The plasmid PE which harbored the whole hexon-encoding gene of egg drop syndrome virus(EDSV) was identified and the hexon protein gene was obtained from it by restriction endonucleases.The gene was then directionally inserted into the PphI/KpnI sites of pQE32 and fused with the 6×His gene.This recombinant plasmid was transformed into E.coli M15 for expression and induced with IPTG.It was demonstrated by SDS-PAGE and Western blot that one expressed protein,110kD in size,could be specifically recognized by polyclonal anti-EDSV serum.The objective product was principally soluble and accounted for 21% of the total cellular proteins.The protein was used to detect the existence of antiEDSV IgG in 41 clinical chicken sera by agar diffusion test,and the result was in accordance with that when EDSV was used as antigen.These results showed that the expressed hexon recombinant protein can be used as diagnostic antigen of EDSV.
According to the nucleotide sequence of egg drop syndrome virus(EDSV)AV-127 strain (a foreign standard isolate)from the GenBank,a pair of oligonucleotide primers was designed and synthesizedWith use of polymerase chain reaction(PCR),the hexon-encoding gene fragment was amplified from the genome of EDSV SG9301 strain that was isolated from Sichuan Province of China and the amplified fragment was directly inserted into pMD-T vectorThe recombinant plasmid harboring the hexon-encoding gene was identified by digestion of restriction endonucleases and PCRThen,a positive clone was sequencedThe 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 (an attenuated strain)Comparison with AV-127 strain indicated that there was 9985% homology at the nucleotide levelThe homology of the deduced amino acid sequence with AV-127 strain and AAv-2 strain was over 98%,which indicated that the hexon protein was conservativeBut comparatively,the homology of the hexon protein of SG9301 strain with AV-127 strain was higher(9978%),the homology of the hexon protein of AAV-2 strain with AV-127 strain and SG9301 strain were 9868% and 9846% respectivelyComparison of nucleotides and amino acids suggested that the biological character is consistent with the homologies of nucleotide and amino acid of the hexon(Namely,the homology of the hexon of virulence strain with virulence strain was higher than that of virulence strain with attenuated strain),the hexon was one of the most conserved viral proteins among EDSV
