摘要
鸭瘟(Duck Plague, DP),又称鸭病毒性肠炎(Duck Enteritis),是由鸭瘟病毒引起的鸭、鹅和其他雁形目禽类动物发生的一种以血管损伤,组织出血,消化道黏膜糜烂,淋巴器官受损和全身实质性器官退行性病变为特征的急性、接触性、败血性传染病。鸭瘟病毒(Duck Plague Virus, DPV),又称鸭肠炎病毒(Duck Enteritis Virus, DEV),或鸭疱疹病毒1型(Anatid herpesvirus1, AnHV-1),属疱疹病毒科未定种的疱疹病毒。本研究在实验室前期构建的DPV CHv毒株基因文库的基础上,首次发现并鉴定了DPVCHv株gD基因(GenBank登录号：EU195085),并对gD基因开展系列研究,获得以下结果：
1.鸭瘟病毒gD基因发现、克隆及生物信息学分析根据本实验室构建的DPV基因组文库,随机挑取文库中的重组质粒测序,并对测定的序列进行手工和计算机拼接,寻找病毒基因组的开放阅读框架(open reading frames, ORF)。拼接出的其中一个ORF1269采用多种网上生物信息学数据库及软件进行生物信息学分析。结果表明该ORF属疱疹病毒gD基因家族,为DPV的gD基因(GenBank登录号EU195085)。 gD基因完整编码区(ORF)全长1269bp,C+G共557bp,含量为44%。编码的gD蛋白含有422个氨基酸,分子量为47.5kD,理论等电点(PI)为6.66。进化树分析gD基因与与猫疱疹病毒-1型(FeHV-1)海豹疱疹病毒1型(PhoHV-1)以及犬疱疹病毒亲缘性较高；而gD蛋白与鸡传染性喉气管炎病毒(ILTV、GaHV-1)的gD亲缘性最高。gD具有一个特征性的信号肽位于氨基酸序列1-22位；在7-29,360-382氨基酸之间有一跨膜区；gD蛋白主要定位于内质网中(44.4%),其次在胞质、分泌小囊泡、生物膜外(包括细胞膜)、线粒体和细胞核中呈相同分布(11.1%)。gD有4个N-糖基化位点和9个O-糖基化位点。gD富含无规卷曲(Coil),含量高达59.72%；其次为p-折叠(Strand),含量为24.64%；而α-螺旋(Helix)含量较低占15.64%。
2.鸭瘟病毒gD基因胞外区原核表达、产物纯化及其抗体制备根据发现的DPVgD基因序列,设计一对引物扩增DPV gD基因胞外区并将其克隆至pGEM-T载体中,经酶切和DNA测序鉴定正确后,将gD胞外区基因插入到pET32a载体的MscⅠ和HindⅢ之间,成功构建重组表达质粒pET32a-gD。将pET32a-gD转化感受态宿主菌E.coli BL21(DE3), IPTG诱导后经SDS-PAGE电泳检测出大小约为30kD的重组蛋白,重组蛋白以可溶和不可溶包涵体两种形式表达。Western blot检测发现该蛋白能与DPV阳性血清发生特异性反应。大量表达gD蛋白,使用Ni-NTA亲和层析进行纯化,将纯化后的gD胞外区蛋白与弗氏佐剂混合后免疫家兔三次,使用ELISA方法测定血清中抗gD抗体滴度。抗gD抗体滴度可达1：256000；采集免疫家兔血清,使用Protein A亲和层析纯化收获抗gD蛋白抗体IgG,为后续研究奠定基础。
3.鸭瘟病毒gD基因真核表达载体构建及在COS-7细胞中的瞬时表达根据DPVgD基因序列,设计一对特异引物,用PCR方法从DPV基因组中扩增目标基因全序列,将其正向插入真核表达载体pEGFP-N1和pcDNA3.1(+)多克隆位点NheⅠ和HindⅢ酶切位点之间,成功构建了重组真核表达质粒pEGFP-N1-gD和pcDNA3.1(+)-gD,脂质体介导转染至COS-7细胞中,采用激光共聚焦显微镜直接观察、Western-blotting及间接免疫荧光方法检测gD蛋白的表达。激光共聚焦显微镜下可见转染了pEGFP-N1-gD质粒的COS-7细胞质内及细胞膜上出现荧光蛋白的点状聚集现象,显示gD主要定位于细胞膜上。Western-blotting显示gD在COS-7细胞中表达产物的分子量为55KD左右,比gD预测分子量要大,为gD糖基化修饰后成熟糖蛋白；间接免疫荧光检测发现在COS-7细胞中的胞浆、核内及细胞膜上均出现荧光蛋白的点状聚集现象,显示gD主要定位于细胞膜上
4.鸭瘟病毒gD基因的转录及表达分析及gD蛋白在宿主细胞中的亚细胞定位研究常规方法制备鸭胚成纤维细胞,细胞长成单层后,接种病毒DPV CHv株,分别于接毒后Oh(未接毒细胞,空白对照组)、2h、4h、6h、8h、12h、24h、48h、60h采集样品,进行(1)细胞总RNA提取；(2)细胞总蛋白提取,(3)细胞固定。分别进行(1)荧光定量PCR检测gD基因转录时相；(2) Western-blotting检测gD蛋白表达时相；(3)间接免疫荧光检测gD亚细胞定位。结果表明：DPVgD基因在病毒感染鸭胚成纤维细胞后2h开始转录,8h开始表达,12h后转录量和表达量迅速增加后稍下降但仍维持较高水平至60h；该基因在DEF细胞中的表达产物呈现高丰度现象,主要为分子量55Kd的成熟糖蛋白.而gD蛋白在DEF细胞中的定位是一个动态变化过程,最早在接种后8h观察到定位于胞浆,12h见除胞浆外还定位于细胞核膜上,24h主要定位于细胞膜,48h在细胞核核膜、细胞膜及胞浆中均有定位。持续至60h后由于细胞融合崩解至定位不规则。该定位特征与疱疹病毒糖蛋白相同。检测到的时相定位与疱疹病毒糖蛋白的合成过程及定位基本相似,证实了DPVgD功能与其他疱疹病毒gD功能的相似性。
5. DPV gD胞外区蛋白作为包被抗原检测鸭瘟抗体间接ELISA建立和应用本试验以pET32a为载体原核表达的重组DPVgD蛋白作包被抗原,进行间接ELISA检测DPV抗体的研究；并对该方法进行了优化和应用。采用方阵滴定实验确定重组表达蛋白与血清的最佳稀释度,最适包被重组抗原蛋白浓度为100ng/孔(1μg/mL,100μL/孔)包被后37℃1h,然后于4℃条件下过夜,最佳血清稀释度为1：80，0.5%的明胶PBST作封闭液,阴阳性的临界值(cut off值)为阴性样本OD值的平均值(x)+3SD。用建立的间接ELISA检测方法对收集到的DPV阳性血清、DHBV阳性血清、DHV阳性血清以及GpV阳性血清作1：80稀释,ELISA测定发现其只与DPV阳性血清发生反应。与本室自制的DPV全病毒ELISA试剂盒平行检测临床样本血清90份,吻合率为94%,检出率差异不显著。结果表明建立的gD-ELISA检测方法具有很好的特异性和敏感性,可以用于DPV感染的诊断和疫苗免疫后的抗体监测。
6.基于DPV gD基因的PCR检测方法建立和应用依据DPV gD基因无信号肽片段序列设计引物,建立了检测DPVgD基因PCR方法并进行了应用。特异性试验中,从正常DEF细胞、鸭胚尿囊液及DHV. DHBV. GpVDNA中均未扩增出阳性条带,同时对同为疱疹病毒的HSV-1, HSV-2和BV来说,可从病毒DNA中能扩增出条带,说明了gD蛋白在同科病毒中的同源性,而扩增出的片段大小并不与gD基因预计扩增片段大小相同,说明了gD基因PCR引物及方法的特异性；敏感性试验结果显示能检测出lpg左右的DPV DNA;应用该PCR方法对DPV感染病料肝脏、脾脏、胸腺、淋巴结和法氏囊等免疫相关器官组织进行检测,均能扩增出与预期大小一致以及序列吻合的特异性条带,正常组织中未检出阳性条带。证实该方法可应用于临床DPV的检测。
Duck plague (DP), also called duck virus enteritis (DVE), is an acute, contagious and lethal septicemia herpes virus infection of ducks,geese,and swans. It had causes great economical loss in domestic ducks and wild waterfowls. Duck Plague Virus (DPV), also called Duck Enteritis Virus (DEV), or Anatid herpesvirus1(AnHV-1), is an unassigned species in the FAMILY HERPESVIRIDAE. Only few research in the fields of duck plague virus genomics and gene express were performed. Based on the DPV CHv DNA gene library constructed in our laboratory, DPV glycoprotein D (gD) gene sequence was first identified (Gene Bank Accession No. EU195085), a series of studies were conducted and results got as follows:
l.The discovery, Cloning and Molecular Characterization of duck plague virus gD gene According to the CHv strain of duck plague virus (DPV CHv) DNA gene library constructed in our laboratory, A complete open reading frame (ORF1269) found was homology to herpes virus glycoprotein D gene. Sequence analysis confirmed that the ORF1269is DPV gD gene. Bioinformatics analysis showed that DPV gD gene is1269bp in length, encoded a422amino acid. Its molecular weight was45kD and the isoelectric point (PI) was6.66. DPV gD gene is closer to Felid herpesvirus1(FeHV-1), Phocid herpesvirus1(PhoHV-1) and Canid herpesvirus1(CaHV-1) than others in genetic relationship, while gD is closer to Gallid herpesvirus1(GaHV-1). Signal peptide located at positions laa-22aa. While transmembrane helix at7aa-29aa and360aa-382aa. There are4N-linked glyeosylation sites,9O-linked glyeosylation sites and25Phosphorylation site in DPV gD. The main location site in cell is endoplasmic reticulum, about44.4%, and the others are cytoplasm, vacuolar, extracellular, including cell wall, mitochondrial and nuclear, about11.1%each.
2. Cloning, prokaryotic expression, purification and antibody preparation of Duck plague virus gD gene According to the Duck plague virus gD gene sequence found, Extracellular region of DPV gD gene about729bp was amplified and cloned into pGEM-T vector. After confirmed with restriction enzyme digestion and DNA sequencing. The gD gene fragment were sub cloned into Msc I and HindⅢ site of prokaryotic expression vector pET-32a (+).Recombinant plasmid pET-32a-gD was constructed successfully. Plasmid PET32a-gD was transformed into competent host bacteria E. coli BL21(DE3). After induction with IPTG, size of approximately30kD recombinant protein was detected by SDS-PAGE electrophoresis。Recombinant protein expressed as two forms: the soluble and insoluble inclusion body. The results of Western blotting demonstrated that the protein can specifically react with anti-DPV serum. The recombinant gD protein was purified by using Ni-NTA affinity chromatography. Immunized rabbit with purified gD extracellular region protein mixed with Freund's adjuvant three times, the ELISA method was used to measure the titers of anti-gD antibody in the serum. The titer is1:256000. Collected the immuned rabbit serum, anti-gD IgG was purified by using Protein A affinity chromatography,
3. Construction of Duck plague virus gD gene eukaryotic expression vector and its transient expression in COS-7cells According gene sequence of DPVgD, a pair of specific primers was designed, the whole gene sequence was amplified from the DPV genome. Insert the fragment between the cloning sites NheⅠ and HindⅢ of the eukaryotic expression vector pEGFP-N1and pcDNA3.1(+), then the Recombinant eukaryotic expression plasmid pEGFP-N1-gD and pcDNA3.1(+)-gD were successfully constructed. then the Recombinant eukaryotic expression plasmids were transfected in COS-7cells Liposome-mediated method, using laser scanning confocal microscope,Western-blotting, indirect immunofluorescence method were used to observe the expression of gD protein. The fluorescent protein punctate aggregated in cytoplasm and cell membrane of COS-7transfected plasmid pEGFP-Nl-gD, mainly located in the cell membrane. Data of Western-blotting showed molecular weight of the gD expressed in COS-7cells was about55KD, a little larger than molecular weight of gD predicted. The fluorescent protein punctate aggregated in cytoplasm,nuclease and cell membrane of COS-7transfected plasmid pcDNA3.1(+)-gD,mainly located in the cell membrane.
4.The transcription and expression analysis of duck plague virus gD and the subcellular localization of gD protein in the DPV-infected DEF cells
DEF cells prepared as conventional methods used. CHv strains inoculated whenthe cells grow as a monolayer. Samples were collected respectively after inoculated Oh (blank control group),2h,4h,6h,8h,12h,24h,48h,60h. and dealed with(1) extracting total cellular RNA,(2) extracting total cellular proteins,(3) fixing cell. Then used the sample for (1) detecting gD gene transcription phase by Fluorescent quantitative PCR,(2) detecting gD gene expression phase by Western-blotting,(3) detecting gD subcellular localization of gD protein by indirect immunofluorescence method. The results show that the gene has begun to transcript from2h after DPV infection,8h began to express. Transcription and expression increased rapidly after12h then continued to60h. The expression product of gD gene in the DEF cells appeared high abundance, molecular weight of mainly mature glycoprotein is55kD. the subcellular localization of gD protein in DEF cells is a dynamic process. localized in the cytoplasm from8h after DPV infection, after12h in nuclear membrane, after24h in cell membrane, after48h in nuclear membrane, the cell membrane and cytoplasm. Continued to60h, the location is irregular because of cell fusion and disintegration. The location feature just the same herpes virus glycoprotein, Confirmed the similarity of DPVgD function with other herpes virus gD function.
5. Establishment and Application of an Indirect Recombinant Glycoprotein D-based Enzyme-linked Immunosorbent Assays (ELISA) An indirect ELISA for detecting antibody of duck plague was developed using purified Recombinant gD as antigen. anti-DPV sera were used to carry ELISA.Data of chessboard titration showed that the fitful concentration of antigen is100ng per well (1μg/mL,100μL/well), and of serum, is1:80. Results of crossing test and interdiction test demonstrated specificity and sensitivity of the method. The right coating condition is in37℃1h, then4℃overnight. 5g/L gelatine PBST is the fitful block solution. The indirect gD-ELISA was specific and sensitive according to the detection of DHV, DHBV and GpV. Statistical data of90clinical samples showed that there was no difference between two detection systems: gD-ELISA and DPV-ELISA. The recombinant protein gD has high immunoreactivity. gD-based indirect ELISA method can be used in diagnosis of DPV in clinic.
6Development and application of a Polymerase Chain Reaction to Detect DPV gD gene According to the DPV gD gene fragment lack of signal peptide, a PCR assay was developed. A specific DNA fragment about1009bp was obtained from DNA of DPV CHv strain and DPV vaccine. There are no specific fragments were amplified from normal DEF,duck embryo allantoic fluid, duck hepatitis virus, duck hepatitis B virus and gosling plague virus., at the same time,some brands varies in length can be amplified from DNA of herpes virus HSV-1,HSV-2and BV, demonstrated that gD is Congeneric in herpes virus homology. The length of the amplified fragment is not the same as the gD gene expected, indicating the specificity of the PCR primers and methods. Susceptibility test results show that about lpg DPV DNA can be detected. Application of the PCR method of to detect, the right length and specific fragment can be amplified from DPV infected tissue liver, spleen, thymus, lymph nodes and Bursa of Fabricius.The positive bands were not detected in the normal tissues. Confirmed that the method can be used in clinical detection of DPV.
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