【作者】 李慧昕；

【导师】 孔宪刚；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2007， 博士

【摘要】 鸭病毒性肠炎(Duck viral enteritis，DVE)，又名鸭瘟(Duck plague，DP)，是由鸭肠炎病毒(Duck enteritis virus，DEV)引起的鸭、鹅及多种雁形目禽类的一种急性、热性、败血性传染病，曾在多个国家和地区发生流行，给养鸭业造成巨大的经济损失。DEV是疱疹病毒科成员，第八次病毒分类报告将其划分为疱疹病毒科未分类病毒。由于DEV分子生物学研究相对滞后，其基因组组成和结构未知，在很大程度上限制了DEV致病机制及该病在防控方面的研究。本研究根据本实验室提交至GenBank上的DEV clone-03部分基因组序列，选择在疱疹病毒中较为保守的囊膜蛋白UL27、主要衣壳蛋白UL19以及在绝大多数疱疹病毒成员基因组排列中保守的基因簇(UL22-TK-UL24)进行测序。选取同源性较高的α-疱疹病毒同源蛋白作为参考毒株，应用DNAStar软件对5个基因进行序列比对，分析各个基因编码蛋白的保守区。结果表明，DEV clone-03 UL19和UL27保守，UL22次之，TK具有胸苷激酶特征性结构域：ATP结合结构域和核苷结合结构域。与HSV-1相似，UL24也存在5个保守位点。UL22和UL27编码的蛋白具有跨膜糖蛋白典型特征，UL22具有7个糖基化位点，6个跨膜区，在N末端1～21位氨基酸为信号肽序列，符合疱疹病毒gH糖蛋白的特点；UL27有9个潜在的糖基化位点，3个跨膜区，符合疱疹病毒gB糖蛋白的特点。此外，根据5个基因编码氨基酸序列进行系统发育进化分析，发现DEV clone-03与马立克病毒属亲缘关系较近，但又形成独立分支。根据这5个基因比较结果，建议将鸭肠炎病毒归类为疱疹病毒科、α-疱疹病毒亚科。为了鉴定DEV clone-03结构基因，将DEV clone-03 UL19基因分成UL19-N、UL19-M、UL19-C三段进行原核表达并制备抗血清。同时表达UL27基因中段UL27-M和UL6基因的UL6-1和UL6-2二段，应用Western blot和间接免疫荧光方法检测6个重组蛋白的单因子血清与DEV clone-03全病毒的反应性。结果表明UL19-N、UL19-M和UL19-C三段蛋白制备的抗血清均能够与全病毒发生特异性反应，在大约150kDa处检测到特异性蛋白条带，大小与DNAStar软件预测的DEVclone-03 UL19蛋白大小一致，说明DEV clone-03 UL19是病毒的结构蛋白，大小约为150kDa，结合与其他疱疹病毒同源蛋白共有的保守区，初步认为DEV clone-03 UL19可能是病毒的衣壳蛋白。应用Western blot方法检测UL27，未能够在DEV clone-03中检测到预期大小的蛋白条带，但是，应用间接免疫荧光方法检测，UL27-M抗血清能够与全病毒发生抗原抗体反应，说明DEVclone-03 UL27是病毒的组成成分，结合与其他疱疹病毒同源蛋白共有的保守区，以及UL27具有跨膜糖蛋白特征，推测DEV clone-03 UL27可能是病毒的囊膜糖蛋白。应用UL6-2蛋白抗血清检测DEV全病毒，在大约88kDa处检测到特异性蛋白条带，与DNAStar软件预测DEV clone-03 UL6蛋白大小一致，结合其他研究报道，初步认为DEV clone-03 UL6是病毒的结构蛋白。应用DNAStar软件对上述检测抗原性较好的DEV clone-03 UL19蛋白从蛋白的亲水性、抗原性以及表面可及性进行表位预测，三段蛋白均存在抗原表位优势区，因此选取UL19-C段蛋白制备单克隆抗体，拟对UL19抗原表位进行初步定位。将UL19-C蛋白作为抗原免疫6-8周龄的BALB／c小鼠，制备单克隆抗体，经过3次克隆，分别采用间接ELISA、Western blot和间接免疫荧光方法筛选，最后得到4株(185、3A1、4F9、6F6)稳定分泌抗体的杂交瘤细胞株。将DEV clone-03 UL19-C分成两段进行表达，N端命名为UL19-C1、C端命名为UL19-C2，二者有16个氨基酸的重叠。Western blot检测，4株单克隆抗体均能够识别UL19-C1蛋白，而不能够与UL19-C2发生特异性反应，说明获得的4株单克隆抗体都是针对UL19-C1蛋白的，即在DEV clone-03 UL19蛋白C端921～1162位氨基酸的片段内有能够被4株单克隆抗体识别的抗原表位。更多还原

【Abstract】 Duck viral enteritis (DVE), also known as duck plague, is an acute, heat, haemorrhagic and lethal disease. The disease is caused by duck enteritis virus (DEV), which was epidemic in many countries and regions and caused economic losses. DEV is a member of of family Herpesviridae. According to the report of Eighth International Committee on Taxonomy of Virus (ICTV), DEV was assigned as member of family Herpesviridae, unclassified virus. The genomic structure of DEV was still unknown due to the molecular biology of the virus lagging behind other herpesvirus, which limited the research on pathogenesis of the virus and control of the disease.According to the genomic sequences of DEV clone-03 submitted in GenBank, we selected the conserved genes of glycoprotein UL27, major capsid protein UL19 and the conserved gene cluster of UL22-TK-UL24 as targeted genes for cloning. Homologues of alphaherpesviruses were used as reference strains to analyze the conserved motifs of five proteins. Multiple alignment showed that UL19 and UL27 were more conserved, however, UL22 showed week level homology. The TK possessed the signature motifs of thymidine kinase: one is ATP binding mogif and the other is nucleoside binding motif. Similar to HSV-1, DEV clone-03 UL24 also contained 5 conserved sites. Protein UL22 and UL27 were typical transmembran glycoprotein, seven N-linked glycosylation sites and six transmemebrane regions were predicted in UL22 protein, and nine N-linke glycosylation sites and three transmemebrane regions existed in UL27 protein. In addition, UL22 protein contained signal peptide sequence, which located at the 1～20 amino acids. Phylogenetic analysis on the five proteins showed that DEV clone-03 had closer relationship with genus Mardivirus but showing a far relationship with Iltovirus. From the tree view of the five proteins, although all five proteins showed closely relationship with genus Mardivrus, each protein formed separate branch. We suggest that DEV should be classified into family Herpesviridae, subfamily Alphaherpesvirinae.To identify the structural genes of DEV clone-03, the UL19, UL27 and UL6 were cloned and expressed in prokaryotic system and the proteins were used to immunize the rabbit to acquire antiserum. Western blot and indirect immunity fluorenscence methods were used to detect the reaction of antiserum to DEV. The result showed that the antiserum of UL19-N, UL19-M and UL19-C could react with DEV and recognized a specific band with molecular weight of approximately 150kDa. The size of specific protein band was consistent with the prediction. Together with the information that DEV UL19 possessed conserved region with the homologues of alphaherpesviruses, we considered UL19 as a structural protein of DEV clone-03. UL19 may function as capsid protein. The UL27 protein could not be detected by Western blot but could be detected by indirected immuno fluorenscence. The result showed that antiserum of UL27-M could react with DEV. We predicted that UL27 was composition of DEV clone-03. We presumed that UL27 may be glycoprotein of DEV because UL27 contained conserved region that existed in other alphaherpesviruses and had the proterties of transmembrane glycoprotein. The antiserum of UL6 could react with DEV clone-03, and a specific protein band with a molecular weight of 88kDa was detected, which was consistent with our prediction. We thought UL6 as a structure protein of DEV clone-03.The antigenic epitopes of DEV UL19 were predicted from the aspect of hydrophilicity, antigenic and surface probability plot by using DNAStar software. The result showed that the amino fragment, middle fragment and carboxyl fragment possessed antigenetic epitopes, so we randomly select the UL19-C fragment to identify antigenic epitopes of DEV clone-03 UL19. The UL19-C protein was taken as antigen to prepare monoclonal antibody. After three times clone, we acquired 4 strains (1B5, 3A1, 4F9, 6F6) stable hybridoma cells. DEV UL19-C was truncated into two overlapped fragment to express in prokaryotic system, we named the amino terminal as UL19-C1 and the carboxyl terminal as UL19-C2. Western blot detection showed that 4 monoclonal antibody strains could not recognize UL19-C2 but UL19-C1. The 4 monoclonal antibody strains were specific for epitopes of UL19-C1. There were antigenic epitopes in 921～1162 amino acid of DEV clone-03 UL19 carboxyl terminal fragment, which could be recognized specifically by 4 monoclonal antibody strains.更多还原