本文選題：鴨瘟病毒中國(guó)強(qiáng)毒株 + 基因組解析��； 參考：《四川農(nóng)業(yè)大學(xué)》2015年博士論文

【摘要】：鴨瘟(duck plague)又稱鴨病毒性腸炎(duck virus enteritis),是鴨、鵝和其他雁形目禽類的一種急性、熱性、敗血性傳染病。本病發(fā)病快、死亡率高,是目前世界各國(guó)水禽業(yè)危害最嚴(yán)重的傳染病之一。該病的病原鴨瘟病毒(Duck Plague virus, DPV)是皰疹病毒科(Herpesvirales)、α皰疹病毒亞科(Alphaherpesvirinae)、馬立克氏病毒屬(Mardivirus)的成員之一。但由于DPV的研究起步晚,其分子生物學(xué)研究遠(yuǎn)遠(yuǎn)落后于其它皰疹病毒。一段時(shí)間以來(lái)對(duì)于DPV的研究多集中于流行病學(xué)、診斷和防治等方面；與其分子生物學(xué)特性相關(guān)的基礎(chǔ)研究,如病毒的基因組結(jié)構(gòu)、物理圖譜、病毒基因功能以及病毒在機(jī)體細(xì)胞和組織中的復(fù)制及致病機(jī)理、病毒感染過(guò)程以及機(jī)體抗病毒感染機(jī)制等多方面研究均有許多不明之處。因此本論文的主要目的就是希望對(duì)本實(shí)驗(yàn)室測(cè)序獲得的鴨瘟病毒基因組序列進(jìn)行解析；并通過(guò)構(gòu)建一個(gè)可以對(duì)DPV進(jìn)行細(xì)菌遺傳學(xué)操作的平臺(tái),為DPV的基因功能研究提供技術(shù)手段的支持,此外利用該平臺(tái)用兩步RED重組敲除UL55基因,驗(yàn)證此平臺(tái)的可行性并初步闡釋UL55功能。主要研究?jī)?nèi)容如下：1鴨瘟病毒中國(guó)強(qiáng)毒株基因組信息解析對(duì)本實(shí)驗(yàn)室測(cè)序獲得的鴨瘟中國(guó)強(qiáng)毒株的基因組序列進(jìn)行生物信息學(xué)分析。鴨瘟病毒中國(guó)強(qiáng)毒株基因組組成為雙股線狀雙鏈DNA:UL-IRS-US-TRS,是典型的D型皰疹病毒基因組結(jié)構(gòu)。DPV CHv基因組全長(zhǎng)162,175 bp,GC含量為44.89%,包括潛在的76個(gè)能編碼功能蛋白的ORF。BLAST搜尋相似性序列發(fā)現(xiàn)五條與DPV CHv同期或之后提交的其他鴨瘟病毒毒株全基因組序列,他們間具有高度同源性,進(jìn)化樹(shù)分析顯示六株DPV病毒按地域和毒力差異進(jìn)行分類,DPV CHv處于進(jìn)化樹(shù)的中間位置。對(duì)基因組編碼區(qū)的ORF進(jìn)行掃描分析發(fā)現(xiàn),六株DPV在UL、US區(qū)域分別有23、5個(gè)ORF在核苷酸水平上存在整個(gè)ORF的缺失、部分序列插入、缺失等情況出現(xiàn),這些突變的產(chǎn)生可能是造成六株不同來(lái)源毒株毒力和地理差異的主要原因。而DPV基因組中不存在核苷酸序列插入或缺失的53個(gè)ORF在氨基酸水平上高度同源,多為DPV基因組的保守性基因,不受病毒傳代致弱及地域差異的影響,編碼蛋白大多為與病毒DNA代謝相關(guān)的結(jié)構(gòu)蛋白。密碼子使用模式分析結(jié)果顯示其在基因組密碼子的使用上偏向A/T結(jié)尾的密碼子,其密碼子使用模式主要受突變壓力的影響。將其與人類、大腸桿菌、酵母的密碼子使用模式進(jìn)行比較,結(jié)果表明鴨瘟病毒的密碼子使用模式與酵母系統(tǒng)更為接近。2鴨瘟病毒UL55基因生物信息學(xué)分析DPV UL55基因由561bp核苷酸組成,包括一個(gè)完整的開(kāi)放性閱讀框,編碼一個(gè)由186個(gè)氨基酸組成的20.7981kDa蛋白質(zhì)。UL55蛋白沒(méi)有信號(hào)肽及跨膜區(qū)。密碼子分析顯示UL55基因偏向A/T結(jié)尾的密碼子,密碼子使用模式與人類最接近。UL55蛋白整體表現(xiàn)出親水性,抗原表位主要集中在相應(yīng)親水區(qū)域的無(wú)規(guī)則卷曲,其功能與病毒的裝配、出芽、成熟和釋放相關(guān)�；诤塑账嵝蛄械倪M(jìn)化樹(shù)分析顯示DPV屬于馬立克氏病毒基因?qū)俚囊粏T。3鴨瘟病毒UL55基因的克隆、原核表達(dá)及多克隆抗體制備通過(guò)將UL55基因通過(guò)克隆到表達(dá)載體pET32a(+)上實(shí)現(xiàn)對(duì)UL55蛋白的原核表達(dá),試驗(yàn)表明重組UL55基因能在大腸桿菌BL21(DE)中進(jìn)行融合表達(dá)。通過(guò)優(yōu)化誘導(dǎo)表達(dá)條件,UL55重組蛋白能在37℃條件下,通過(guò)0.8mmM的IPTG誘導(dǎo)表達(dá)4h產(chǎn)生大量非可溶形式的包涵體蛋白。將包涵體蛋白通過(guò)裂解后進(jìn)行純化及復(fù)性處理再與兔抗DPV CHv多克隆抗體進(jìn)行免疫印跡反應(yīng),結(jié)果表明純化復(fù)性后的UL55蛋白具有與天然蛋白相似的免疫反應(yīng)活性。復(fù)性后的UL55蛋白免疫兔子制備的高免血清能夠與UL55重組蛋白發(fā)生良好的免疫反應(yīng)。4原核表達(dá)UL55蛋白作為抗原檢測(cè)DPV血清的間接ELISA方法的建立本方法是基于純化的重組UL55原核表達(dá)蛋白建立的可以檢測(cè)DP血清的間接ELISA法,該方法特異性強(qiáng),對(duì)抗鴨病毒性肝炎病毒(DHV)、鴨疫里默氏菌(RA)、鴨大腸桿菌(E.coli)、鴨源沙門氏菌(Salmonella)、腫頭性出血癥病毒和鴨源流感病毒的陽(yáng)性血清進(jìn)行檢測(cè),結(jié)果均為陰性；該方法的對(duì)酶標(biāo)板內(nèi)或板間重復(fù)試驗(yàn)顯示變異系數(shù)均小于10%,能檢出經(jīng)1：6400倍稀釋的DPV弱毒疫苗免疫鴨的陽(yáng)性血清。將其與經(jīng)典的中和試驗(yàn)及DPV全病毒包被的ELISA同時(shí)檢測(cè)50份感染了DPV的臨床鴨血清樣本,發(fā)現(xiàn)其對(duì)DPV IgG的檢出率介于中和試驗(yàn)和DPV-ELISA之間。由于其制備方法簡(jiǎn)便、操作簡(jiǎn)單,特異性、靈敏性較高。且不存在散毒的風(fēng)險(xiǎn),具有良好的應(yīng)用前景,為進(jìn)一步組裝成試劑盒奠定了基礎(chǔ)。5鴨瘟病毒UL55基因轉(zhuǎn)錄、表達(dá)時(shí)相分析以β-actin基因作為內(nèi)參基因,用相對(duì)熒光定量方法對(duì)UL55基因在體外感染宿主細(xì)胞中的轉(zhuǎn)錄情況進(jìn)行了分析。轉(zhuǎn)錄時(shí)相分析結(jié)果表明UL55基因在轉(zhuǎn)錄后的0-8h內(nèi)處于一個(gè)較低的水平；12h后開(kāi)始迅速增加直至在感染后36h達(dá)到轉(zhuǎn)錄峰值,之后開(kāi)始逐步下降,直到感染后的60h仍然可以檢測(cè)到大量轉(zhuǎn)錄的UL55基因,UL55基因的轉(zhuǎn)錄過(guò)程可以被核酸抑制劑更昔洛韋抑制,說(shuō)明UL55基因是嚴(yán)格依賴于DNA合成的γ2基因。Western-blot分析體外感染宿主細(xì)胞中UL55基因的表達(dá)時(shí)相表明UL55蛋白的表達(dá)水平也表現(xiàn)出相似的規(guī)律,進(jìn)一步佐證了UL55基因?yàn)棣?基因的結(jié)論。6細(xì)菌人工染色體重組鴨瘟病毒拯救系統(tǒng)平臺(tái)構(gòu)建通過(guò)多步克隆構(gòu)建了含TK基因同源臂、報(bào)告基因EGFP和細(xì)菌人工染色體核心功能元件的重組鴨瘟病毒轉(zhuǎn)移載體pUC18/EGFP-TKAB-BAC11,將其與DPV CHv病毒核酸共轉(zhuǎn)染獲得了重組病毒DPV CHv-BAC-G。利用EGFP報(bào)告基因的指示作用,通過(guò)8輪噬斑純化獲得了純化的重組病毒。提取環(huán)化時(shí)期的重組病毒DPV CHv-BAC-G,電擊轉(zhuǎn)化至DH10B細(xì)胞中,獲得了能夠在細(xì)菌中復(fù)制的重組病毒轉(zhuǎn)化子。將克隆化的病毒質(zhì)粒pBAC-DPV轉(zhuǎn)染至宿主細(xì)胞DEF,成功拯救出重組病毒DPV CHv-BAC-G。拯救出的重組病毒可以以細(xì)菌和病毒兩種形式存在,能夠?qū)崿F(xiàn)在細(xì)菌和宿主細(xì)胞內(nèi)的同時(shí)復(fù)制,有利于利用原核系統(tǒng)成熟的基因操作手段研究原本僅能在細(xì)胞水平研究的鴨瘟病毒,成功建立細(xì)菌人工染色體重組鴨瘟病毒拯救系統(tǒng)平臺(tái)。7重組鴨瘟病毒UL55基因缺失株的構(gòu)建及其體外生物學(xué)特性研究在構(gòu)建的細(xì)菌人工染色體重組鴨瘟病毒拯救系統(tǒng)平臺(tái)基礎(chǔ)上,利用大腸桿菌細(xì)胞內(nèi)的兩步RED重組技術(shù)構(gòu)建UL55基因缺失株及其回復(fù)突變株。構(gòu)建的回復(fù)突變株克隆能夠通過(guò)轉(zhuǎn)染DEF細(xì)胞獲得拯救產(chǎn)生與親本相同的致細(xì)胞病變,酶切圖譜與親本株DPV CHv-BAC-G無(wú)差異,是為真正的回復(fù)突變株�？瞻咴囼�(yàn)、一步生長(zhǎng)曲線和致病性比較結(jié)果表明,構(gòu)建的UL55缺失株和回復(fù)突變株與預(yù)期結(jié)果一致,能產(chǎn)生與親本株DPV CHv-BAC-G相似細(xì)胞病變效應(yīng)、形成形態(tài)及大小相似的空斑、在感染細(xì)胞中展現(xiàn)出相同的增殖周期。8 DPV UL55基因在感染宿主細(xì)胞體內(nèi)的定位分析以制備的兔抗UL55多克隆抗體作為一抗,用間接免疫熒光的方法檢測(cè)UL55基因編碼蛋白在感染細(xì)胞內(nèi)的動(dòng)態(tài)分布。結(jié)果顯示,UL55蛋白最早在感染后的5.5h內(nèi)開(kāi)始在細(xì)胞質(zhì)大量表達(dá),并隨著時(shí)間的推移表達(dá)量逐漸增多,其表達(dá)量在感染后的22.5h達(dá)到峰值。之后UL55蛋白的表達(dá)量逐步下降,并從細(xì)胞質(zhì)內(nèi)的散在分布顆粒逐漸形成熒光斑向核膜周邊轉(zhuǎn)移,大量存在于核膜兩極。之后隨著時(shí)間的推移,UL55蛋白的熒光逐漸消失,推測(cè)其隨著病毒復(fù)制周期的結(jié)束和細(xì)胞的崩解而消失。9 DPV UL55與UL26.5基因在感染宿主細(xì)胞體內(nèi)的定位分析UL55蛋白和UL26.5蛋白在細(xì)胞內(nèi)的共定位結(jié)果顯示,UL26.5和UL55蛋白在感染細(xì)胞內(nèi)鄰接并部分重疊,但當(dāng)DPV不表達(dá)UL55蛋白時(shí),UL26.5蛋白的定位沒(méi)有變化,表明UL55基因的缺失并不影響UL26.5在核內(nèi)的定位和其功能的發(fā)揮,預(yù)示著UL26.5蛋白形成的核衣殼的裝配由包括UL55在內(nèi)的多個(gè)蛋白完成,UL55蛋白參與病毒的裝配,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.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65
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本文編號(hào)：1986755