Cloning And Expression Of Hexon Gene From Egg Drop Syndrome Virus Detected In HeBei And Analysis Of Immunogenicity

A strain of egg drop syndrome virus (EDSV) was detected from chicks which suffered from egg drop syndrome (EDS) in HeBei and provisionally named as HeBei strain (EDSV-HB). A full length DNA clone for hexon gene of the virus was constructed, and complete nucleotide sequence of the gene was determined. And then the expression of the hexon-encoding gene and the immunogenic of hexon protein were studied in this paper.According to the nucleotide sequence of egg drop syndrome virus AV-127 strain from the GeneBank, a pair of primers specifically to amplify DNA of hexon gene of EDSV-HB strain was designed using Premer5.0 software. PCR was performed for viral DNA extracted from the genome of EDSV-HB strain using these primers and the amplified fragment was inserted into pBS-T vector. These DNAs were transformed into competent Escherichia Coli TOP10 cells. Then the recombinant plasmid was identified by digestion of restriction endonucleases and PCR methods. The complete nucleotide sequence of the gene was determined. The result showed that the recombinant plasmid contained the complete sequence of the hexon-encoding gene and the hexon-encoding gene was 2733 base pair long which encoded 910 amino acids, identical with AV-127 strain and AAV-2 strain.The nucleotide and deduced amino acid sequences were analyzed by computer program (DNAstar) using available data from data base (GeneBank). Nucleotide homology and phylogenetic analysis were undertaken among HeBei strain and other strains of adenovirus. Comparison with AV-127 strain indicated that there was 99.5% homology at the amino acids level. Comparison with AAV-2 strain showed that there was 98.4% homology at the amino acid level, which indicated that the hexon protein was conservative. Comparison of nucleotide sequence and deduced amino acid sequences of hexon gene among other adenovirus revealed the homology was very lowly, ranged from 24.0% to 59.8%.The cloned genomic DNA was subcloned into prokaryotic expression vector PET-32a-(+). The recombinant expressing plasmid was identified by restriction enzymes and sequence analysis. The results indicated that the fragment was correctly inserted into the PET-32a. One recombinant plasmid named pET-H was constructed which expressed a 110KDa protein in E.coli BL21, The result of SDS-PAGE and Western blotting indicate the expressed product could react with EDSV positive serum.After the conditions were optimized, the recombinant protein was expressed on a larger scale. Chickens were immuned with purified recombinant protein, and all "the chickens produced relatively supernal antibody. The results showed that the recombinant protein expressed in E.coli still maintained some antigenicity of EDSV antigenes. It provided the basis for further studying on recombinant vaccine against EDS.