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凡纳滨对虾网格重链蛋白与WSSV结构蛋白在体外的相互作用
王中一1,2, 刘庆慧1,2, 黄 倢1,2
1.青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 农业部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071;2.水产科学国家级实验教学示范中心 上海海洋大学水产与生命学院 上海 201306
摘要:
根据凡纳滨对虾(Litopenaeus vannamei)网格重链蛋白(CHC)的2个功能结构域Clathtin Propel Repeat (LvCHC1)和Clathrin Heavy Chain Repeat Homology (LvCHC2),分别设计2对特异性引物,扩增目的片段,并克隆至pBAD/gⅢA载体上,以E.coli Top10为宿主菌,在阿拉伯糖的诱导下获得LvCHC1和LvCHC2重组蛋白。以Co2+亲和层析方法,获得纯化的LvCHC1和LvCHC2蛋白,并经质谱分析验证。采用Far-Western方法分析LvCHC1和LvCHC2蛋白与白斑综合征病毒(WSSV)结构蛋白VP26、VP28N和VP37的作用,结果显示,LvCHC1和LvCHC2与VP28N没有结合作用,但都能与VP26和VP37结合,其中与VP26的结合作用较强。表明网格蛋白介导的内吞途径在WSSV侵染过程中起到了一定的作用。本研究可为深入研究WSSV入侵机制提供依据。
关键词:  凡纳滨对虾  网格重链蛋白  WSSV  体外作用
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In vitro Interaction Between Domain of Clathrin Heavy Chain in Litopenaeus vannamei and WSSV Structural Proteins
WANG Zhongyi,LIU Qinghui,HUANG Jie
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071
Abstract:
Clathrin heavy chain protein (CHC), a conserved protein only found in eukaryotes, is one of the essential components of clathrin. White spot syndrome virus (WSSV) infection is one of the main diseases in aquaculture. It belongs to the Whispovirus genus of the Nimaviridae family. WSSV, a rod-shaped, non-occluded baculovirus with large doubled-stranded DNA, can infect shrimps such as penaeid shrimp and crayfish. WSSV has a wide range of hosts in crustacean with high infection and mortality rate, and it causes up to 100% mortality within 3~7 days, which causes great economic loss in aquaculture industry. Recent research showed that the clathrin mediated endocytosis path regulates WSSV infection in the Cherax quadricarinatus hematopoietic cells. However, whether the clathrin heavy chain protein plays an important way in the clathrin mediated endocytosis pathway is unclear. To address this, two primers were designed to clone the LvCHC1 and LvCHC2 according to the two structural domains: clathrin propel repeat (LvCHC1) and clathrin heavy chain repeat homology (LvCHC2) of Litopenaeus vannamei CHC. LvCHC1 and LvCHC2 were cloned into prokaryotic expression vector pBAD/gⅢA and transformed into E. coli TOP 10. The recombinant LvCHC1 and LvCHC2 were successfully obtained by inducing with L-arabinose. The pure LvCHC1 and LvCHC2 were acquired using Co2+ affinity chromatography purification. Mass spectrometry analysis showed the correctness of the recombinant LvCHC1 and LvCHC2. Far-Western blot results indicated that LvCHC1 and LvCHC2 interacted with VP26 and VP37 with higher banding activity with VP26, and that LvCHC1 and LvCHC2 did not bind to VP28N. Taken together, the results indicated that clathrin heavy chain-mediated endocytosis is required for WSSV infection.
Key words:  Litopenaeus vannamei  Clathrin heavy chain protein  White Spot Syndrome Virus  Interaction in vitro