本文選題：α屬冠狀病毒　切入點：S蛋白　出處：《復(fù)旦大學(xué)》2011年碩士論文

【摘要】：冠狀病毒(Coronaviruses, CoV)為有包膜的正鏈RNA病毒,分為α、β和γ三個屬。CoV的RNA基因組發(fā)生重組后,相關(guān)基因的突變突破種屬障礙,實現(xiàn)跨宿主感染。CoV的S蛋白識別細(xì)胞受體并介導(dǎo)膜融合,是病毒感染宿主細(xì)胞的關(guān)鍵因子。S蛋白S1結(jié)構(gòu)域的受體識別區(qū)(Receptor binding domain, RBD)直接參與了受體的識別,該區(qū)域的氨基酸變異會導(dǎo)致病毒的種屬嗜性和感染特性的變化。由于α屬CoV的受體為氨肽酶N(Aminopeptitase N, APN),其氨基酸序列高度保守。因此,S蛋白的變異在很大程度上決定了CoV的種屬特異性。本論文選擇犬冠狀病毒(Canine coroanvirus, CCoV)、豬傳染性胃腸炎病毒(Transmissible gastroenteritis virus, TGEV)和貓冠狀病毒(Feline coronavirus, FCoV)三種α屬冠狀病毒為模型,研究三種病毒在不同宿主來源細(xì)胞上的感染特性。并構(gòu)建嵌合異源S基因的重組病毒,探討CoV種屬間傳播的分子機制。 首先,用CCoV、FCoV和TGEV分別感染犬A72、貓FCWF和豬ST細(xì)胞,通過噬斑實驗等方法分析病毒復(fù)制與細(xì)胞病理變化(Cytopathic effect, CPE)差異。研究發(fā)現(xiàn)：三種病毒在其天然宿主來源的細(xì)胞上均良好增殖,CPE出現(xiàn)的時間較早,細(xì)胞病變明顯并且典型,噬斑的大小和形狀較一致,病毒滴度峰值出現(xiàn)在感染后12小時左右。在犬A72和貓FCWF細(xì)胞上三種病毒的噬斑形態(tài)存在差異：TGEV在A72細(xì)胞上的適應(yīng)性不如CCoV和FCoV;CCoV和TGEV對FCWF細(xì)胞的病變小于FCoV。在豬ST細(xì)胞上,CCoV幾乎不引起明顯的CPE,而FCoV出現(xiàn)CPE較晚,二者均不能形成明顯噬斑和有效增殖。另外,CCoV、FCoV和TGEV均不能在小鼠L2細(xì)胞上增殖,同樣β屬MHV也不能感染A72、FCWF和ST細(xì)胞。上述結(jié)果說明同屬冠狀病毒容易突破宿主障礙,但不同屬間不容易突破。 其次,將CCoV、FCoV和TGEV的S基因分別克隆到載體pMH54中替換MHV的S基因,未能獲得重組病毒。但將三個病毒的S基因膜外區(qū)替換MHV的S基因膜外區(qū),結(jié)果顯示相應(yīng)的重組MHV復(fù)制特性與野生型相似。此結(jié)果證明α屬CoV的細(xì)胞感染性差異與S基因膜外區(qū)有關(guān),而膜內(nèi)區(qū)可能通過與病毒其它蛋白的作用限制S蛋白裝配到病毒粒子上。進一步制備α屬CoV的S蛋白N端高變區(qū)替代的重組病毒,從而更精確地對決定屬內(nèi)跨宿主感染的S蛋白的氨基酸定位是必要的。
[Abstract]:Coronaviruses (CoV) is a positive strand RNA virus with envelope. After recombination of RNA genome of 偽, 尾 and 緯, the mutation of related genes breaks through the obstacle of species, and the S protein of transhomologous infection. CoV recognizes cell receptor and mediates membrane fusion.Receptor binding domain (RBDs), the receptor recognition region of S1 domain of S protein, is a key factor of virus infection in host cells. The amino acid variation in this region will lead to the change of species eosinophilia and infection characteristics.The amino acid sequence of 偽 -genus CoV is highly conserved because its receptor is aminopeptidase N(Aminopeptitase N, APNN.Therefore, the variation of S protein determines the species-specificity of CoV to a great extent.The recombinant virus containing chimeric heterologous S gene was constructed to study the molecular mechanism of interspecies transmission of CoV.Firstly, CCoV FCoV and TGEV were used to infect canine A72, cat FCWF and porcine St cells respectively. The difference of viral replication and cytopathic effectt (CPE) was analyzed by plaque assay.CCoV did not cause significant CPE in St cells, but CPE appeared late in FCoV, neither of them could form plaque and proliferate effectively.In addition, CCoV FCoV and TGEV could not proliferate on L2 cells, and neither 尾 MHV could infect A72 FCWF and St cells.These results suggest that the coronavirus of the same genus can easily break through the host barrier, but it is not easy to break through among different genera.Secondly, the S gene of CCoV FCoV and TGEV were cloned into the vector pMH54 to replace the S gene of MHV, and the recombinant virus was not obtained.However, the S gene extracellular region of MHV was replaced by the S gene extracellular region of the three viruses. The results showed that the replication characteristics of the corresponding recombinant MHV were similar to those of the wild type.The results showed that the difference in cell infectivity of 偽 -genus CoV was related to the extracellular region of S gene, and the intramembrane region might restrict the assembly of S protein to virus particles by interaction with other proteins of the virus.Furthermore, it is necessary to prepare the recombinant virus substituted for the N-terminal hypervariable region of S protein of 偽 -genus CoV, so as to more accurately locate the amino acid of S protein which determines the intrageneric cross host infection.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R373.9

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