凡纳滨对虾RAS与WSSV-VP26的相互作用

RAS from Litopenaeus vannamei Interacts with WSSV-VP26 in vitro

中文关键词: 凡纳滨对虾  RAS蛋白  WSSV  互作

英文关键词: Litopenaeus vannamei  RAS  WSSV  Interaction

中文摘要:

      白斑综合征病毒(WSSV)是对虾养殖中主要的病原之一，病原与宿主作用是介导病毒感染的重要过程。RAS蛋白是Ras基因分泌的保守蛋白，为小G蛋白家族的一员，普遍存在于从酵母菌到哺乳动物的真核细胞中，具有偶联受体和效应系统传递跨膜信号的功能，在细胞增殖和分化中起双重调节的作用，但关于RAS与WSSV的作用尚不明确。本研究将凡纳滨对虾(Litopenaeus vannamei) Ras基因克隆至pBAD/gⅢA表达载体上，以E. coli Top10为宿主菌，在L-阿拉伯糖的诱导下获得RAS重组蛋白。以Co2+亲和层析方法，获得纯化的RAS蛋白，质谱分析显示，该蛋白为凡纳滨对虾RAS。采用Far-western和ELISA检测方法分析RAS与WSSV结构蛋白VP26、VP28N和VP37的相互作用。Far-western结果显示，RAS与VP26有明显的结合作用，ELISA实验结果显示，RAS与VP26蛋白的相互作用随RAS量的增加而增强。本研究表明，RAS参与WSSV侵染过程，为进一步研究WSSV侵染机制提供了理论基础。

英文摘要:

      White spot syndrome virus (WSSV) is one of the most harmful pathogens in shrimp farming, which has great impacts by causing economic losses in aquaculture industry. It belongs to the Whispovirus genus of the Nimaviridae family. However, how the virus enter the cell is not very clear. Interaction between the virus and host is important for viral infection. Recently, some researchers found that GTP-binding proteins, such as Rab5, Rab6, and Rab7 interacted with infectious hypodermol and hematopoietic necrosis virus (IHHNV) and WSSV. However, the studies did not reveal the interaction between RAS protein and WSSV. The RAS protein belongs to the GTP-binding proteins and exists in eukaryotes from yeast to humans. Being a membrane protein, RAS becomes post-translationally modified after synthesis and is transported via the endoplasmic reticulum and Golgi complex route. Thus, in this study, the RAS of Litopenaeus vannamei was cloned. Then, it was ligated with prokaryotic expression vector pBAD/gⅢA using T4 DNA ligase, transformed into E.coli TOP10, and induced with L-arabinose. Pure RAS protein was acquired using Co2+ affinity chromatography purification. Mass spectrometry analysis showed the recombinant protein was RAS of L. vannamei. Far-western and ELISA assays were applied to determine the interaction of RAS with WSSV-VP26, WSSV-VP28N, and WSSV-VP37. The Far-western assay showed that the RAS protein interacted with VP26. The ELISA assay indicated that the interaction between RAS and VP26 grew stronger with the increasing concentration of RAS. Taken together, the RAS protein was responsible for WSSV infection. This study could provide the basis for research on the mechanism of WSSV invasion.