利用BAC克隆构建PRV UL21缺失株及其生物学特性研究

发布时间：2018-03-17 22:33

本文选题：伪狂犬病毒　切入点：UL21　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：伪狂犬病毒(Pseudorabies virus,PRV),又叫做奥叶兹基氏病病毒(Aujeszky's disease virus)、猪疱疹病毒Ⅰ型(Suid herpesvirus 1),属于疱疹病毒科的α疱疹病毒亚科,是世界上广泛存在的一种病原物。伪狂犬病毒的天然宿主分布广泛,它能够感染除高等灵长类以外的大多数哺乳动物,最主要的天然宿主是猪。伪狂犬病毒能引起母猪的呼吸道炎症、流产、死胎,以及仔猪的死亡,在我国每年都能造成严重的经济损失。伪狂犬病毒基因组长约为150Kb,其中包括长独特区、短独特区、内部重复序列和末端重复序列。伪狂犬病毒基因组能够编码70种蛋白质,分为结构蛋白和非结构蛋白两种。pUL21位于伪狂犬病毒的内层被膜,在疱疹病毒科中诸多病毒都存在且序列保守,应该是维持其毒力的重要基因。UL21基因被认为参与了病毒粒子的包装过程,其缺失株在复制过程中出现大量的未加工的基因组串联体,而且不含基因组的衣壳比例也大大增加。目前有研究表明,修复UL21突变的PRV Bartha株在神经细胞中的逆向轴突感染的速度增加,表明pUL21可能与病毒运输有关。细菌人工染色体(bacteria artificial chromosome,BAC)是基于天然存在于大肠杆菌中的质粒F因子设计的一种能够容纳大片段DNA插入序列的一种载体。BAC一经问世,便被广泛用于构建真核生物基因组文库。BAC文库有稳定性强、插入片段长、复制快、突变容易的优点。BAC用于克隆疱疹病毒基因组始于1997年,其首次应用于小鼠巨细胞病毒[1],自此疱疹病毒的研究翻开了新的篇章。由于通过BAC能够在大肠杆菌中利用原核重组系统对病毒基因组进行突变,省去了构建重组毒时复杂繁琐的过程,也节省了大量的时间,一时间该技术纷纷被用于各种疱疹病毒的基因编辑。本研究出于研究UL21缺失对PRV增殖影响和新建立的BAC平台构建突变株效率的目的,构建了UL21缺失株rPRV-ΔUL21及其回复突变株rPRV-ΔUL21R,并通过空斑大小比较实验研究其生物学特性。在构建rPRV-ΔUL21时先将扩增的Kan表达盒电转化含有pPRV Ea的GS1783,完成第一次Red重组;再分别诱导I-SceⅠ和Red操纵子表达,完成第二次重组。从两次重组的PCR验证来看,重组效率不是很稳定,但每一轮都能挑到阳性克隆,而且并没有受到质粒残留的影响。该结果说明BAC基因编辑平台稳定可靠,能够短时间内构建重组病毒,为病毒基因功能研究节省大量的时间精力。最后western blot验证结果表明,rPRV-ΔUL21完全不表达UL21蛋白,说明PRV的UL21缺失株的构建非常成功。随后进行的空斑大小比较实验结果显示,rPRV-ΔUL21感染MDBK细胞上所产生的空斑大小明显小于PRV Ea株,但是减小的幅度并不大。这说明UL21的确影响PRV病毒在培养细胞上的增殖,但是其功能并不是复制必需的。为了确保UL21基因的突变没有影响与其重叠的lncRNA CTO-L的表达,本研究中采用了real time PCR来检测感染病毒后12h的PK-15细胞中CTO-L的含量。检测结果表明,rPRV-ΔUL21中对UL21的突变不仅基本没有改变CTO-L的序列,同时也没有影响其表达量。本研究在构建重组毒时采用了新构建的细菌人工染色体基因编辑平台,摸索出了一套正确的使用方法,方便后续其它成员构建重组病毒研究基因功能。对UL21缺失病毒生理特性的研究则进一步完善了对UL21基因功能的相关研究,加深了我们对UL21,尤其是对其与CTO-L之间关系的理解。
[Abstract]:Pseudorabies virus (Pseudorabies, virus, PRV), also called Aujesky's disease virus (Aujeszky's disease virus), porcine herpesvirus (Suid herpesvirus 1), alpha herpesvirus belongs to the herpes virus, is a pathogen widely exist in the world. The natural host of pseudorabies virus is widely distributed and it can infect most mammals in higher primates other than the natural host is the most important. Swine pseudorabies virus can cause respiratory tract inflammation, sow abortion, stillbirth, and piglet mortality, can cause serious economic losses every year in our country. Pseudorabies virus genome is about 150Kb, including long unique region, short unique region, internal repeats and terminal repeats. Pseudorabies virus genome encoding 70 proteins, the inner layer is divided into structural and non structural protein two.PUL21 in pseudorabies virus In the film, Herpesviridae in many viruses and conserved sequences,.UL21 gene is an important gene should maintain its virulence is thought to participate in the process of packing of virus particles, the mutant during replication not processing large genomic repeats, and does not contain the genome capsid is greatly increased. The proportion of at present, studies have shown that the repair of UL21 mutant PRV strain Bartha in nerve cells in retrograde axonal infection rate increased, indicating that pUL21 may be involved in viral transport. Bacterial artificial chromosome (bacteria artificial, chromosome, BAC) is based on a plasmid naturally found in Escherichia coli F factor design of a carrier can accommodate.BAC the large fragment of DNA insertion sequence was developed, it is widely used in the construction of the eukaryotic genome library of.BAC library has strong stability, long insert, copy fast, easy to jump The advantages of.BAC used for cloning the herpes virus genome in 1997, its first application in murine cytomegalovirus [1] herpes virus, since this study opened a new chapter. Through the BAC to the viral genome mutation using the Prokaryotic Recombinant system in Escherichia coli, the process to construct the recombinant virus, complex and cumbersome, but also save a lot of time, a time of the technology have been used in various gene editing of herpes simplex virus. Construct the mutant efficiency for the purpose of studying the effect of UL21 deficiency on the proliferation of PRV and the new BAC platform in this research, construction of the UL21 mutant rPRV- UL21 and its mutant strains of rPRV- UL21R, and the plaque size a comparative experimental study on its biological characteristics. In the construction of rPRV- Delta UL21 first amplified Kan expression cassette containing pPRV Ea GS1783 transformation, the completion of the first recombinant Red respectively induced I-Sce 1; And the expression of Red operon, completed second restructuring. From PCR to verify the two reorganization, the recombination efficiency is not very stable, but each round to pick up the positive clones, but was not affected by plasmid residue. The results suggest that BAC gene editing platform is stable and reliable, able to construct recombinant virus within a short period of time, save a large amount of time and energy for gene function. Finally western blot verification results show that the rPRV- UL21 did not express UL21 protein, indicating the construction of PRV UL21 mutant was very successful. Plaque size followed by comparison of experimental results show that rPRV- UL21 infection of plaque size produced by MDBK cells was less than PRV Ea strain, but the magnitude is not large. This shows that UL21 does reduce the effect of PRV on the proliferation of the virus in cultured cells, but its function is not essential for replication. In order to ensure that the UL21 mutation does not affect the The expression of lncRNA CTO-L and its rings overlap, this study uses real time PCR to detect the content of CTO-L 12h in PK-15 cells after infection. The detection results show that the rPRV- UL21 mutation in the UL21 sequence not only basically no change in CTO-L, but also did not affect its expression. The research in construction recombinant virus was used to construct new bacterial artificial chromosome gene editing platform, worked out a set of proper use method, convenient for other members of construction of recombinant virus gene function. Research on the physiological characteristics of UL21 deletion virus is to further improve the relevant research on UL21 gene function, our understanding of the UL21, especially the understanding of the relationship with CTO-L.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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