【作者】 沈婵娟；

【导师】 程安春； 汪铭书；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2010， 博士

【摘要】 1鸭瘟病毒U151基因的序列特征分析及密码子偏爱性分析根据本实验室构建的鸭瘟病毒(DPV) DNA文库,并结合NCBI中的开放阅读框(ORF) Finder和BLAST工具进行分析,一个完整的ORF被鉴定出来。该ORF是DPV UL51基因(GenBank No.DQ072725),大小为759bp,编码一种皮层蛋白,并且含有一个在α-疱疹病毒亚科中保守的区域。进化关系分析揭示DPV UL51基因与马立克属疱疹病毒UL51基因的进化关系最近。之后,对该基因编码蛋白序列进行了一系列生物信息学分析,结果显示,该蛋白含有4个潜在的磷酸化位点、,1个潜在的酰基化位点和4个潜在的线性B细胞表位,表明它可能是一种磷酸化和酰基化的蛋白,并且能够诱导较强的免疫反应；同时,该蛋白不含任何的N-糖基化位点、跨膜区域、信号肽和核定位信号,却含有高尔基体定位信号,表明它可能定位于胞浆的高尔基体中。对DPV UL51基因密码子偏爱性分析的结果揭示,该基因对同义密码子中第三位为A和T的密码子有强烈的偏爱性,并且原核表达系统可能更适合该基因的表达。这些结果为进一步研究DPV UL51基因的特性和功能提供了理论支持。2 DPV UL51基因的克隆、原核表达、产物纯化及其多克隆抗体的制备根据本实验室提供的DPV UL51基因序列,采用primer5.0软件设计一对特异性引物,运用PCR方法从DPV基因组DNA中扩增UL51基因,并将其克隆至pMD18-T载体中,经EcoRⅠ和XhoⅠ双酶切、PCR和DNA测序鉴定正确后,将该基因正向插入原核表达载体pET28a(+)的EcoRⅠ和XhoⅠ位点之间,成功构建了重组表达质粒pET28a-UL51。将该表达质粒转化到表达宿主菌BL21(DE3)中,用IPTG诱导,表达出了大小约为34KD的重组UL51蛋白,并且主要以包涵体形式存在；经过对诱导剂IPTG浓度、诱导温度和诱导时间的优化,确定了该重组表达质粒的最佳诱导条件为0.8mmol/L IPTG、37℃条件下诱导4h。将表达产物用镍柱梯度亲和层析纯化后,高纯度的表达蛋白与等量弗氏佐剂混合作为免疫原,四次免疫家兔,获得了抗重组UL51蛋白高免血清。将该血清经辛酸-硫酸铵粗提和High Q阴离子交换柱层析纯化后,得到了特异性强的兔抗重组UL51蛋白抗体IgG,为进一步研究DPV UL51基因的特性和功能奠定了基础。3 DPV UL51基因在感染宿主细胞中的转录和表达特征本试验应用实时荧光定量RT-PCR和Western blotting法检测DPV UL51基因在鸭胚成纤维细胞(DEF)中的转录和表达情况。结果表明：该基因在DPV感染后2h时已经开始转录,8h开始表达,48h转录和表达量都达到最高峰,之后逐渐降低；该基因在DEF细胞中的表达产物分子量为34KD；并且该基因表达产物是病毒粒子的一种组成成分。该研究为阐明DPVUL51基因的功能提供了有用的数据。4 DPV UL51蛋白在病毒感染细胞中的定位通过间接免疫荧光技术检测DPV UL51蛋白在病毒感染细胞内的定位特性,结果显示,最早可在感染后8h的胞浆中检测到特异性荧光点,24-36 h有大量绿色荧光团块聚集于近核区域,之后随着细胞病变的增强,胞核和胞浆中的绿色荧光均开始减弱。用胶体金免疫电镜法检测DPV UL51蛋白在DPV感染细胞中的亚细胞定位,结果显示,DPV UL51蛋白主要位于胞浆空泡中的病毒粒子和一些膜结构上；此外,在感染早期,DPV UL51蛋白大量累积于高尔基复合体膜上,其后通过某种未知机理被运送到细胞膜附近。该研究也为阐明DPVUL51蛋白的功能奠定了基础。5 DPV UL51基因真核表达载体的构建及其在COS-7细胞中的瞬时转录和表达特性根据DPV UL51基因序列设计一对特异性引物,用PCR扩增并克隆至pMD18-T载体上,经双酶切和测序鉴定后,再将该目的片段亚克隆到pcDNA3.1(+)真核表达载体上,得到重组质粒pcDNA3.1-UL51,通过脂质体介导将其转入COS-7细胞；应用实时荧光定量RT-PCR、Western blotting和间接免疫荧光法检测该基因在COS-7细胞中的转录、表达和定位情况。结果表明：该基因在转染后6h时已经开始转录,12 h开始表达,24 h转录和表达量都达到最高峰,之后逐渐降低；并且该基因在COS-7细胞中得表达产物的分子量为33 KD。间接免疫荧光法显示该基因的表达产物早期聚集于近核区域,晚期定位于胞浆和胞核中。该研究也为阐明DPV UL51基因的特性和功能提供了有用的数据。6用免疫组化方法检测DPV UL51蛋白在人工感染鸭组织中的定位和动态分布采用DPV强毒CHv株人工感染30日龄鸭,于攻毒后于不同时间采集法氏囊、胸腺、脾、哈德氏腺、肝、胰、食管、腺胃、小肠(包括十二指肠、空肠和回肠)、大肠(包括盲肠和直肠)、脑、肾、肺、心、肌肉组织或器官,用建立的基于重组UL51蛋白抗体的间接免疫荧光和免疫酶组化法,分别检测DPV UL51蛋白在鸭体组织中的定位和动态分布。结果表明,DPV UL51蛋白主要分布在法氏囊、胸腺、脾脏、肝脏、食道、和肠道中,并且主要定位于淋巴细胞、网状细胞、巨噬细胞和上皮细胞的胞浆中。本研究不仅有助于理解在急性DPV感染时,DPV的致病机理,同时也为α-疱疹病毒UL51蛋白同源物的定位和分布的研究奠定了基础。7基于重组UL51蛋白的间接EL ISA法和胶体金免疫层析试纸条法检测鸭瘟病毒抗体的研究和应用DPV感染给世界上养鸭国家和地区造成了重大的经济损失。DPV特异性抗体的监测是评价DPV弱毒疫苗效果和制定合理的免疫程序的关键。因此,本研究中,基于纯化的重组UL51蛋白,我们分别建立了一种间接ELISA法(UL51-ELISA)和一种免疫层析试纸条法(UL51-ICS)来检测DPV血清抗体。首先对UL51-ELISA法的反应条件进行优化,结果表明,最适重组UL51蛋白包被量为2.5μg/100μL,最佳的血清稀释倍数为1：200,最佳的酶标二抗稀释倍数为1：2000；用建立的UL51-ELISA法对鸭病毒性肝炎病毒(DHV)、鸭疫里默氏菌(RA)、鸭大肠杆菌(E.coli)的阳性血清进行检测,结果均为阴性,特异性好；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1：3200倍稀释的DPV阳性血清。UL51-ICS是基于膜层析原理,并以胶体金标记的重组UL51蛋白和胶体金标记的羊抗兔IgG混合物共同作为示踪剂的一种方法。该ICS法将纯化的重组UL51蛋白抗原包被于检测线(T),兔IgG包被于质控线(C)。经优化和筛选,确定了UL51-ICS法中的重组UL51蛋白、兔IgG、胶体金标记的重组UL51蛋白和胶体金标记的羊抗兔IgG的最佳工作浓度分别为2mg/mL、lmg/mL、2mg/mL和2mg/mL。用建立的UL51-ICS法分别对非DPV的鸭源病原体阳性血清进行检测,结果均为阴性,特异性好；并能检出经1：128倍稀释的DPV阳性血清。同时,该法也具有良好的批内及批间重复性和较好的稳定性,制备好的试纸条至少可在4℃或25℃保存1年；为了评价UL51-ELISA和UL51-ICS法的效果,我们同时用UL51-ELISA、UL51-ICS、包被全病毒的ELISA法(DPV-ELISA)和中和试验(NT)四种方法对110份地方鸭血清进行检测,结果显示,与DPV-ELISA和NT相比,本研究建立的UL51-ELISA法与UL51-ICS法都有较高的特异性、敏感性和很高的的符合率,并且成本低,适于在现场或实验室进行DPV感染的血清学监测。8基于抗重组UL51蛋白抗体的抗原捕获ELISA法和胶体金免疫层析试纸条法检测DPV的研究和应用本研究中,我们使用纯化的大鼠抗重组UL51蛋白多克隆抗体和兔抗重组UL51蛋白多克隆抗体,分别建立了一种抗原捕获ELISA法(AC-ELISA)和一种胶体金免疫层析试纸条(ICS)法来检测DPV。使用建立的抗原捕获AC-ELISA方法,可以检测到1：640倍稀释的阳性DPV细胞培养液中的DPV；检测含DHV的鸭胚尿囊液、含RA的菌体培养液和含E. coli菌体培养液等结果均为阴性。使用建立的ICS法,可以检测到1：80倍稀释的阳性DPV细胞培养液中的DPV；检测含DHV的鸭胚尿囊液、含RA的菌体培养液和含E. coli菌体培养液,结果均为阴性。为了评价AC-ELISA和ICS方法的效果,我们同时用AC-ELISA、ICS和常规PCR三种方法对10份人工感染DPV强毒后的病鸭泄殖腔棉拭子样品进行检测,结果显示,与PCR方法相比,本研究建立的AC-ELISA法和ICS具有较高的特异性、敏感性和符合率,适于在现场或实验室进行DPV感染的检测。更多还原

【Abstract】 1 Sequence analysis and codon bias analysis of duck plague virus UL51 gene According to duck plague virus (DPV) DNA library constructed in our laboratory, and combining together with the analysis of open reading frame (ORF) Finder tool and BLAST tool of NCBI, a complete ORF (GenBank accession number DQ072725) of DPV was identified as a tegument protein encoding the DPV UL51 gene with a size of 759 bp, and contains a conserved domain of Alphaherpesvirinae UL51. Then, phylogenetic tree analysis revealed that the DPV UL51 gene was evolutionarily closer to Mardivirus genus of the Alphaherpesvirinae subfamily. After that, the UL51 gene was analyzed by a series of bioinformatics tools. The results showed that the DPV UL51 protein contained fourteen potential phosphorylation sites, one potential palmitoylation site and four potential linear B-cell epitopes, suggesting that it might be a phosphorylated and palmitoylated protein, and be able to induce a very strong immune response. Meanwhile, the DPV UL51 protein does not contain any N-linked glycosylation sites, transmembrane helix, signal peptides, and nuclear location signals (NLS), but it contains Golgi apparatus location signals, suggesting that it might localize to the Golgi apparatus of the cytoplasm. Besides, the results of codons usage bias analysis of the DPV UL51 gene showed that, codon of the gene was strong bias towards the synonymous codons with A and T at the third codon position, and the prokaryotic expression system might be more suitable for the expression of the gene. These results provided theoretical supports for the further study on the characterization and function of DPV UL51.2 Cloning, prokaryotic expression, purification and polyclonal antibody preparation of DPV UL51 gene A pair of primers was designed based on the DPV UL51 gene sequence identified by our laboratory. The UL51 gene fragment was amplified from the genome of DPV by PCR, and cloned into pMD18-T vector and sequenced. Then the gene from the pMD18-UL51 vector with two restriction enzymes (EcoR I and XhoⅠ) digestion was subcloned into the prokaryotic expression vector pET-28a (+) to generate the recombinant plasmid pET28-UL51, which was identified by two restriction enzymes (EcoR I and XhoⅠ) digestion, and then transformed into E. coli BL21(DE3) strain and expressed under isopropylβ-D-1-thiogalactopyranoside (IPTG) induction. SDS-PAGE analysis showed that the induced expressed protein is about 34 KD, and mostly exists in inclusion examined by soluble analysis. Through optimization test it was found that the optimal condition was 0.8mmol/L IPTG as inductor, duration of 4 hours at 37℃. The fusion protein was purified by Ni2+-NAT column affinity chromatography, and used to immunize rabbits to generate the UL51 antiserum. Its antibody titer tested by agar gel precipitation was up to 1:32. The UL51 antibody IgG was subsequently obtained by using caprylic acid and ammonium sulfate precipitation and High-Q anion-exchange chromatography. These researches will provide a basis for further functional analysis of the DPV UL51 gene.3 The transcription and expression characteristics of DPV UL51 gene in DPV-infected cells In this study, we determined the transcription and expression characteristics of DPV UL51 gene by real-time quantitative RT-PCR and western blotting. The results indicated that DPV UL51 gene transcripts appeared at 2 h post infection (PI), and its expression products were first detected at 8 h PI, and then both of the transcripts and expression products were up to a peak at 48 h PI, thereafter both of them were reduced; A 34 KD protein in DEF cells was detected, and the UL51 protein was a component of DPV virions. This study provides serviceable datum for elucidating characteristics and functions of the DPV UL51 gene.4 Subcellular localization of DPV UL51 protein in DPV-infected host cells The results of indirect immunofluorescence technique showed that specific fluorescence appeared in cytoplasm as early as 8 hour PI and a great deal of specific fluorescence concentrated in the juxtanuclear region from 24 to 36 h PI,but there after the specific fluorescence became to weaken followed by the appearance of cytopathic effect. Transmission immunoelectron microscopy analysis revealed that DPV protein (pUL51) was mainly associated with cytoplasmic virions and also with some membranous structure near the pUL51-specific immuno-labeling intracellular virion in the cytoplasmic vesicles; moreover, the pUL51 efficiently accumulated in the Golgi apparatus at first, and then was sent to the plasma membrane from the Golgi by some unknown mechanism. In a word, these experimental results lay a foundation for further study on the function of DPV UL51 protein.5 Construction of the DPV UL51 gene eukaryotic expression vector and transient transcription and expression of the gene in COS-7 cells Based on the sequence of DPV UL51 gene to design a pair of primers, the gene was amplified by PCR, and cloned into pMD18-T vector. After restriction enzyme digestion and sequence analysis, it was subcloned into the pcDNA3.1 eukaryotic expression vector to generate the recombinant plasmid pcDNA3.1-UL51, which was transfected into the COS-7 cells by Lipofectin method; then, its transcription, expression and localization were determined by real-time qualiticative RT-PCR, western blotting and indirect immunofluorescence. The results indicated that its transcripts appeared at 6 h post transfection (PT), and its expression products were first detected at 12 h PT, and then both of the transcripts and expression products were up to a peak at 24 h PT, thereafter both of them were reduced; A 33 KD protein in COS-7 cells was detected, and the UL51 protein was localized to the perinuclear regions at 12 h PT, and to the nucleus and cytoplasm at later times. This study provides serviceable datum for elucidating characteristics and functions of the DPV UL51 gene.6 The localization and dynamic distribution properties of DPV UL51 protein in experimentally DPV-infected ducks detected by immunohistochemistry staining Fifty-eight 30-day-old DPV-free ducks were intramuscularly inoculated with the pathogenic DPV CHv strain as infection group, and two ducks were selected as pre-infection group.18 different tissues (bursa of Fabricius, thymus, spleen, Harders glands, liver, pancreas, esophagus, glandularis ventriculus, duodenum, jejunum, ileum, caecum, rectum, cerebrum, kidney, lung, myocardium, muscle) were collected from DPV-infected ducks at sequential time points, and prepared for indirect immunofluorescence staining and immunoperoxidase staining. The results showed that in acute DP cases, DPV pUL51 was mainly distributed in the bursa of Fabricius, thymus, spleen, liver, esophagus and intestine, and was mostly localized in the cytoplasm of lymphocytes, reticulum cells, macrophages and epithelial cells. The research will be not only useful for understanding the pathogenesis of this DP, but also for studying the localization and distribution properties of alphaherpesvirus pUL51 homologs.7 Development and application of an indirect ELISA and an immunochromatographic strip test based on recombinant UL51 protein for detecting antibody against DPV DPV infection causes substantial economic losses to the worldwide duck-producing areas. The monitoring of DPV-specific antibodies is a key to evaluate the effect of weak-DPV vaccine and develop rational immunization programs. Thus, in this study, based on a purified recombinant UL51 protein, an indirect ELISA (UL51-ELISA) and an immunochromatographic strip (UL51-ICS) test were developed for detecting the DPV serum antibodies. The optimum conditions for UL51-ELISA were determined. The results demonstrated that the optimized evaluation could be obtained when the recombinant UL51 protein concentration is 2.5μg/100μL, the dilution of the examined serum is 1:200, and the enzyme linked antibody dilution is 1:2000. Other duck infected pathogen specific positive antiserum, such as duck hepatitis virus (DHV), duck riemirella anatipestifer (RA) and duck E.coli, were employed as negative controls and showed negative results. The coefficients of intra-assay and inter-assay variation were less than 10% and could detect DPV positive antiserum with a dilution of 1:3200. The UL51-ICS test is based on membrane chromatography, and uses both the recombinant UL51 protein conjugated with colloidal gold and goat anti-rabbit IgG conjugated with colloidal gold as the tracers. In the UL51-ICS, the purified recombinant UL51 protein was used as the capture reagent at the "test line", and the purified rabbit IgG was used as the capture reagent at the "control line". The optimum conditions for UL51-ICS test were determined. The results demonstrated that the optimized evaluation could be obtained when the recombinant UL51 protein concentration is 2mg/mL, the rabbit IgG concentration is lmg/mL, the recombinant UL51 protein conjugated with colloidal gold concentration is 2mg/mL, and the goat anti-rabbit IgG conjugated with colloidal gold concentration is 2mg/mL. Other duck infected pathogen specific positive antiserum were employed as negative controls and showed negative results. The reproducibility of intra-assay and inter-assay were good. The strips were stable for one year at 4℃or 25℃, and could detect DPV positive antiserum with a dilution of 1:128. To evaluate the effect of the UL51-ELISA and the UL51-ICS test,110 duck serum samples collected from several duck flocks were simultaneously tested by the UL51-ELISA, UL51-ICS, the whole DPV antigen as coated antigen ELISA method (DPV-ELISA) and neutralization test (NT). The results of detections revealed that, compared with the DPV-ELISA and NT, both the UL51-ELISA and UL51-ICS test have higher specificity, higher sensitivity, highest coincidence, low cost, and are suitable for the serological surveillance of DPV infection in the field or in the laboratory.8 Development and application of an antigen capture ELISA method and an immunochromatographic strip test based on anti-recombinant UL51 protein polyclonal antibody for detecting DPV antigen An antigen capture enzyme-linked immunosorbent assay (AC-ELISA) and an immunochromatographic strip (ICS) test were developed with the purified rat anti-UL51 recombinant protein polyclonal antibody and rabbit anti-UL51 recombinant protein polyclonal antibody for detecting DPV. For AC-ELISA, the 1:640 diluted viruses in the DPV-infected cells can be observed, while other pathogens such as DHV, RA, E.coli can not be detected by the AC-ELISA. For ICS, the 1:80 diluted viruses in the DPV-infected cells can be observed, while other pathogens such as DHV, RA, E.coli can not be detected by the AC-ELISA, too. Ten cloaca cotton pledget samples of ducks which were experimentally infected with virulent DPV CHv, were assayed. The results of detections revealed that, compared with the PCR, both the AC-ELISA and ICS test have higher specificity, higher sensitivity, higher coincidence, and can be used to diagnose DPV in the field or in the laboratory.更多还原