MDBK细胞牛病毒性腹泻病毒互作蛋白的筛选和初步鉴定

发布时间：2017-12-27 06:13

本文关键词：MDBK细胞牛病毒性腹泻病毒互作蛋白的筛选和初步鉴定　出处：《黑龙江八一农垦大学》2017年硕士论文　论文类型：学位论文

【摘要】：牛病毒性腹泻病毒(Bovine Viral Diarrhea Virus,BVDV)使全球养牛业每年都遭受巨大的经济损失,其在分类上属于黄病毒科、瘟病毒属。BVDV生物型被分为细胞病变(CP)和非细胞病变(NCP)。母牛在妊娠的前三个月感染NCP BVDV将会产出持续性感染(PI)的小牛,这种牛对BVDV产生免疫耐受,终身带毒,传染其他动物。目前,国外主要采用疫苗免疫、检疫和净化来防控该病。由于该病毒的致病机理非常复杂,针对病毒感染和传播的机制研究是当今的热点。大量研究表明E2蛋白是BVDV非常重要的一个囊膜蛋白,为了揭示该病的致病机理,有必要研究BVDV及其E2蛋白在不同宿主细胞上的受体蛋白,确定BVDV与宿主细胞的互作机制,为防治该病奠定理论基础。本研究首先通过克隆BVDV结构蛋白E2的全基因,之后成功构建了真核质粒p Fast-E2和r Bacmid-E2,并将r Bacmid-E2成功转染sf9细胞,最终获得能够稳定表达E2蛋白的重组杆状病毒,经Western Blotting验证后,在43.6k Da位置出现清晰的目的条带。然后以MDBK细胞为模式细胞,将表达的E2蛋白作为pull-down实验的“诱饵”,钓出与之发生相互作用的宿主蛋白。此外,通过从PI牛中分离得到一株NCP型BVDV,并利用该毒株进行病毒铺覆蛋白印记实验(VOPBA)和免疫共沉淀实验(Co-IP),候选蛋白进行质谱鉴定,并对鉴定结果进行GO分析。经上述三种方法筛选获得30种候选蛋白,这些蛋白从功能上分为5大类,分别为构成细胞骨架成分、RNA结合功能、能量结合功能、蛋白结合功能以及离子通道结合功能。排除外来污染和未鉴定出种类的蛋白之后,经过GO分析和查阅相关文献,最终确定出4种最具有后续研究价值的互作蛋白,分别是波形蛋白、α-辅肌动蛋白、肌动蛋白和微管蛋白。由于质谱鉴定结果中CD46的独特性肽段仅检测到一个,因此,对已知的BVDV唯一受体CD46分子在MDBK细胞(体外感染)以及淋巴和单核细胞(体内感染)上的表达情况进行实时荧光定量PCR检测。结果显示,BVDV的感染能引起MDBK细胞和淋巴细胞CD46表达量的增加,但对单核细胞影响不明显。综上所述,本研究成功的获得能够稳定表达BVDV E2蛋白的重组杆状病毒,并筛选出可能与BVDV存在相互作用的候选宿主蛋白。并且BVDV能够诱导MDBK和牛外周血淋巴细胞CD46基因转录水平的上调。以上研究对BVDV的致病机理探索和新受体的发现都具有理论指导意义。
[Abstract]:Bovine viral diarrhea virus (Bovine Viral Diarrhea Virus, BVDV) the global cattle industry every year suffered huge economic losses, which belongs to the family Flaviviridae, classified in the genus pestivirus. The BVDV biologic type is divided into cytopathic (CP) and non cytopathic (NCP). Cows infected with NCP BVDV in the first three months of pregnancy will produce persistent infection (PI) calf, which is tolerant to BVDV, life-long, and infect other animals. At present, vaccine immunization, quarantine and purification are used in foreign countries to prevent and control the disease. Because the pathogenic mechanism of the virus is very complex, the research on the mechanism of virus infection and transmission is a hot spot today. A large number of studies show that E2 protein of BVDV is a very important membrane protein, in order to reveal the pathogenic mechanism of the disease, it is necessary to study the BVDV receptor protein and E2 protein in different host cells, determine the mechanism of interaction between BVDV and host cells, the foundation laid for the prevention and treatment of disease. This study first by cloning of BVDV structural protein E2, after the successful construction of eukaryotic plasmid P Fast-E2 and R Bacmid-E2, and R Bacmid-E2 was transfected into the Sf9 cells, can obtain recombinant baculovirus expressing E2 protein stability by Western Blotting, after verification, appear clear target band in 43.6k Da. Then using MDBK cells as model cells, the expressed E2 protein was used as the "bait" of the pull-down experiment to catch the host protein that interacted with it. In addition, a NCP BVDV was isolated from PI cattle, and the virus was covered by protein labeling test (VOPBA) and co immunoprecipitation (Co-IP). The candidate proteins were identified by mass spectrometry and GO analysis was performed. 30 candidate proteins were screened out by the above three methods. These proteins were functionally divided into 5 categories, which are cytoskeletal components, RNA binding function, energy binding function, protein binding function and ion channel binding function. After excluding foreign contamination and unidentified proteins, we identified 4 kinds of interacting proteins, which are the most valuable GO proteins, vimentin, alpha actin, actin and tubulin. Because only one of the CD46 unique peptides was detected in mass spectrometry, the expression of the known BVDV unique receptor CD46 molecule in MDBK cells (in vitro infection) and lymph and mononuclear cells (infection in vivo) was detected by real-time quantitative PCR. The results showed that the infection of BVDV could increase the expression of CD46 in MDBK cells and lymphocytes, but had no obvious effect on mononuclear cells. In conclusion, we successfully obtained recombinant baculovirus expressing BVDV E2 protein and screened candidate host proteins that might interact with BVDV. And BVDV can induce the up regulation of CD46 gene transcriptional level in MDBK and bovine peripheral blood lymphocytes. These studies have theoretical significance for the exploration of the pathogenesis of BVDV and the discovery of new receptors.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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