本文選題：偽狂犬病病毒(PRV)　切入點：EPO　出處：《廣西大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：偽狂犬病(Pseudorabies,PR)是由偽狂犬病病毒(Pseudorabies Virus,PRV)引起多種家畜與野生動物以發(fā)熱,奇癢,腦脊髓炎,流產(chǎn)及死亡為主要特征的急性傳染病。豬是該病的貯存宿主和主要傳染源,豬感染PRV后常表現(xiàn)為哺乳仔豬死亡、育肥豬生長停滯及妊娠母豬繁殖障礙。該病在歐洲、亞洲等多個國家呈蔓延趨勢,已給世界養(yǎng)豬業(yè)帶來了巨大的經(jīng)濟損失,中國尤為嚴(yán)重。PRV屬于雙鏈DNA病毒,大小約150 Kb,可編碼70-100種蛋白質(zhì),其中EP0蛋白是PRV重要的早期蛋白。EP0蛋白包含與鋅指結(jié)構(gòu)類似的環(huán)指結(jié)構(gòu)域,該結(jié)構(gòu)域主要是結(jié)合DNA、RNA及蛋白質(zhì)之間相互作用的區(qū)域,推測它在病毒與宿主相互作用方面具有重要意義。本研究構(gòu)建了 pGBKT7-EP0誘餌質(zhì)粒和PK-15細(xì)胞cDNA文庫,為更好的研究PRV EP0蛋白與宿主細(xì)胞PK-15之間的相互作用機制提供基礎(chǔ)。具體研究內(nèi)容如下:第一部分:構(gòu)建pGBKT7-EP0誘餌質(zhì)粒采用PCR技術(shù),擴增獲得PRVEP0基因,將其克隆至酵母雙雜交載體pGBKT7中,獲得重組誘餌質(zhì)粒pGBKT7-EP0,通過限制性內(nèi)切酶EcoRI與Pst I雙酶切鑒定與基因測序結(jié)果分析,表明成功構(gòu)建誘餌質(zhì)粒pGBKT7-EP0。將該質(zhì)粒轉(zhuǎn)入到Y(jié)2HGold酵母感受態(tài)細(xì)胞中,對其進(jìn)行毒性檢測,自激活效應(yīng)檢測,PCR以及Western-blot檢測,結(jié)果表明誘餌質(zhì)粒表達(dá)的產(chǎn)物對酵母細(xì)胞無毒性作用,無自激活效應(yīng),PCR和Western-blot檢測結(jié)果證實誘餌質(zhì)粒pGBKT7-EP0成功轉(zhuǎn)入酵母細(xì)胞,并且能夠表達(dá)融合蛋白 BD-EP0-Myc。第二部分:PK-15細(xì)胞cDNA文庫構(gòu)建及鑒定選取PRV的宿主細(xì)胞PK-15細(xì)胞來構(gòu)建酵母雙雜交cDNA文庫。本試驗從PK-15細(xì)胞提取總RNA,采用SMART和LD-PCR合成全長的ds cDNA,并對其進(jìn)行均一化和SfiⅠ酶切處理。將得到的dscDNA連接到三框表達(dá)載體中。將這三個重組反應(yīng)產(chǎn)物用電轉(zhuǎn)化方法轉(zhuǎn)入到HST08電轉(zhuǎn)化感受態(tài)細(xì)胞中,構(gòu)建含有三種讀碼框的初級文庫,該文庫庫容分別為3.0X 106、2.0×106和2.0×106 CFU,插入片段均在500bp以上,陽性重組率均大于93%。再將三框初級文庫共同電轉(zhuǎn)化至感受態(tài)細(xì)胞HST08中進(jìn)行擴增,提取質(zhì)粒文庫。最后將質(zhì)粒文庫轉(zhuǎn)化到Y(jié)187酵母感受態(tài)細(xì)胞,得到的cDNA酵母文庫總庫容量為7.5× 105 CFU,基本符合酵母雙雜交cDNA文庫構(gòu)建要求。綜上,本課題成功構(gòu)建了符合酵母雙雜交篩選要求的誘餌質(zhì)粒pGBKT7-EP0和酵母cDNA文庫,為進(jìn)一步研究PRV EPO蛋白與PK-15細(xì)胞蛋白之間的相互作用奠定了基礎(chǔ)。
[Abstract]:Pseudorabies PRV (pseudorabies virus) is an acute infectious disease caused by pseudorabies virus (PRV) in many domestic and wild animals, characterized by fever, itchiness, encephalomyelitis, abortion and death. After infection with PRV, pigs often die of lactation, growth stagnation in fattening pigs and reproductive barriers in pregnant sows. The disease is spreading in many countries such as Europe and Asia, and has brought huge economic losses to the world pig industry. China is especially serious. PRV belongs to double-stranded DNA virus, about 150kb in size, and can encode 70-100 proteins. Among them, EP0 protein is an important early stage protein of PRV. EP0 protein contains ring finger domain similar to zinc finger structure. This domain is mainly a domain of interaction between RNAs and proteins, which is supposed to play an important role in the interaction between virus and host. In this study, pGBKT7-EP0 bait plasmid and cDNA library of PK-15 cells were constructed. The main contents are as follows: the first part: construct pGBKT7-EP0 bait plasmid to amplify and obtain PRVEP0 gene by PCR technique. The recombinant bait plasmid pGBKT7-EP0 was obtained by cloning it into yeast two-hybrid vector pGBKT7. The recombinant bait plasmid pGBKT7-EP0 was identified by restriction endonuclease EcoRI and Pst I, and the results of gene sequencing were analyzed. The results showed that the bait plasmid pGBKT7-EP0 was successfully constructed. The plasmid was transferred into Y2HGold yeast competent cells for toxicity detection, self-activation effect detection and Western-blot detection. The results showed that the product expressed by the bait plasmid had no toxic effect on yeast cells. The results of PCR and Western-blot showed that the bait plasmid pGBKT7-EP0 was successfully transferred into yeast cells. And can express the fusion protein BD-EP0-Myc.part two: PK-15 cell cDNA library was constructed and identified to construct yeast two-hybrid cDNA library with selected host cells PK-15 cells. In this experiment, total RNAs were extracted from PK-15 cells, and SMART and LD-PCR were used to synthesize the full-length cDNA library. The dscDNA was homogenized and digested with Sfi 鈪，

本文編號：1636504