Prokaryotic Expression And Antigenicity Analysis Of Penton,Fiber And Hexon Of Egg Drop Syndrome Virus

Egg Drop Syndrome (EDS-76) was a virulent infectious disease caused by EDSV with the main characteristics of laying shelless or soft shelled eggs and decreasing egg laying, and its occurrence and epidemic in the world caused enormous economy lose to poultry industry.According to the nucleotide sequence of EDSV AV-127 strain from the Genbank, 8 pairs of oligonucleotide primer were designed by using Primer5.0. Using of the polymerase chain reaction(PCR),the target gene fragments were amplified from the genome of EDSV Jing 911 strain,which was amplified in the duck embryonated eggs, then directly were inserted into pMD18-T vector .The recombinant plasmid DNAs were used to transform the competent E.Coli TG1 ce1ls. After the positive colonies harboring the target gene fragments were indentified by the digestion of restriction endonucleases and PCR, they were sequenced. The results indicate that a gene fragment of EDSV Jing 911 strain, including 52/55k,â…¢a, Penton, pâ…¦, pâ…©, pâ…¥, Hexon, EP, 100k, pâ…§and Fiber genes, was obtained, and it was 15715bp.Comparison with AV-127, it indicated that there was high homology between EDSV Jing 911 strain and AV-127 strain at both nucleotide and amino acid level, above 99.00% for other genes except pâ…§and Fiber, and that Penton,Hexon,EP,Pâ…¥and Pâ…©were identical at the amino acid level. So, it was possibe that EDSV Jing 911 strain and AV-127 strain had the same genotype.Then, according to the Phylogenetic trees of Penton,Hexon and EP, it indicated that in the heredity relationship EDSV was closest to OAV287 and BAdV-4.According to the nucleotide sequence of obtained EDSV Jing 911 strain and the reference AV-127 strain, pairs of oligonucleotide primer with two restriction endonucleases sites were designed. Using them, the complete Penton gene,Fiber gene and Hexon gene were amplified by PCR.By sites of the restriction endonucleases EcoRI and Hindâ…¢, Penton-encoding gene, Fiber-encoding gene and Hexon-encoding gene were cloned into the expression vector pET-30a(+).The recombined plasmid named pET-30a-Penton, pET-30a-Fiber and pET-30a-Hexon were transformed into E.Coli. Rosetta and induced by IPTG. The SDS-PAGE analysis show that three expressed fusion proteins with molecular weights of 54.0ku, 69.7ku and 110.0ku. are obtained The Western-blotting, agar diffusion test and Dot-ELISA showed that those proteins have good antigenicity.In this study, part of the nucleotide sequence of EDSV AV-127 strain was cloned, which was a good foundation for the construction of EDSV vector. Three structural proteins are expressed in the procaryotic expression system, and the fusion protein Penton had good antigenicity.All of this is helpful for further studying proteins and establishing a new method of EDSV detection.