The Prokaryotic Expression Of Hexon Gene Of FAdV-4 And Its Application For Immunoassay

Fowl Adenovirus(FAdV)belongs to the family Adenovirus,Aviadenovirus.According to the difference in antigenicity,FAdV can be subdivided into three groups: group I,group II,group III.Fowl Adenovirus group I has congenerous antigen and twelve serotypes.FAdV is susceptible to all day-old Chickens with 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.Fowl Adenovirus serotype 4 group I(FAdV-4)could cause chickens HPS,which is characterized by sudden death of 3 to 5 weeks old broiler chickens and breeders,the pericardium accumulate yellow water samples or gelatin,hepatomegaly,fragile color,renal enlargement,bleeding.Since 2013,the clinical cases of HPS caused by FAdV-4 have been increasing in most domestic chickens in China,resulting in considerable economic losses for the poultry industry.However,establishing a rapid and accurate FAdV serological assay is of great significance.The hexon protein encoded by the Hexon gene of FAdV-4 is the major structural protein of the virus and contains major genus and subgenus-specific antigenic determinants,which gene is the major protective antigen gene.In this study,the Hexon protein of FAdV-4 was selected to predict the region with strong immunogenicity,and primers were designed to amplify the hexon gene partial sequence and cloned into the prokaryotic expression vector pET-32a(+),with the positive plasmid named pET-hexon.The positive plasmid was transformed into Escherichia coli BL21 by kanamycin containing LB medium.The recombination was then induced with IPTG,followed with SDS-PAGE,which analyzed the solubility of the expressed protein.The optimal induction conditions were determined by varying induction time,induction temperature,and IPTG concentration,and verified by western-blot.The results showed that the Hexon protein of FAdV-4 was successfully expressed,the induction temperature was 30°C,the IPTGconcentration was 1mM,and the induction time was 4h.The recombinant protein was mainly expressed in the form of inclusion bodies.Western-blot analysis showed that the recombinant Hexon protein could bind to the mouse anti-His monoclonal antibody and chicken FAdV-4positive serum and showed a specific band,indicating that the protein has better reactogenicity.Hexon recombinant protein expression provides material for the IFA detection method and ELISA detection method for the FAdV-4.The prepared Hexon recombinant protein was immunized into the mice through the neck subcutaneously,and 100?g of each was immunized three times.Then,the serum of the immunized mice was collected to prepare a mouse anti-Hexon multi-antibody antibody.Using the prepared polyclonal antibody as the primary antibody and FITC-labeled goat anti-mouse lgG as the secondary antibody,primary embryonic kidney cells(CEK)infected with FAdV-4were detected by indirect immunofluorescence assay(IFA).The results showed that the polyclonal antibody specifically recognized FAdV-4 infected CEK and the nucleus of infected cells was stained green.The preparation of mouse anti-Hexon multi-antibody antibody has high practical value for the etiological diagnosis of FAdV-4.The recombinant protein was used for coating antigen of indirect ELISA after purified,the optimal conditions was determined as below,the optimal coating concentration was4?g/mL,1:200 for the chicken serum and 60 minutes for the incubation time;the optimal dilution of enzyme-labeled secondary antibody was 1:2500,the incubation time was 45 min,the optimal developing time was 15 min.This study showed that the established ELISA have no response with the positive serum of Newcastle disease Virus,Avian influenza virus,Avian leukosis virus.Marek's disease virus,Reticuloendotheliosis virus and SPF chicken serum,with the OD value lower than 0.312,only reacted with positive serum of anti-FAdV-4.The results of clinical detection showed that the ELISA could be used for surveillance of FAdV antibody level.