Establishment Of A Hexon-based Indirect ELISA For The Detection Of Antibody Against Fowl Adenovirus

Fowl adenovirus(FAdV)are non-enveloped double stranded DNA-virus,which belong to the family Adenovirus,Aviadenovirus.The fowl adenovirus have been grouped into 5 species(FAdV-A to FAdV-E)with 12 serotypes(FAdV-1 to 8a and 8b to 11)based on the restriction enzyme digest pattern and serum cross-neutralization test.FAdV is susceptible to all day-old chickens with a worldwide distribution,normally,chickens infected with FAdV was characterized as subclinical symptoms,while acute infections were responsible for inclusion body hepatitis(IBH),hydropericardium syndrome(HPS)and gizzard erosion.Since 2013,the clinical cases of IBH and HPS have been increasing in most domestic chickens,with mortality approaching 80%,resulting in considerable economic losses.However,effective vaccines and medicines is not available as well as reliable serological detection methods.In this study,conserved hexon gene was selected and cloned into pGEX-6P-1,pET-32a and pCold I vector for prokaryotic expression,then recombinant protein was purified and used for the establishment of an indirect ELISA for the detection of antibody against fowl adenovirus.1.Prokaryotic expression and purification of 4 epitopes from hexonIn this study,4 conserved epitopes of hexon were selected and cloned into prokaryotic expression vector pGEX-6P-1 after alignment with 5 species of fowl adenovirus,with the positive recombinant plasmid named GST-hexonl,GST-hexon2,GST-hexon3 and GST-hexon4 respectively.The recombination was then induced with ImM IPTG,followed with SDS-PAGE and Western blot,the results revealed that recombinant protein was mostly in the pellet,and only GST-hexonl could be recognized by the FAdV positive serum.Then hexon 1 was cloned into pET-32a and pCold I for soluble trial,the SDS-PAGE analysis showed that recombinant protein was still in the pellet,therefore,recombinant protein was purified from the the pellet with the final concentration of 0.5mg/mL.The results of SDS-PAGE and Western blot revealed that recombinant protein purified well and have good reaction with positive serum,which provided materials for establishment of the indirect ELISA method for detection of antibody against fowl adenovirus.2.Establishment of an indirect ELISA for detection of antibody against fowl adenovirusThe recombinant protein was used for coating antigen of indirect ELISA after purified,the optimal conditions was determined as below,the optimal coating concentration was 6.2?g/mL,1:400 for the chicken serum and 60 minutes for the incubation time;the optimal dilution of emzyme-labeled secondary antibody was 1:50000,the incubation time was 45min,the optimal developing time was 10min.The cutoff of the ELISA was OD(?)0.173.The specificity analysis showed that the established ELISA have no response with the positive serum of Newcastle disease virus,Avian leukosis virus.Marek's disease virus,Infectious bronchitis,Infectious bursal disease virus,Avian influenza virus and SPF chicken serum,with the OD value lower than 0.1548.The results of clinical detection showed that the ELISA could be used for surveillance of FAdV antibody level,consider all the detection results,the ELISA method established in this study have some value for detection of antibody against fowl adenovirus,as well as immune surveillance.