本文選題：日本囊對蝦 + 白斑綜合征病毒　； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：類泛素化(Neddylation)是一個(gè)蛋白翻譯后進(jìn)行修飾的可逆的過程。NEDD8(neural precursor cell expressed,developmentally down-regulated 8,神經(jīng)前體細(xì)胞表達(dá)發(fā)育下調(diào)蛋白-8),可以與靶蛋白上賴氨酸殘基相互結(jié)合,發(fā)生Neddylation。Neddylation修飾也是通過E1激活酶、E2結(jié)合酶和E3連接酶的參與,對蛋白進(jìn)行類泛素化修飾,從而促進(jìn)蛋白的降解或調(diào)控靶蛋白的功能。去類泛素化(Deneddylation)和類泛素化為互逆的反應(yīng)。Deneddylation是由COP9信號復(fù)合體(Constitutive photomorphogenesis 9 signalosome,CSN)介導(dǎo)來完成的。CSN由八個(gè)亞基構(gòu)成。CSN5,其中的第五個(gè)亞基,具有不同于其他七個(gè)亞基的明顯特點(diǎn)。CSN5具有催化活性位點(diǎn)和鋅離子結(jié)合位點(diǎn),CSN5在CSN發(fā)揮其功能的過程中占據(jù)關(guān)鍵位置。然而,其在抗病毒免疫中的作用尚不清楚。在本論文中,我們分別對日本囊對蝦體內(nèi)的CS7個(gè)亞基(CSN1-CSN2和CSN4-CSN8)進(jìn)行了鑒定和克隆。我們分析了 CSN的每個(gè)亞基在日本囊對蝦中的組織分布和WSSV刺激的表達(dá)模式。結(jié)果顯示,MjCSN1-2和MjCSN4-6在對蝦的組織中是組成型的分布。MjCSN7和MjCSN8,在對蝦的組織也都是表達(dá)的,除去肝胰腺組織。WSSV對對蝦進(jìn)行刺激時(shí),MjCSN1,MjCSN2,MjCSN4和MjCSN7,MjCSN8的表達(dá)水平明顯的降低,而MjCSN5的表達(dá)水平是呈現(xiàn)上升的趨勢。通過表達(dá)模式分析,CSN5與其他亞基的表達(dá)情況有明顯不同的。所以,我們選擇了對蝦體內(nèi)的CSN5亞基(命名為MjCSN5)作為下一步研究的重點(diǎn)。我們在大腸桿菌中進(jìn)行了對MjCSN5重組蛋白的表達(dá)。抗體就是使用了該重組蛋白。為了研究MjCSN5的功能,設(shè)計(jì)進(jìn)行了 RNA干擾和過表達(dá)實(shí)驗(yàn)。MjCSN5能夠成功的敲除降低后,WSSV在對蝦內(nèi)的復(fù)制水平是上升的。過表達(dá)MjCSN5與穿膜肽融構(gòu)的重組蛋白,WSSV復(fù)制量是下降的。死亡率實(shí)驗(yàn)證明,與對照組相比,注射重組蛋白的實(shí)驗(yàn)組對蝦的成活率明顯的提高。進(jìn)一步的機(jī)理研究表明,這種作用可能是通過抑制NF-κB途徑來實(shí)現(xiàn)的。綜上所述,CSN5在對蝦體內(nèi)顯示出抗病毒的功能,本研究可以為白斑綜合征疾病的控制提供新的理論和方法。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S945.4

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本文編號：2007147