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对虾Rab GTPase在抗病毒免疫中的作用以及对虾白斑综合症病毒(WSSV)膜蛋白与对虾蛋…

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6T+A$t8[ fU H1F0对虾Rab GTPase在抗病毒免疫中的作用以及对虾白斑综合症病毒(WSSV)膜蛋白与对虾蛋白的互作研究
2JqNz{ _0Function Analysis of Rab GTPase in Shrimp Antiviral Immunity and Interaction between White Spot Syndrome Virus (WSSV) Envelope Proteins and Shrimp Proteins

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作者】吴文林 【导师】章晓波; 徐洵
9D2_)cLh)pR0【作者基本信息】厦门大学,生物化学与分子生物学,2006年,博士

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H%I"`DOaB0【中文摘要】 对虾白斑综合症病毒(WSSV)是危害养殖对虾的最主要病原,其致病能力强,传播范围广,给世界对虾养殖业造成了巨大的损失,然而至今仍无有效的防治方法。本论文开展了对虾免疫信号因子Rab蛋白在对虾免疫过程中的作用机理研究,同时探索WSSV病毒膜蛋白在感染过程中的作用,从病毒和宿主两方面入手,为建立有效的防治方法提供科学依据。通过3’RACE和5’RACE获得对虾Rab基因(命名为PjRab)的cDNA全长,其ORF为636bp,编码212个氨基酸。序列分析发现,PjRab具备Rab蛋白的基本特征,但在C-端与已知的Rab蛋白不同,可能是一种新的Rab蛋白,这是首次在海洋无脊椎动物体内克隆到该基因。对该基因进行了原核表达,并得到了有活性的表达产物。RT-PCR和Western blot分析发现,PjRab在对虾组织中均有转录和翻译。Real-time PCR分析发现,在受到病毒刺激后PjRab基因表达上调,说明PjRab参与了对虾的免疫过程。通过GST-pull down、质谱等方法,发现PjRab蛋白与肌动蛋白(actin)、原肌球蛋白(tropmoyosin)以及对虾白斑综合症病毒的膜蛋白...更多VP466之间存在互作。体外和体内试验证明,PjRab与actin之间存在相互作用。采用抗体抑制Rab蛋白、RNAi抑制Rab基因表达、体外注射mRNA等方法,结果都证明Rab参与了血细胞的吞噬过程。因此,Rab蛋白参与抗病毒免疫的一条可能途径是,通过与actin的互作,调控血细胞的吞噬活性。这是动物中首次发现这一新途径,我们的研究结果拓展了对小G蛋白在免疫反应中的作用的认识。采用抗体中和试验发现,WSSV被VP28、VP68、VP281和VP466的抗体中和后能延缓对虾的死亡,PCR进一步证实了这一结果。因此,这4种病毒膜蛋白在WSSV的感染过程中起重要作用。为了进一步了解膜蛋白的作用,选择VP466进行研究。通过FITC标记、GST pull-down、质谱分析,发现了VP466的互作因子——ATP合成酶。克隆了对虾ATP合成酶基因的完整阅读框,该基因ORF含有1431bp,编码477个氨基酸。昆虫细胞内的互作试验表明,VP466与ATP合成酶存在相互作用。  还原

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【英文摘要】 White spot syndrome virus (WSSV) is one of the most devastating pathogens. It has spread to shrimp-farming areas around the world with a high infection and mortality rate and caused tremendous economic loss. However, up to date, there is no efficient method to control the virus. This study aimed at the mechanisms of Rab GTPase in shrimp immune and the functions of envelope proteins in WSSV infection. These works on the virus and host would be helpful for shrimp disease control.In this investigation, a Rab gene (named as PjRab gene) was obtained from Peneaus japonicus shrimp by 3’RACE and 5’RAC. The ORF of PjRab gene contained 636 bp enconding 212 amino acids. PjRab protein exhibited high homology with Rabs from other species, but was different from other Rabs in C-terminus, thereby constituting a unique member of the Rab GTPase family. This is the first Rab gene obtained from marine invertebrates. The gene was expressed in E. coli with GTP binding activity. The results of RT-PCR and We...更多stern blot indicated that PjRab gene was ubiquitously expressed in shrimp tissues. Real-time PCR revealed that the PjRab gene was up-regulated after WSSV infection suggesting that the PjRab protein might play a role in shrimp immune response against virus infection. It was found that the PjRab protein was interacted with shrimpβ-actin, tropmyosin and VP466 of WSSV as revealed by GST pull-down and mass spectrometry assays. The interaction between PjRab and actin was confirmed in vitro & in vivo. The results of antibody neutralization and RNAi assays showed that PjRab was involved in phagocytosis of shrimp hemocytes. Therefore, the regulation of hemocyte phagocytosis through interaction with actin might be a putative passway for Rab protein in antiviral immunity. This first finding of Rab involved in immune respone in animal extended the knowledge of small GTPases.Antibody neutralization showed that the virus infection could be significantly delayed or neutralized by antibodies against VP28, VP68, VP281 and VP466. These results were further confirmed by quantitative PCR. It could be concluded that the viral envelope proteins VP28, VP68, VP281 and VP466 played important roles in  还原水利论文`FA0}0Ysq?

"Ak @!Hn1HTa0【中文关键词】 对虾; Rab; 对虾白斑综合症病毒
$glP`(s!N,s0【英文关键词】 shrimp; Rab; white spot syndrome virus (WSSV)水利论文6F&M Br\%NY
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TAG: 对虾 综合症 膜蛋白 GTPase Rab
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