COP9信号亚基5在日本囊对虾中的抗病毒功能研究

发布时间：2018-06-11 23:29

本文选题：日本囊对虾 + 白斑综合征病毒　；参考：《山东大学》2017年硕士论文

【摘要】：类泛素化(Neddylation)是一个蛋白翻译后进行修饰的可逆的过程。NEDD8(neural precursor cell expressed,developmentally down-regulated 8,神经前体细胞表达发育下调蛋白-8),可以与靶蛋白上赖氨酸残基相互结合,发生Neddylation。Neddylation修饰也是通过E1激活酶、E2结合酶和E3连接酶的参与,对蛋白进行类泛素化修饰,从而促进蛋白的降解或调控靶蛋白的功能。去类泛素化(Deneddylation)和类泛素化为互逆的反应。Deneddylation是由COP9信号复合体(Constitutive photomorphogenesis 9 signalosome,CSN)介导来完成的。CSN由八个亚基构成。CSN5,其中的第五个亚基,具有不同于其他七个亚基的明显特点。CSN5具有催化活性位点和锌离子结合位点,CSN5在CSN发挥其功能的过程中占据关键位置。然而,其在抗病毒免疫中的作用尚不清楚。在本论文中,我们分别对日本囊对虾体内的CS7个亚基(CSN1-CSN2和CSN4-CSN8)进行了鉴定和克隆。我们分析了 CSN的每个亚基在日本囊对虾中的组织分布和WSSV刺激的表达模式。结果显示,MjCSN1-2和MjCSN4-6在对虾的组织中是组成型的分布。MjCSN7和MjCSN8,在对虾的组织也都是表达的,除去肝胰腺组织。WSSV对对虾进行刺激时,MjCSN1,MjCSN2,MjCSN4和MjCSN7,MjCSN8的表达水平明显的降低,而MjCSN5的表达水平是呈现上升的趋势。通过表达模式分析,CSN5与其他亚基的表达情况有明显不同的。所以,我们选择了对虾体内的CSN5亚基(命名为MjCSN5)作为下一步研究的重点。我们在大肠杆菌中进行了对MjCSN5重组蛋白的表达。抗体就是使用了该重组蛋白。为了研究MjCSN5的功能,设计进行了 RNA干扰和过表达实验。MjCSN5能够成功的敲除降低后,WSSV在对虾内的复制水平是上升的。过表达MjCSN5与穿膜肽融构的重组蛋白,WSSV复制量是下降的。死亡率实验证明,与对照组相比,注射重组蛋白的实验组对虾的成活率明显的提高。进一步的机理研究表明,这种作用可能是通过抑制NF-κB途径来实现的。综上所述,CSN5在对虾体内显示出抗病毒的功能,本研究可以为白斑综合征疾病的控制提供新的理论和方法。
[Abstract]:Ubiquitin is a reversible process of post-translational modification. NEDD8 / neural precursor cell expressed developmental down-regulated 8, a down-regulation of neural precursor cell expression protein -8, can bind to lysine residues on target proteins. Neddylation.Neddylation is also involved in E1-activator E2 binding enzyme and E3 ligase to modify proteins by Ubiquitin, thus promoting the degradation of proteins or regulating the function of target proteins. Deneddylation) and ubiquitin conversion are mediated by COP9 signaling complex Constitutive photomorphogenesis 9 signalosome CSNs. CSN consists of eight subunits, the fifth of which, CSN5 has catalytic activity site and zinc ion binding site CSN5 plays a key role in the process of CSN performing its function. However, its role in anti-viral immunity is unclear. In this paper, we identified and cloned CSN1-CSN2 and CSN4-CSN8 from 7 subunits of Penaeus japonicus respectively. We analyzed the tissue distribution of each subunit of CSN in Penaeus japonicus and the expression pattern of WSSV stimulation. The results showed that MjCSN1-2 and MjCSN4-6 were constitutively distributed in shrimp tissues. MjCSN7 and MjCSN8 were also expressed in shrimp tissues. The expression levels of MjCSN1MjCSN2MjCSN4 and MjCSN7MjCSN8 were significantly decreased when the hepatopancreatic tissue. The expression level of MjCSN 5 was on the rise. The expression patterns of CSN 5 were different from those of other subunits. Therefore, we selected CSN 5 subunit (named MjCSN 5) as the focus of further research. We expressed MjCSN 5 recombinant protein in E. coli. The antibody is the use of the recombinant protein. In order to study the function of MjCSN5, we designed RNA interference and overexpression experiment. MjCSN5 can successfully knock out and reduce WSSV replication level in shrimp is increased. The replication of recombinant protein WSSV which overexpressed MjCSN5 and transmembrane peptide was decreased. The mortality test showed that the survival rate of prawns injected with recombinant protein was significantly higher than that of control group. Further studies indicate that this effect may be achieved by inhibiting NF- 魏 B pathway. In conclusion, CSN5 showed antiviral function in prawns. This study could provide a new theory and method for the control of leukoplakia syndrome.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S945.4

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