Screening And Identification The Dominant Epitopes On Penton Of EDSV

Egg Drop Syndrome (EDS-76), also known as Duck aviadenovirus1(DAV-1), is an infections disease caused by EDSV, which is characterized by laying of thin shell eggs or non-shell eggs.This virus was found frist time in1976, subsequent, it was epidemic in occident and it was found in many countries on the whole world, caused considerable economic losses to the poultry industry. Penton is one of the important structural protein of EDSV; Penton has good immunogenicity and antigenicity, it has neutralization activity, The Penton contains all necessary components for viral attachment and entry into the host cell. In this study, the recombinant plasmid pET-30a-Penton was used as the templates for PCR, by the means of overlapping peptides expressed in E.coli combination with Western blot to identify the dominant epitopes region of Penton of EDSV. At the same time, the dominant epitope regions lay a foundation for the research of epitope vaccin, diagnostic reagents.In order to identify the dominant epitope regions on Penton of EDSV, we prepared the anti-EDSV positive sera of ducks, and designed a set of partially overlapping fragments of84-97aa which overlay the extracellular region of Penton. Overlapping part is15-20amino acids, each protein was synthesized6pairs of primers. Then PCR products were cloned into the expression vector pET-30a and then these fragments confirmed by sequencing. Each fragment was expressed in Escherichia coli Rosetta(DE3)pLysS. The PA (1-87aa) was detected by Western blot with positive serum against EDSV.4overlapping fragments which overlay1to87aa, these fragmentis are29-33aa, Overlapping part is12-13amino acids, we screend the epitopes secondly. The results of Western blot showed that PA-1(1-33aa), PA-2(21-49aa) and PA-3(37-68aa) have good antigenicity. With the purpose of identification the dominant epitope regions on Penton of EDSV, we screend the epitopes thirdly. The results of Western blot showed that PA-H-1(1-17aa)、PA-H-4(22-39aa)、PA-H-5(30-47aa). PA-H-6(38-53aa)and PA-H-8(51-68aa)have good antigenicity. At the basic, three pairs of oligonucleotides overlaying PA-H-1, three pairs of oligonucleotides overlaying PA-H-8were designed respectively. Each pairs of oligonucleotides was cloned into the expression vector pET-32a. The expression proteins were purified to analyze the antigenicity by Western blot. PA-H-1.、PA-H-1.2、PA-H-8.1were incubated with the positive serum of EDSV. For the purpose of decreasing the numbers of amino acid, we screened the epitopes sequentially. The result of the fifth and sixth identification of the epitopes showed that the region of M1ESFVPPPR9、 V5PPPRVF11and H53SDYFT58are the domain epitope region of Penton of EDSV.