Molecular Characterization And Identification Of Partially Immunodomiant Epitope Regions Of Duck Enteritis Virus Ul27 Gene

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.