【作者】 刘倩；

【导师】 康翠洁；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2015， 硕士

【摘要】 海洋生物是人类食物的重要来源。对虾是其中一种重要的海水经济养殖动物,然而对虾养殖业易受到多种细菌和病毒的感染,遭受到巨大损失。因此,研究对虾先天免疫机理,不仅可以为防治虾病提供基础理论指导,还能与模式生物的相关研究进行比较,具有重要的理论意义与应用价值。细胞是最小的生命系统组成单位,在其内部的正常活动与秩序的维持中,胞内的衔接蛋白(Adaptor protein)起到不可忽视的作用。其正常的免疫活动,如信号转导,细胞免疫等等,都依赖于接头蛋白才能有条不紊的进行,因此,研究对虾接头蛋白在免疫过程中的功能对于我们进一步了解对虾的先天免疫机制和细胞中衔接蛋白的功能有重要的作用。Syntenin蛋白是一种含有两个串联的PDZ(首次在在突触后密度蛋白PSD-95,DlgA和ZO-1中报道的重复序列)结构域的多功能的胞质内接头蛋白,能够参与到细胞粘附,迁移,分化,凋亡以及在脊椎动物的病毒的免疫反应过程中。但是,目前关于Syntenin蛋白在无脊椎动物的病毒免疫和细菌免疫方面的功能和具体作用机理没有研究。本文选取了日本囊对虾(Marsupenaeus japonicus)做为实验动物,克隆得到了Syntenin的同源基因,命名为MjSyn,并研究了该基因及其编码的蛋白在对虾免疫反应中的功能及其作用机理。MjSyn的cDNA全长共1223bp,其完整开放阅读框为963 bp,共编码320个氨基酸。结构域分析发现MjSyn共包含两个PDZ结构域,一个N端区域和一个C端区域。MjSyn在对虾各个组织中均有分布,并且在受到金黄色葡萄球菌(Staphylococcus aureus),鳗弧菌(Vibrio anguillarum)和白斑综合症病毒(white spot syndrome virus, WSSV)刺激后,在不同时间点均有不同程度的上调表达。研究发现,通过RNAi方法干扰对虾体内MjSyn的表达量后,对虾对于细菌感染的清除能力下降了。利用血细胞吞噬荧光细菌的方法,表明干扰MjSyn影响了细胞对于细菌的吞噬能力,进而影响了对虾对细菌的清除能力。通过进一步筛选吞噬相关基因,我们发现干扰MjSyn后,日本对虾中的Calnexin(MjCnx)的mRNA和蛋白的表达都受到抑制,这表明MjSyn可能通过影响calnexin相关途径来影响细胞的吞噬过程。在对虾抗病毒免疫方面的研究中,我们发现干扰MjSyn后,病毒蛋白VP28在对虾体内的复制减少。而过表达MjSyn后,病毒蛋白VP28的复制增加。体外pull-down和体内Co-IP实验证明,全长MjSyn可以结合病毒的囊膜蛋白VP24、 VP28和VP26。全长蛋白中,结构域1+2与结构域C(CTD)都能够分别结合这三种病毒蛋白。另外,实验发现对虾受到病毒刺激后,MjSyn向细胞膜迁移,呈极化分布,F-actin的迁移趋势与MjSyn相吻合,因此两者的共定位增加。值得注意的是,在病毒刺激后的血淋巴中能够检测到分泌出细胞外的MjSyn及VP28蛋白。这些结果表明,MjSyn通过结合病毒蛋白和调控F-actin的分布,促进病毒释放的过程来参与对虾的病毒免疫。更多还原

【Abstract】 Mariculture organisms are important sources of human food. Among them, kuruma shrimp Marsupenaeus japonicus is an important aquaculture industry species. However, the shrimp aquaculture industry suffered the diseases that caused by bacteria and virus in the past decade. Study on the innate immunity system of shrimp will provide both theoretical and practical support to this industry. As the smallest living system, cell relies on various proteins to organize life activities in order. Among them, adaptor proteins play a significant role in connection and transport. The functional study of adaptor proteins in shrimp will help to understand the mechanism of innate immunity better.Syntenin is a multifunctional cytosolic adaptor protein, contributing to cell migration, proliferation, attachment and apoptosis, as well as immune response to virus in vertebrate. However, the function of syntenin in the anti-bacteria and anti-virus response of invertebrate remains unclear. In this study, we identified a syntenin-like gene (MjSyn) from the kuruma shrimp (Marsupenaeus japonicus), detected its function in the immunity of shrimp. The full-length MjSyn was 1223 bp with a 963 bp open reading frame encoding 320 amino acids. The deduced MjSyn proteins contained two atypical PDZ domains (sequence repeat that firstly reported in postsynaptic density protein or PSD-95, DlgA and ZO-1 protein), an N-terminal domain and a C-terminal domain. Reverse transcription PCR showed that MjSyn was expressed in all tested tissues. Quantitative real-time RT-PCR analysis revealed that MjSyn transcripts in hemocyte, gill and intestine were significantly induced at various time points after infection with Staphylococcus aureus, Vibrio anguillarum and white spot syndrome virus (WSSV). Knockdown the expression of MjSyn by RNA interference led to significant decrease of phagocytic ability and increased bacteria number in vivo of shrimp. Moreover, the expression of MjCnx, a cytoplasma and membrane location lectin chaperone protein was inhibited in the MjSyn knock down shrimp, indicated a possible calnexin related way. Taken together, MjSyn participating in the bacteria clearance response of kuruma shrimp provides a new insight into the function of this kind of important adaptor protein.At the same time, we found that the replication of WSSV in shrimp decreased when MjSyn was knocked down, while over-expression of MjSyn led to an increasing expression of VP28. Pull-down assay in vitro showed that full-length MjSyn and truncated PDZ1+2 and CTD could bind to the envelope proteins VP24, VP26 and VP28, indicating a role of anti-viral immune activity via binding to virus envelop proteins. In addition, MjSyn in cytoplasm clustered onto the cell membrane and distributed in polarization when challenged with WSSV. The F-actin movement tendency was collaborate with MjSyn, indicating a connection between MjSyn and F-actin in response to WSSV infection. It is worth noting that secreted MjSyn and VP28 can be detected in hemolymph after WSSV challenge. In summary, our results provide novel evidence that MjSyn plays an important role in shrimp innate immune system.更多还原