【作者】 张强；

【导师】 王金星；

【作者基本信息】 山东大学 ， 生物工程（专业学位）， 2017， 硕士

【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。更多还原

【Abstract】 Neddylation is a reversible post-translational modification and the ubiquitin-like protein NEDD8(neural precursor cell expressed developmentally down-regulated 8)is conjugated to lysine residues in the target protein.Like Ubiquitination,Neddylation requires the activating enzyme El,conjugating enzyme E2 and ligase enzyme E3,and then modified the target proteins specificly.The reverse reaction to neddylation is Deneddylation.The COP9(Constitutive photomorphogenesis 9)signalosome(CSN)mediated the process of Deneddylation.CSN is composed of eight subunits(CSN1-8).The fifth subunit of COP9 signalosome,CSN5,have obvious characteristics compared with the other seven subunits,the catalytic active sites and zinc ion binding sites.CSN5 played an important role in the function of CSN.However,the function of CSN5 in antiviral immunity is not clear.In this paper,we identified 7 subunits(CSN1-2 and CSN4-8)of COP9 signalosome in shrimp.Their tissue distribution and expression patterns in kuruma shrimp Marsupenaeus japonicus challenged by white spot syndrome virus(WSSV)were analyzed.The result showed that MjCSNl-2 and MjCSN4-6 were existed in all tested tissues,MjCSN7 and MjCSN8 were existed in all tested tissues except in hepatopancreas.After WSSV challenged,the expression level of MjCSNl,MjCSN2,MjCSN4 and MjCSN7,MjCSN8 were highly decreased,but the expression level of MjCSN5 was conspicuously increased.We found that the expression level of CSN5 was obviously different from other subunits post challenge by WSSV.Therefore,we focused our study on the CSN5(designated as MjCSN5)in the shrimp.MjCSN5 was recombinantly expressed in Escherichia coli and its polyclonal antibody was prepared.RNA interference and overexpression of MjCSN5 were also perfored.After knockdown of CSN5,the amount of WSSV In shrimp was obviously increased.When injected the recombinant protein of MjCSN5 with membrane penetrating peptide into shrimp,WSSV replication was inhibited and the survival rate of shrimp was significantly improved comparied with the control.These results indicate that CSN5 can inhibit the replication of WSSV in shrimp,and might be used in shrimp aquaculture for the white spot syndrome disease control.更多还原