鸭Viperin蛋白抗新城疫病毒机制的初步研究

发布时间：2018-05-28 02:43

本文选题：鸭Viperin + 新城疫病毒　；参考：《中国农业科学院》2015年硕士论文

【摘要】：2001年首次报道Viperin是一种受干扰素、病毒等刺激诱导的基因。近年来,已经证实Viperin不仅能被多种因素诱导而大量表达,而且还具有广谱的抗病毒和抗细菌等作用,甚至还参与调节细胞信号通路的功能。由此可见,Viperin在天然免疫中发挥非常重要的角色。随着对Viperin的深入和全面研究,逐渐揭示Viperin的抗多种不同病毒、抗细菌等作用机制。本研究从长白鸭的外周淋巴细胞中扩增鸭Viperin基因并利用生物信息学方法对其进行详尽地基因分析。基于Viperin参与抗病毒作用,通过鸭Viperin蛋白抗NDV的试验研究,以揭示鸭Viperin抗NDV的分子机制。本研究首先采集鸭抗凝血并分离其外周血淋巴细胞,然后将外周淋巴血细胞提取鸭的总RNA反转录成cDNA。根据已公布的鸭的全基因组中RSAD2序列(登录号NW_004676922.1以及XM 005018580.1)设计相应的扩增引物,最终获得了鸭Viperin ORF序列。根据获得的鸭Viperin基因序列,使用生物信息学软件对扩增的鸭Viperin序列进行详尽的功能分析。结果表明,鸭Viperin的蛋白结构与其它已公布物种的Viperin蛋白非常相似,都包括N端区域、中心区域和C端区域。根据Viperin的基因序列,将Viperin基因克隆到pGEX 4T-1原核表达载体,分别利用原核表达方法表达并纯化Viperin的融合蛋白。将该融合蛋白免疫1月龄家兔,检测血清抗体水平,成功制备出兔抗Viperin的血清。早期研究已证实多种病毒可诱导Viperin的表达,于是我们开展鸭Viperin在体内体外水平的表达分析。一方面,通过制备并培养鸭的原代胚胎细胞(DEF),利用不同剂量poly(I:C)和NDV感染DEF后检测Viperin的表达。结果表明,poly(I:C)和NDV均能刺激诱导Viperin的表达。另一方面,以2周龄长白鸭的为动物材料,使用用NDV感染鸭来检测Viperin在不同脏器的分布。结果发现,正常生理情况下,不同脏器的Viperin含量具有较大的差异,Viperin主要分布于血液,其次是肺脏和脾脏,肾脏和肌肉较低；以对照组相比,在NDV感染后,不同脏器Viperin的表达都有不同程度的增加。该结果表明,NDV在细胞水平和鸭体上均能够增强Viperin表达。为了进一步确定鸭Viperin发挥抗病毒的关键区域,我们构建了Viperin的缺失片段(包括N端和C端缺失,分别命名为Vip△N和Vip△C)以及设计抑制鸭Viperin表达的siRNA序列。将鸭的Viperin基因(Viperin, Vip△N和Vip△C)克隆到pCAGGS真核表达载体上并转染DEF细胞；利用siRNA技术,转染siRNA质粒于DEF细胞,在转染后24小时后使用NDV感染DEF,在感染后不同时间内收集样品以检测NDV的病毒复制情况。结果表明,过表达鸭Viperin的情况下,能显著的抑制NDV感染,试验组的NDV滴度明显低于对照组；转染siRNA质粒后能抑制Viperin的表达,并有利于NDV复制。另外,从过表达鸭VipAN和Vip△C结果看,截短体的抗病毒作用已经显著降低,N端缺失后的Viperin还具有有限的抗病毒作用,而C端缺失后则导致Viperin抗病毒作用的完全丧失。这表明,Viperin的C端区域是其发挥抗病毒作用的关键区域。综上所述,本研究研究了鸭Viperin基因的生物特性和探究该蛋白的生物学功能,发现poly(I:C)和NDV等因素均能诱导Viperin的表达,说明鸭Viperin与其它物种Viperin具有类似的特征；Viperin蛋白能够发挥显著的抗NDV的作用,进一步发现Viperin的C端是其发挥抗病毒的关键区域。
[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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.32

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