢA连接,获得重组表达载体pBAD/gⅢA-T3L,转化入大肠杆菌TOP 10中,用L-阿拉伯糖(L-Arab)诱导并经钴离子亲和层析纯化得到重组蛋白。质谱分析显示,纯化的重组蛋白为FcT3。用地高辛将FcT3L标记并与WSSV作用,Far-western分析显示,FcT3L与VP26结合。ELISA实验结果显示,FcT3L与VP26蛋白的相互作用随VP26量的增加而增强。推测FcT3通过与VP26作用介导病毒核衣壳的入胞过程。
关键词:  中国明对虾  Tetraspanin-3  WSSV  相互作用
DOI:10.11758/yykxjz.20150609
分类号:
基金项目:国家重点基础研究发展计划(2012CB114401)、泰山学者"建设工程专项经费"和农业部科研杰出人才和创新团队专项经费共同资助
Interaction of Tetraspanin-3 in Fenneropenaeus chinensis with WSSV in vitro
GUAN Guangkuo,LIU Qinghui,HUANG Jie
Abstract:
White spot syndrome virus (WSSV) is a widespread and disastrous viral pathogen of cultured shrimp that infects many different species of crustaceans. Tetraspanin-3 protein belongs to the tetraspanin superfamily, which plays an important role in signal transduction and immune process. Increasing evidence has shown that tetraspanins play important roles in virus invasion, penetration and fusion events. Antibody to the large extracellular loop (LEL) of Tetraspanin-3 of Fenneropenaeus chinensis can block the WSSV infection. However, whether the F. chinensis Tetraspanin-3 is involved directly with WSSV remained largely unknown. In this research, we cloned the LEL fragment of Tetraspanin-3 from shrimp F. chinensis (FcT3L) and ligated it with prokaryotic expression vector pBAD/gⅢA to get recombinant expression vector pBAD/gⅢA-FcT3L. After transformed into E. coli (TOP 10) and induced using the L-arabinose, we successfully obtained the pure recombinant protein using Co2+ affinity chromatography purification. Mass spectrometry analysis showed the correctness of the recombinant protein. Moreover, FcT3L was labeled with DIG and incubated with WSSV and the results showed that FcT3L can interact with VP26. Furthermore, recombinant VP26, VP28N, VP28C and VP37 were separated by SDS-PAGE and transferred to PVDF membranes. After incubating with DIG-labeled FcT3L, a positive band was shown with VP26. To confirm the interaction of FcT3L with VP26, FcT3L was coated with 96-well plates and different amount of VP26 was added. ELISA assay showed that the interaction between FcT3L and VP26 grew stronger with the increasing concentration of FcT3L. Results suggested that VP26 might bind to the Tetraspanin-3 and act to help the WSSV diffuse and penetrate into the cytoplasm. The detailed mechanism needs further investigation.
Key words:  Tetraspanin-3  Fenneropenaeus chinensis  WSSV  Interaction