Cloning And Expression Of The Hexon Gene Of Avian Adenovirus And Development Of Elisa Based On Antigen Of Recombinant Proteins And Quantitative Real-Time PCR Method For Detecting Avian Adenovirus

Fowl adenoviruses belong to the Adenovifidae and the Aviadenovirus.The fowl adenoviruses are subdivided into three groups,which the groupⅠcomprised of 12 serotypes of avian adenoviruses and that shared a common group antigen.The fowl adenoviruses have been associated with inclusion chicken embryo lethal orphan(CELO)virus and body hepatitis(IBH)virus and hydropericardium syndrome(HPS)virus.All the poultry could be infected fowl adenoviruses.The fowl adenovirus infections exhibit liver damage,aplastic anemia,hemorrhage,minor respiratory disorders and dropped egg production.A pair of specific primers were designed and synthesized according to hexon gene sequences of CELO from GenBank,which a antigenicity gene of hexon PCR amplify from CELO strain.The analysis using DNAstar proteins oftware showed that the deduced amino acid sequence from the CELO hexon gene had good hydrophobicity and antigenicity.The product ligated with PGEX-4T-1 Prokaryotic expression Vector,and transformed into Escherichia DH5a competent cell.The recombination was induced with 0.2mmol/L of IPTG.The results of SDS-PAGE analysis revealed that the molecular weight of the expressed product was 72.24 kD.Western-blotting analysis showed that the Prokaryotic expression hexon recombinant protein could be specifically recognized by the positive sera of avian infected with FAV.In additional,antigenicity hexon protein was Prokaryotic expressed.The inclusion body of protein was purified by urea and Triton X-100.The concentration of the protein was 30μg/mL.The purity of urea-purified fusion protein was 85%.Achieves a higher density and purity,which showed that the experimental exploration could effective purify the recombinant protein.The protein was used for coating antigen of indirect enzyme-linked immunosorbent assay(ELISA)after purified.The optimal conditions was determined.It was shown that the optimal of the coating concentration of the protein was 10μg/mL,the optimal dilution of serum sample was 1:200,the optimal dilution of enzyme-labeled goat-chicken IgG was 1:5000.The recombinant protein showed no reaction with antiserum to NDV,AIV, EDSV,ARV.It indicated the indirect ELISA had good specifity for detetion of FAV antibody in serum.Our research had developmented of ELISA method,and the method detected the serums which were infected with FAV.The result showed that ELISA was the sensitivity in FAV antibody detection.The last,according to the FAV hexon gene sequences available in GenBank, a pair of primers was designed for establish a SYBR GreenⅠquantitative real-time PCR method for Avain Adenovirus GroupⅠ.To establish the standard curve,the DNA plasmid served as a standard.The analysis of specificity and sensitivity and repeatability was also carried out.The results showed the linear range of Cp value was with a good correlation efficient.None of the negative control samples showed false-positive reactions when done in duplicates.The detection limit of the assay was 100 copies per reaction.The results showed that the present real time PCR assay was of high specificity and sensitivity.It took only five hours from viral DNA extraction to complete the detection.The assay may therefore offer a rapid,economic and reliable method for detecting of the Avain Adenovirus GroupⅠpathogen.