本文選題：鴨Viperin + 新城疫病毒�。� 參考：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：2001年首次報道Viperin是一種受干擾素、病毒等刺激誘導(dǎo)的基因。近年來,已經(jīng)證實Viperin不僅能被多種因素誘導(dǎo)而大量表達(dá),而且還具有廣譜的抗病毒和抗細(xì)菌等作用,甚至還參與調(diào)節(jié)細(xì)胞信號通路的功能。由此可見,Viperin在天然免疫中發(fā)揮非常重要的角色。隨著對Viperin的深入和全面研究,逐漸揭示Viperin的抗多種不同病毒、抗細(xì)菌等作用機(jī)制。本研究從長白鴨的外周淋巴細(xì)胞中擴(kuò)增鴨Viperin基因并利用生物信息學(xué)方法對其進(jìn)行詳盡地基因分析�；赩iperin參與抗病毒作用,通過鴨Viperin蛋白抗NDV的試驗研究,以揭示鴨Viperin抗NDV的分子機(jī)制。本研究首先采集鴨抗凝血并分離其外周血淋巴細(xì)胞,然后將外周淋巴血細(xì)胞提取鴨的總RNA反轉(zhuǎn)錄成cDNA。根據(jù)已公布的鴨的全基因組中RSAD2序列(登錄號NW_004676922.1以及XM 005018580.1)設(shè)計相應(yīng)的擴(kuò)增引物,最終獲得了鴨Viperin ORF序列。根據(jù)獲得的鴨Viperin基因序列,使用生物信息學(xué)軟件對擴(kuò)增的鴨Viperin序列進(jìn)行詳盡的功能分析。結(jié)果表明,鴨Viperin的蛋白結(jié)構(gòu)與其它已公布物種的Viperin蛋白非常相似,都包括N端區(qū)域、中心區(qū)域和C端區(qū)域。根據(jù)Viperin的基因序列,將Viperin基因克隆到pGEX 4T-1原核表達(dá)載體,分別利用原核表達(dá)方法表達(dá)并純化Viperin的融合蛋白。將該融合蛋白免疫1月齡家兔,檢測血清抗體水平,成功制備出兔抗Viperin的血清。早期研究已證實多種病毒可誘導(dǎo)Viperin的表達(dá),于是我們開展鴨Viperin在體內(nèi)體外水平的表達(dá)分析。一方面,通過制備并培養(yǎng)鴨的原代胚胎細(xì)胞(DEF),利用不同劑量poly(I:C)和NDV感染DEF后檢測Viperin的表達(dá)。結(jié)果表明,poly(I:C)和NDV均能刺激誘導(dǎo)Viperin的表達(dá)。另一方面,以2周齡長白鴨的為動物材料,使用用NDV感染鴨來檢測Viperin在不同臟器的分布。結(jié)果發(fā)現(xiàn),正常生理情況下,不同臟器的Viperin含量具有較大的差異,Viperin主要分布于血液,其次是肺臟和脾臟,腎臟和肌肉較低；以對照組相比,在NDV感染后,不同臟器Viperin的表達(dá)都有不同程度的增加。該結(jié)果表明,NDV在細(xì)胞水平和鴨體上均能夠增強(qiáng)Viperin表達(dá)。為了進(jìn)一步確定鴨Viperin發(fā)揮抗病毒的關(guān)鍵區(qū)域,我們構(gòu)建了Viperin的缺失片段(包括N端和C端缺失,分別命名為Vip△N和Vip△C)以及設(shè)計抑制鴨Viperin表達(dá)的siRNA序列。將鴨的Viperin基因(Viperin, Vip△N和Vip△C)克隆到pCAGGS真核表達(dá)載體上并轉(zhuǎn)染DEF細(xì)胞；利用siRNA技術(shù),轉(zhuǎn)染siRNA質(zhì)粒于DEF細(xì)胞,在轉(zhuǎn)染后24小時后使用NDV感染DEF,在感染后不同時間內(nèi)收集樣品以檢測NDV的病毒復(fù)制情況。結(jié)果表明,過表達(dá)鴨Viperin的情況下,能顯著的抑制NDV感染,試驗組的NDV滴度明顯低于對照組；轉(zhuǎn)染siRNA質(zhì)粒后能抑制Viperin的表達(dá),并有利于NDV復(fù)制。另外,從過表達(dá)鴨VipAN和Vip△C結(jié)果看,截短體的抗病毒作用已經(jīng)顯著降低,N端缺失后的Viperin還具有有限的抗病毒作用,而C端缺失后則導(dǎo)致Viperin抗病毒作用的完全喪失。這表明,Viperin的C端區(qū)域是其發(fā)揮抗病毒作用的關(guān)鍵區(qū)域。綜上所述,本研究研究了鴨Viperin基因的生物特性和探究該蛋白的生物學(xué)功能,發(fā)現(xiàn)poly(I:C)和NDV等因素均能誘導(dǎo)Viperin的表達(dá),說明鴨Viperin與其它物種Viperin具有類似的特征；Viperin蛋白能夠發(fā)揮顯著的抗NDV的作用,進(jìn)一步發(fā)現(xiàn)Viperin的C端是其發(fā)揮抗病毒的關(guān)鍵區(qū)域。
[Abstract]:In 2001, Viperin was first reported as a gene stimulated by interferon, virus and other stimuli. In recent years, it has been proved that Viperin not only can be induced by a variety of factors, but also has broad spectrum of antiviral and anti bacterial effects, and even participates in regulating the function of cell signaling pathway. Thus, Viperin can be found in natural immune system. With a thorough and comprehensive study of Viperin, Viperin has been gradually revealed to resist a variety of different viruses and anti bacteria mechanisms. This study amplified the duck Viperin gene from the peripheral lymphocytes of the long white duck and carried out a detailed genetic analysis by bioinformatics. Based on Viperin, it was involved in the antiviral activity. Using the experimental study of duck Viperin protein resistance to NDV, the molecular mechanism of duck Viperin resistance to NDV was revealed. This study first collected ducks' anticoagulant and separated the peripheral blood lymphocytes. Then, the total RNA of the peripheral lymphoblastic blood cells extracted from the peripheral lymphoblastic cells was transcribed into cDNA. according to the RSAD2 sequence of the published duck genome (login NW_004676922.1). And XM 5018580.1) designed the corresponding amplification primers and finally obtained the duck Viperin ORF sequence. According to the obtained duck Viperin gene sequence, the amplified duck Viperin sequence was detailed functional analysis using the bioinformatics software. The results showed that the protein structure of duck Viperin was very similar to the Viperin protein of its published species. Including the N terminal region, the central region and the C end region, the Viperin gene was cloned to the pGEX 4T-1 prokaryotic expression vector according to the gene sequence of Viperin, and the fusion protein of Viperin was expressed and purified by the prokaryotic expression method respectively. The fusion protein was immunized 1 month old rabbits and the serum anti body level was detected. The serum of Rabbit anti Viperin was successfully prepared. The study has confirmed that a variety of viruses can induce the expression of Viperin, so we carry out the expression analysis of duck Viperin in vivo and in vitro. On the one hand, the expression of Viperin is detected by the preparation and culture of the original embryo cells (DEF) of ducks and DEF in different doses of poly (I:C) and NDV. The results show that both poly (I:C) and NDV can stimulate the inducement of Vip. The expression of Erin. On the other hand, the distribution of Viperin in different organs was detected by using NDV infected ducks as animal materials for 2 weeks old long white ducks. The results showed that the Viperin content of different organs was significantly different in normal physiological conditions, Viperin was mainly distributed in the blood, followed by the lungs and spleen, and the kidney and muscle were lower. The expression of Viperin in different organs increased in varying degrees after NDV infection. The results showed that NDV was able to enhance the expression of Viperin on both the cell level and the duck body. In order to further determine the key region of the duck Viperin to play the antivirus, we constructed the missing fragments of Viperin (including the N end and C end deletion, named Vip, respectively. Delta N and Vip Delta C) and the siRNA sequence designed to inhibit the expression of duck Viperin. The Viperin gene of ducks (Viperin, Vip Delta N and Vip Delta C) was cloned into the pCAGGS eukaryotic expression vector and transfected into the cells. The plasmid was transfected to the cells by using the technique and the transfected 24 hours after the transfection was used to collect samples at different time after infection. The results showed that the virus replication of NDV was detected. The results showed that the overexpression of duck Viperin could significantly inhibit NDV infection, and the NDV titer of the test group was significantly lower than that of the control group. The transfection of siRNA plasmid could inhibit the expression of Viperin and be beneficial to the replication of NDV. In addition, the antiviral effect of the truncated duck VipAN and Vip Delta C was observed. The Viperin has a limited antiviral effect after the deletion of the N terminal, and the deletion of the C end leads to the complete loss of the antiviral effect of the Viperin. This indicates that the C terminal region of Viperin is the key area for its antiviral effect. In summary, this study has studied the biological characteristics of the duck Viperin gene and the exploration of the protein's birth. It is found that poly (I:C) and NDV can induce the expression of Viperin, indicating that duck Viperin has similar characteristics to other species Viperin; Viperin protein can play a significant role in anti NDV, and further found that C end of Viperin is the key area for its antiviral activity.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.32

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