Analysis Of Advantage Region Of Epitopes On Hexon Of EDSV

Egg Drop Syndrome (EDS-76) is an infectious disease caused by EDSV, which is characterized by laying of non-shell eggs, soft shell eggs or thin shell eggs. The occurrence and prevalence of EDSV in the world brought about an enormous economy lose to poultry industry. There needs to be an accurate, convenient diagnostic method for diagnosis. Meanwhile, it needs analyze the viral antigen to select the suitable peptides as subunits vaccine for disease prevention. In this study, the recombinant plasmid pMD18-T-Hexon was used as the templates for PCR in next procedure.In order to map the antigenic epitopes on Hexon protein of EDSV, a set of partially overlapping fragments of 150-170aa which overlay the whole protein were designed. Overlapping part is 15-20 amino acids. 6 pairs of primers were synthesized. The PCR products were cloned into expression vector pET-30a and then these fragments were confirmed by sequencing. Each fragment was expressed in E.coli. and purified by elution from sodium dodecyl sulphate(SDS)polyacrylamide gels. The antigenicity of these fused protein were determined by western blot with viral infected sera. Linear immunodominant B-cell epitopes were primarily found in three fragments: HB(153~306), HD(443~618), HE(598~769). 6 overlapping fragments which overlay 153 to 306aa and 13 overlapping fragments which overlay 443 to 769aa were further expressed in E. coli as well. The further results of mapping Hexon protein immunodominant epitopes showed that the fragments HB-3(193~229), HB-4(216~255), HDE-3(491~530), HDE-5(539~577), HDE-9(633~671)and HDE-10(657~696) were positive. The short peptide HB-34 was dissected into five overlapping fragments, HDE-3 was dissected into three overlapping fragments, HDE-5 was dissected into three overlapping fragments, HDE-910 was dissected into five overlapping fragments each with overlapping of five amino acids. A pair of oligonucleotide fragments was synthesized for each short peptide. After phosphorylation and annealing, the products were cloned into expression vector pET-32a. The further results of mapping Hexon antigenic protein immunodominant epitopes showed that the eptitopes were in fragments: 204~219, 227~243, 491~506, 550~565, 633~648 and 656~672.Thus structural protein Hexon B-cell epitopes are located on the 204~219, 227~243, 491~506, 550~565, 633~648 and 656~672. The distribution of antigenis epitope on Hexon protein of EDSV can be analyzed on basis of results above. Meanwhile, the antigenic epitopes on Hexon of EDSV were blasted with amino acid on Hexon of other adenovirus by DNAMAN. The results from the study may also be useful for diagnosis of EDSV infecton based on antigenic epitope to develop a new strategy for vaccine design.