鸭瘟病毒中国强毒株基因组解析及UL55基因功能初步研究

发布时间：2018-06-06 13:43

  本文选题：鸭瘟病毒中国强毒株 + 基因组解析　； 参考：《四川农业大学》2015年博士论文

【摘要】：鸭瘟(duck plague)又称鸭病毒性肠炎(duck virus enteritis),是鸭、鹅和其他雁形目禽类的一种急性、热性、败血性传染病。本病发病快、死亡率高,是目前世界各国水禽业危害最严重的传染病之一。该病的病原鸭瘟病毒(Duck Plague virus, DPV)是疱疹病毒科(Herpesvirales)、α疱疹病毒亚科(Alphaherpesvirinae)、马立克氏病毒属(Mardivirus)的成员之一。但由于DPV的研究起步晚,其分子生物学研究远远落后于其它疱疹病毒。一段时间以来对于DPV的研究多集中于流行病学、诊断和防治等方面；与其分子生物学特性相关的基础研究,如病毒的基因组结构、物理图谱、病毒基因功能以及病毒在机体细胞和组织中的复制及致病机理、病毒感染过程以及机体抗病毒感染机制等多方面研究均有许多不明之处。因此本论文的主要目的就是希望对本实验室测序获得的鸭瘟病毒基因组序列进行解析；并通过构建一个可以对DPV进行细菌遗传学操作的平台,为DPV的基因功能研究提供技术手段的支持,此外利用该平台用两步RED重组敲除UL55基因,验证此平台的可行性并初步阐释UL55功能。主要研究内容如下：1鸭瘟病毒中国强毒株基因组信息解析对本实验室测序获得的鸭瘟中国强毒株的基因组序列进行生物信息学分析。鸭瘟病毒中国强毒株基因组组成为双股线状双链DNA:UL-IRS-US-TRS,是典型的D型疱疹病毒基因组结构。DPV CHv基因组全长162,175 bp,GC含量为44.89%,包括潜在的76个能编码功能蛋白的ORF。BLAST搜寻相似性序列发现五条与DPV CHv同期或之后提交的其他鸭瘟病毒毒株全基因组序列,他们间具有高度同源性,进化树分析显示六株DPV病毒按地域和毒力差异进行分类,DPV CHv处于进化树的中间位置。对基因组编码区的ORF进行扫描分析发现,六株DPV在UL、US区域分别有23、5个ORF在核苷酸水平上存在整个ORF的缺失、部分序列插入、缺失等情况出现,这些突变的产生可能是造成六株不同来源毒株毒力和地理差异的主要原因。而DPV基因组中不存在核苷酸序列插入或缺失的53个ORF在氨基酸水平上高度同源,多为DPV基因组的保守性基因,不受病毒传代致弱及地域差异的影响,编码蛋白大多为与病毒DNA代谢相关的结构蛋白。密码子使用模式分析结果显示其在基因组密码子的使用上偏向A/T结尾的密码子,其密码子使用模式主要受突变压力的影响。将其与人类、大肠杆菌、酵母的密码子使用模式进行比较,结果表明鸭瘟病毒的密码子使用模式与酵母系统更为接近。2鸭瘟病毒UL55基因生物信息学分析DPV UL55基因由561bp核苷酸组成,包括一个完整的开放性阅读框,编码一个由186个氨基酸组成的20.7981kDa蛋白质。UL55蛋白没有信号肽及跨膜区。密码子分析显示UL55基因偏向A/T结尾的密码子,密码子使用模式与人类最接近。UL55蛋白整体表现出亲水性,抗原表位主要集中在相应亲水区域的无规则卷曲,其功能与病毒的装配、出芽、成熟和释放相关。基于核苷酸序列的进化树分析显示DPV属于马立克氏病毒基因属的一员。3鸭瘟病毒UL55基因的克隆、原核表达及多克隆抗体制备通过将UL55基因通过克隆到表达载体pET32a(+)上实现对UL55蛋白的原核表达,试验表明重组UL55基因能在大肠杆菌BL21(DE)中进行融合表达。通过优化诱导表达条件,UL55重组蛋白能在37℃条件下,通过0.8mmM的IPTG诱导表达4h产生大量非可溶形式的包涵体蛋白。将包涵体蛋白通过裂解后进行纯化及复性处理再与兔抗DPV CHv多克隆抗体进行免疫印迹反应,结果表明纯化复性后的UL55蛋白具有与天然蛋白相似的免疫反应活性。复性后的UL55蛋白免疫兔子制备的高免血清能够与UL55重组蛋白发生良好的免疫反应。4原核表达UL55蛋白作为抗原检测DPV血清的间接ELISA方法的建立本方法是基于纯化的重组UL55原核表达蛋白建立的可以检测DP血清的间接ELISA法,该方法特异性强,对抗鸭病毒性肝炎病毒(DHV)、鸭疫里默氏菌(RA)、鸭大肠杆菌(E.coli)、鸭源沙门氏菌(Salmonella)、肿头性出血症病毒和鸭源流感病毒的阳性血清进行检测,结果均为阴性；该方法的对酶标板内或板间重复试验显示变异系数均小于10%,能检出经1：6400倍稀释的DPV弱毒疫苗免疫鸭的阳性血清。将其与经典的中和试验及DPV全病毒包被的ELISA同时检测50份感染了DPV的临床鸭血清样本,发现其对DPV IgG的检出率介于中和试验和DPV-ELISA之间。由于其制备方法简便、操作简单,特异性、灵敏性较高。且不存在散毒的风险,具有良好的应用前景,为进一步组装成试剂盒奠定了基础。5鸭瘟病毒UL55基因转录、表达时相分析以β-actin基因作为内参基因,用相对荧光定量方法对UL55基因在体外感染宿主细胞中的转录情况进行了分析。转录时相分析结果表明UL55基因在转录后的0-8h内处于一个较低的水平；12h后开始迅速增加直至在感染后36h达到转录峰值,之后开始逐步下降,直到感染后的60h仍然可以检测到大量转录的UL55基因,UL55基因的转录过程可以被核酸抑制剂更昔洛韦抑制,说明UL55基因是严格依赖于DNA合成的γ2基因。Western-blot分析体外感染宿主细胞中UL55基因的表达时相表明UL55蛋白的表达水平也表现出相似的规律,进一步佐证了UL55基因为γ2基因的结论。6细菌人工染色体重组鸭瘟病毒拯救系统平台构建通过多步克隆构建了含TK基因同源臂、报告基因EGFP和细菌人工染色体核心功能元件的重组鸭瘟病毒转移载体pUC18/EGFP-TKAB-BAC11,将其与DPV CHv病毒核酸共转染获得了重组病毒DPV CHv-BAC-G。利用EGFP报告基因的指示作用,通过8轮噬斑纯化获得了纯化的重组病毒。提取环化时期的重组病毒DPV CHv-BAC-G,电击转化至DH10B细胞中,获得了能够在细菌中复制的重组病毒转化子。将克隆化的病毒质粒pBAC-DPV转染至宿主细胞DEF,成功拯救出重组病毒DPV CHv-BAC-G。拯救出的重组病毒可以以细菌和病毒两种形式存在,能够实现在细菌和宿主细胞内的同时复制,有利于利用原核系统成熟的基因操作手段研究原本仅能在细胞水平研究的鸭瘟病毒,成功建立细菌人工染色体重组鸭瘟病毒拯救系统平台。7重组鸭瘟病毒UL55基因缺失株的构建及其体外生物学特性研究在构建的细菌人工染色体重组鸭瘟病毒拯救系统平台基础上,利用大肠杆菌细胞内的两步RED重组技术构建UL55基因缺失株及其回复突变株。构建的回复突变株克隆能够通过转染DEF细胞获得拯救产生与亲本相同的致细胞病变,酶切图谱与亲本株DPV CHv-BAC-G无差异,是为真正的回复突变株。空斑试验、一步生长曲线和致病性比较结果表明,构建的UL55缺失株和回复突变株与预期结果一致,能产生与亲本株DPV CHv-BAC-G相似细胞病变效应、形成形态及大小相似的空斑、在感染细胞中展现出相同的增殖周期。8 DPV UL55基因在感染宿主细胞体内的定位分析以制备的兔抗UL55多克隆抗体作为一抗,用间接免疫荧光的方法检测UL55基因编码蛋白在感染细胞内的动态分布。结果显示,UL55蛋白最早在感染后的5.5h内开始在细胞质大量表达,并随着时间的推移表达量逐渐增多,其表达量在感染后的22.5h达到峰值。之后UL55蛋白的表达量逐步下降,并从细胞质内的散在分布颗粒逐渐形成荧光斑向核膜周边转移,大量存在于核膜两极。之后随着时间的推移,UL55蛋白的荧光逐渐消失,推测其随着病毒复制周期的结束和细胞的崩解而消失。9 DPV UL55与UL26.5基因在感染宿主细胞体内的定位分析UL55蛋白和UL26.5蛋白在细胞内的共定位结果显示,UL26.5和UL55蛋白在感染细胞内邻接并部分重叠,但当DPV不表达UL55蛋白时,UL26.5蛋白的定位没有变化,表明UL55基因的缺失并不影响UL26.5在核内的定位和其功能的发挥,预示着UL26.5蛋白形成的核衣壳的装配由包括UL55在内的多个蛋白完成,UL55蛋白参与病毒的装配,但并不是必需。
[Abstract]:Duck plague (duck plague), also known as duck viral enteritis (duck virus enteritis), is an acute, hot, septic infectious disease of duck, goose and other wild goose fowl. It is one of the most serious infectious diseases in waterfowl industry all over the world. The pathogenic duck plague virus (Duck Plague virus, DPV) is one of the herpes disease. Herpesvirales, alpha herpes virus subfamily (Alphaherpesvirinae), one of the members of the Marek's virus (Mardivirus). But because of the late start of the study of DPV, its molecular biology is far behind the other herpes viruses. For a period of time, the study of DPV has focused on epidemiology, diagnosis and Prevention and so on. The basic research related to the biological characteristics of the virus, such as the genome structure of the virus, the physical map, the function of the virus gene, the replication and pathogenesis of the virus in the body and tissue of the body, the process of virus infection and the mechanism of the virus infection of the body, are not clear. The main purpose of this paper is to hope that the main purpose of this paper is to hope. The genome sequence of the duck plague virus was analyzed by the sequencing of the laboratory, and a technical support for the genetic function of DPV was provided by constructing a platform that could carry out the genetic operation of DPV. In addition, the platform was reorganized by two step RED to knock out the UL55 basis, and the feasibility of the platform was verified and the UL55 was explained. The main research contents are as follows: 1 the genome information analysis of the Chinese strong virulent strain of duck plague virus was analyzed by bioinformatics analysis of the genome sequence of the Chinese strong strain of duck plague. The genome of the duck plague virus in China was composed of two strand linear double stranded DNA: UL-IRS-US-TRS, which was a typical D herpes virus genome. The total length of the genomic.DPV CHv was 162175 BP, and the GC content was 44.89%, including the ORF.BLAST search similarity sequence of the potential 76 coding functional proteins. Five of the whole genome sequences of the other duck plague virus strains were presented at the same time or after DPV CHv, and they were highly homologous. The phylogenetic tree analysis showed that six DPV viruses were located in the region. DPV CHv is in the middle of the evolutionary tree, and the ORF of the genome coding region is scanned and analyzed. The six DPV in UL and US regions have 23,5 ORF at the nucleotide level, which exist in the absence of the whole ORF, the partial sequence insertion and the deletion, which may cause six different strains. The main reason for the virulence and geographical difference of the source strain is that the 53 ORF in the DPV genome does not have the high homology at the amino acid level, most of which are conserved genes of the DPV genome, which are not affected by the weak and regional difference of the virus, and most of the encoded proteins are the structural proteins related to the metabolism of the virus DNA. The code analysis results show that the codon used in the genomic codon uses the codon that ends to the A/T. The use mode of the codon is mainly influenced by the mutation pressure. The codon usage pattern is compared with the codon usage pattern of human, Escherichia coli and yeast. The results show that the codon usage pattern of the duck plague virus is more connected with the yeast system. The bioinformatics analysis of the UL55 gene of the near.2 duck plague virus DPV UL55 gene consists of 561bp nucleotides, including a complete open reading frame, encoding a 20.7981kDa protein.UL55 protein composed of 186 amino acids, without signal peptide and transmembrane region. Codon analysis shows that UL55 based codon that ends to A/T, codon use The pattern and human closest to the.UL55 protein epitopes the hydrophilicity, and the antigen epitopes mainly focus on the irregular curls in the corresponding hydrophilic regions. Their function is related to the assembly, bud, maturation and release of the virus. The nucleotide sequence based evolutionary tree analysis shows that DPV belongs to a member of the.3 duck plague virus UL55 gene of the Marek's virus gene. Cloning, prokaryotic expression and polyclonal antibody preparation can express the prokaryotic expression of UL55 protein by cloning the UL55 gene through the expression vector pET32a (+). The experiment shows that the recombinant UL55 gene can be fused in the BL21 (DE) of Escherichia coli. By optimizing the induced expression conditions, the UL55 recombinant protein can pass the IPTG of 0.8mmM at 37 C. A large number of insoluble forms of inclusion body proteins were induced by induced expression of 4H. The inclusion body protein was purified and retreated after lysis, and then immunoblotting with Rabbit anti DPV CHv polyclonal antibody. The results showed that the purified UL55 protein had a similar immune response to natural protein. After refolding, the UL55 protein was immune to rabbit. The preparation of high serum free serum can produce a good immune response to the recombinant protein of UL55,.4 prokaryotic expression UL55 protein as an indirect ELISA method for antigen detection of DPV serum. This method is based on the indirect ELISA method based on purified recombinant UL55 prokaryotic expression protein to detect DP sera. This method is specific and against duck virus specificity. Hepatitis virus (DHV), RA, duck Escherichia coli (E.coli), Salmonella duck source (Salmonella), swollen cephaemia virus and duck influenza virus positive serum were detected. The results were all negative, and the coefficient of variation was less than 10% in the enzyme labelled plate or between plates, and it can be detected by 1:6400 times dilution. The positive serum of DPV weakly toxic vaccine was immunized with duck, and 50 clinical duck serum samples infected with DPV were detected simultaneously with the classical neutralization test and the ELISA of DPV whole virus envelope. The detection rate of DPV IgG was between neutralization test and DPV-ELISA. The preparation method was simple, simple, specific and high sensitivity. The risk of detoxification has a good prospect of application. The transcription of the base.5 duck plague virus UL55 gene is established for the further assembly of the kit. The expression phase analysis takes the beta -actin gene as the internal reference gene. The transcription of the UL55 gene in the host cells infected in vitro is analyzed by relative fluorescence quantitative method. The results showed that the UL55 gene was at a lower level in the post transcriptional 0-8h; after 12h, it began to increase rapidly until the peak of 36h after infection, and then gradually decreased until the 60H of the infected 60H could still detect a large number of transcriptional UL55 genes, and the UL55 gene transfer process could be inhibited by ganciclovir, a nucleic acid inhibitor, It is indicated that the UL55 gene is strictly dependent on the DNA synthesis of gamma 2 gene.Western-blot to analyze the expression of UL55 gene in the infected host cells in vitro, indicating that the expression level of UL55 protein is also similar, and further testified that the UL55 gene is the gamma 2 gene of the recombinant duck plague virus rescue system platform construction of the.6 bacterial artificial chromophore. The recombinant duck plague virus transfer vector pUC18/EGFP-TKAB-BAC11 containing the TK gene, EGFP and the core functional component of the bacterial artificial chromosome, was constructed by multistep cloning, and co transfected with DPV CHv virus nucleic acid was co transfected to obtain the directive function of the recombinant virus DPV CHv-BAC-G. using the EGFP reporter gene, and obtained by 8 rounds of plaque purification. The recombinant virus was purified. The recombinant virus DPV CHv-BAC-G was extracted from the cyclization period, and the electric shock was converted into DH10B cells. The recombinant virus transformant that could be replicated in the bacteria was obtained. The cloned virus plasmid pBAC-DPV was transfected into the host cell DEF, and the recombinant virus saved by the recombinant virus DPV CHv-BAC-G. could be successfully saved by fine. Bacteria and viruses exist in two forms, which can be replicated at the same time in bacteria and host cells. It is beneficial to study duck plague virus that can only be studied at the cell level by the mature gene operation method of the prokaryotic system, and successfully establish the recombinant duck plague virus rescue system platform.7 for recombinant duck plague virus UL55 gene deficiency. On the basis of the constructed bacterial artificial chromosome recombinant duck plague virus rescue system platform, the two step RED recombination technology in Escherichia coli cells was used to construct the UL55 gene deletion strain and its response mutant. The constructed recovery mutant clon can be saved by transfecting DEF cells. The same mutagenicity of the parent and the parent DPV CHv-BAC-G was no difference between the parent strain and the parent strain. It was the true replying mutant. The one step growth curve and the pathogenicity result showed that the constructed UL55 deletion and the recovery mutant were consistent with the expected results, and could produce similar cell lesion effects with the parent strain of DPV CHv-BAC-G. In the infected cells, the same proliferation cycle is formed, and the same proliferation cycle.8 DPV UL55 gene is displayed in the infected host cells. The Rabbit anti UL55 polyclonal antibody is used as a single antibody, and the dynamic distribution of the UL55 gene encoding egg white in the infected cells is detected by indirect immunofluorescence. The result shows that UL55 protein began to express in cytoplasm as early as 5.5h after infection, and the expression amount gradually increased with time. The expression of 22.5h reached peak value after infection. Then the expression of UL55 protein gradually declined, and the dispersed particles in cytoplasm gradually form fluorescent spots to the periphery of the nuclear membrane. As time goes on, the fluorescence of UL55 protein disappears gradually as time goes on, it is presumed that the.9 DPV UL55 and UL26.5 gene are located in the infected host cells with the end of the virus replication cycle and the disintegration of the cells. The co localization results of the UL55 protein and the UL26.5 protein in the cells of the infected host cells show that UL26.5 and UL55 protein are in the sense. The cells were adjacent and overlapped, but when DPV did not express UL55 protein, the localization of UL26.5 protein did not change, indicating that the deletion of the UL55 gene did not affect the location of UL26.5 in the nucleus and its function, indicating that the assembly of the nucleocapsid formed by the UL26.5 protein was completed by a number of proteins including the UL55, and UL55 protein was involved in the virus. Assembly, but not necessary.
【学位授予单位】：四川农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S852.65
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本文编号：1986755