本文關(guān)鍵詞：MDBK細胞牛病毒性腹瀉病毒互作蛋白的篩選和初步鑒定　出處：《黑龍江八一農(nóng)墾大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 牛病毒性腹瀉病毒 MDBK細胞 蛋白互作 受體 真核表達

【摘要】：牛病毒性腹瀉病毒(Bovine Viral Diarrhea Virus,BVDV)使全球養(yǎng)牛業(yè)每年都遭受巨大的經(jīng)濟損失,其在分類上屬于黃病毒科、瘟病毒屬。BVDV生物型被分為細胞病變(CP)和非細胞病變(NCP)。母牛在妊娠的前三個月感染NCP BVDV將會產(chǎn)出持續(xù)性感染(PI)的小牛,這種牛對BVDV產(chǎn)生免疫耐受,終身帶毒,傳染其他動物。目前,國外主要采用疫苗免疫、檢疫和凈化來防控該病。由于該病毒的致病機理非常復(fù)雜,針對病毒感染和傳播的機制研究是當今的熱點。大量研究表明E2蛋白是BVDV非常重要的一個囊膜蛋白,為了揭示該病的致病機理,有必要研究BVDV及其E2蛋白在不同宿主細胞上的受體蛋白,確定BVDV與宿主細胞的互作機制,為防治該病奠定理論基礎(chǔ)。本研究首先通過克隆BVDV結(jié)構(gòu)蛋白E2的全基因,之后成功構(gòu)建了真核質(zhì)粒p Fast-E2和r Bacmid-E2,并將r Bacmid-E2成功轉(zhuǎn)染sf9細胞,最終獲得能夠穩(wěn)定表達E2蛋白的重組桿狀病毒,經(jīng)Western Blotting驗證后,在43.6k Da位置出現(xiàn)清晰的目的條帶。然后以MDBK細胞為模式細胞,將表達的E2蛋白作為pull-down實驗的“誘餌”,釣出與之發(fā)生相互作用的宿主蛋白。此外,通過從PI牛中分離得到一株NCP型BVDV,并利用該毒株進行病毒鋪覆蛋白印記實驗(VOPBA)和免疫共沉淀實驗(Co-IP),候選蛋白進行質(zhì)譜鑒定,并對鑒定結(jié)果進行GO分析。經(jīng)上述三種方法篩選獲得30種候選蛋白,這些蛋白從功能上分為5大類,分別為構(gòu)成細胞骨架成分、RNA結(jié)合功能、能量結(jié)合功能、蛋白結(jié)合功能以及離子通道結(jié)合功能。排除外來污染和未鑒定出種類的蛋白之后,經(jīng)過GO分析和查閱相關(guān)文獻,最終確定出4種最具有后續(xù)研究價值的互作蛋白,分別是波形蛋白、α-輔肌動蛋白、肌動蛋白和微管蛋白。由于質(zhì)譜鑒定結(jié)果中CD46的獨特性肽段僅檢測到一個,因此,對已知的BVDV唯一受體CD46分子在MDBK細胞(體外感染)以及淋巴和單核細胞(體內(nèi)感染)上的表達情況進行實時熒光定量PCR檢測。結(jié)果顯示,BVDV的感染能引起MDBK細胞和淋巴細胞CD46表達量的增加,但對單核細胞影響不明顯。綜上所述,本研究成功的獲得能夠穩(wěn)定表達BVDV E2蛋白的重組桿狀病毒,并篩選出可能與BVDV存在相互作用的候選宿主蛋白。并且BVDV能夠誘導(dǎo)MDBK和牛外周血淋巴細胞CD46基因轉(zhuǎn)錄水平的上調(diào)。以上研究對BVDV的致病機理探索和新受體的發(fā)現(xiàn)都具有理論指導(dǎo)意義。
[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.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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