【作者】 马波；

【导师】 王君伟；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2008， 博士

【摘要】 鸭病毒肠炎(duck viral enteritis,DVE),又名鸭瘟(duck plague,DP),是由鸭肠炎病毒(duckEnteritis 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。其特征是流行广泛,传播迅速,发病率和死亡率高,给养鸭业造成巨大的经济损失。DEV具有疱疹病毒典型的形态和结构,因此划分为疱疹病毒科,其亚科、属、种分类地位尚未确定。大多数疱疹病毒有12种糖蛋白,gB是最保守的结构糖蛋白之一,在多数疱疹病毒中gB同源物由ul27基因编码。该糖蛋白是病毒复制所必须的,参与病毒吸附、融合穿入及病毒在细胞间的传播,能刺激机体产生中和抗体,介导细胞免疫,是重组疫苗和亚单位疫苗的候选抗原,也是哺乳动物疱疹病毒血清学诊断方法的候选抗原。目前还没有关于DEVul27基因及其编码产物的深入研究。本试验以DEV ul27基因为研究对象,采用PCR技术克隆了DEV C-KCE株ul27基因,并从该基因与其他疱疹病毒的同源性、编码蛋白的结构及抗原性等方面进行了较为详尽的生物信息学分析,在此基础上利用原核表达载体pET-32a在Rosetta(DE3)PlysS宿主菌中分别表达了该基因9个抗原片段,以鸭肠炎病毒高免血清为第一抗体,通过Western blot确定了UL27蛋白的B细胞线性抗原表位优势区,以制备的鼠源抗原肽特异性抗血清进行蚀斑减数试验确定了中和抗原表位的优势区,利用体外淋巴细胞转化试验揭示了外周血抗原特异性T淋巴细胞对DEV的回忆应答规律,通过统计分析初步确定了T淋巴细胞的抗原表位。结果表明:DEV C-KCE株ul27基因完整ORF为3003bp,编码1000个氨基酸,同源性分析表明该基因为α疱疹病毒gB同源物,其遗传进化分析提示DEV与α疱疹病毒马立克氏病毒属亲缘关系最近;ul27基因编码产物具有典型跨膜蛋白结构,推测其信号肽序列位于N端前105个氨基酸以内,胞外区从信号肽裂解位点至832位氨基酸,跨膜区为833～883位氨基酸,胞内区为884～1000位氨基酸;蛋白胞外区存在8个潜在的N糖基化位点,10个保守的半胱氨酸,具有潜在的翻译修饰后的酶解位点,HSPGs结合位点、PACS-1结合基序,符合α疱疹病毒gB的结构特征和功能特征;Western blot结果表明UL27-2、UL27-5、UL27-7、UL27-9抗原肽存在B细胞线性表位,其中UL27-5抗原性最强,UL27-7刺激机体产生的抗体持续时间最长;蚀斑减数试验确定UL27-5、UL27-6、UL27-7、UL7-8、UL27-9、UL27-10抗原肽的抗血清具有中和活性,其中UL27-7、UL27-8是中和表位优势区;体外淋巴细胞转化试验表明,9段抗原肽及其不同组合均具有诱导外周血淋巴细胞发生转化的能力,外周血抗原特异性淋巴细胞在回忆应答中对DEV及UL27抗原肽的反应能力呈现缓慢增强,达到高峰后又快速回落的趋势。经统计分析初步确定DEV UL27 T细胞表位优势区是UL27-6,候选优势区是UL27-4、UL27-10。本研究为明确DEV的分类地位,阐明DEV gB的功能,了解其在机体免疫过程中发挥的作用及其在实践中应用的可能性提供了理论依据和物质基础。更多还原

【Abstract】 Duck viral enteritis(DVE) named duck plague(DP) also,its pathogen is duck enteritis virus (DEV),which is acute,heat and haemorrhagic contagious viral disease in infected birds of the order Anseriformes(ducks,geese and swans).DEV is characterized by epidemic broadly,spread rapidly with high mortality and morbidity,it has been responsible for considerable economic loss to the commercial duck industry.DEV is a member of the herpesviridae according to it has the classical morphology and structure of herpesivirus,but its definite taxonomic classification was unknown.Glycoprotein play the very important role during the course of pathopoiesia.There are 12 glycoprotein in herpesvirus, and gB homologue has been identified in every herpesvirus studied to date,which is the most highly conserved herpesvirus structural glycoprotein.It was encoded by ul27 gene usually. Glycoprotein B is required for herpesvirus infectivity and is involved in virus adsorption, membrane fusion,penetration and cell-cell spread.In addition,gB elicits neutralizing antibodies, cell-mediated immune responses,and has been shown to be a candidate antigen for recombinant, subunit vaccines,serology diagnostic assays in mammalian.There is very limited in study about ul27 genen and UL27 protein of DEV.The DEV ul27gene is the interest of this study,and was cloned frome the C-KCE strain by PCR.The homology of ul27 genen and UL27 protein with other herpesvirus,the structure and the antigenicity of UL27 protein were analysised by bioinformatics.Nine truncated antigenic peptides were expressed in Escherichia coli Rosetta(DE3)PlysS by prokaryotic expression vector pET-32a.The immunodomiannt region of B cell antigen epitope were identified by western blot with the duck serum against DEV.And the immunodomiannt region of neutralization epitope were identified by plaque inhibition test with mice serum against the nine truncated antigenic peptides The rule of the peripheral blood lymphocytes response to UL27 after infection DEV was revealed by LTT.In addition the T cell epitopes were defined primarily by statistics analysis.The results of study as followed:The ORF of ul27 gene from DEV C-KCE strain was consist of 3003 bsae pairs and it enconded a primary translation product of 1000 amino acids.The homology analysis with other herpesvirus indicated ul27 gene was the gB homologue,and Phylogenetic analysis to illustrate the evolutionary relationship of DEV was close to subfamily Alphaherpesvirinae and Mardivirus genus.The DEV UL27 possessed classical structure of transmembane glycoprotein,the putative signal sequence located in the 105 aa of N terminus,the extracellular domain of UL27 from the cleavage site of signal peptide to 832 amino acid long,and transmembane domain from 833 to 884 amino acid long,the cytoplasmic domain from 884 to 1000 amino acid.Bioinformatic analysis of UL27 indicated there were eight potential N-linked glycosylation sites,10 Cys conserved residues,HSPGs binding sites,PACS-1 binding motif in extraceilular domain,all this structure consisted with the structure and function characters of herpesvirus gB.The B cell epitopes were identified in ul27-2、ul27-5、ul27-7、ul27-9 by Western blot,and the ul27-5 was the strongest antigenicity epitope,the antibody against ul27-7 lasted longest.The antibody against ul27-5、ul27-6、ul27-7、ul27-8、ul27-9、ul27-10 derived from the mice have the ability of neutralized DEV by plaque inhibition test,and antibody against ul27-7、ul27-8 show off the greater neutralizing capacity,so ul27-7 and ul27-8 were thought to be region of the immunodomiannt neutralization epitope excist.The nine truncated antigenic peptides displayed the ability to induce the peripheral blood lymphocytes transformation in vitro by lymphocyte transformation test(LTT).The peripheral blood antigen specific lymphocytes presented some tendency in response to the DEV,nine truncated antigenic peptides and their different combination, it was the response ability of peripheral blood antigen specific lymphocytes strengthened slowly then declined rapidly after reaching the peak in recall response.The T cell immunodomiannt epitopes of UL27 located in ul27-6 by statistic analysis primarily,and candidate prevalent epitopes region are ul27-4、ul27-10.The results of this study were very significance for illustration the classify of DEV, understanding the function of DEV gB,revealing the role in immune response of DEV gB,and the possibility to be candidate antigen in vaccine or diagnostic assays.更多还原