Expression Of Fiber Protein Of Serotype 4 Fowl Adenovirus In Baculovirus And Its Application In Antibody Detection

Fowl adenovirus（FAdV）belonging to the Aviadenovirus genus of the family Adenoviridae,is classified into 5 species（FAdV-A-E）with 12 serotypes（1-7,8a,8b,9-11）.Since 2015,the hepatitis-hydropericardium syndrome（HHS）induced by FAdV-4 has been outbreak in most intensive farming areas in China and caused mortality of up to 80%,which restricts the sustainable development of poultry industry in China severely.Given that the inactivated FAdV-4 vaccine has been licensed to control the FAdV-4 in China,an efficient and specific serological diagnostic tool for monitoring the immunological state of the vaccination or diagnosing the infection of FAdV-4 is urgently needed.Among the proteins encoded by FAdV,the Fiber not only carries the important virus-neutralizing and serotype-specific epitopes,but also plays key roles in infection and pathogenesis.In this study,we intended to target Fiber protein of FAdV-4（including Fiber-1 and Fiber-2）for developing an efficient ELISA method for specific detection of antibody against the current circulating FAdV-4 by using the Fiber protein expressed in baculovirus expression system.1.Expression of Fiber protein of FAdV-4 in baculovirus and its purificationIn this study,fiber-1 and fiber-2 gene of FAdV-4（strain SD15）were first cloned into the pFast-Bac-HTA baculovirus vector,respectively.The constructed baculovirus vector were then transfected into competent cells to obtain the shuttle vector,and followed by transfecting Sf9 cells to construct recombinant baculoviruses rBv-fiber-1 and rBv-fiber-2 expressing Fiber-1 and Fiber-2 protein,respectively.The indirect immunofluorescent assay（IFA）identification revealed that the recombinant Fiber-1 and Fiber-2 protein expressed in infected cells could be recognized by FAdV-4 specific antibodies efficiently.The result of Western blot demonstrated that the recombinant Fiber-1 and Fiber-2 protein were sufficiently soluble in lysate supernatant with approximately 55 kDa and 70 kDa size,respectively.The recombinant protein was then purified by Ni+column,and the final concentration of Fiber-1 and Fiber-2 proteins were 0.3 and 0.4 mg/mL,respectively.SDS-PAGE and Western blot analysis showed that they had good purity and reactivity.The expression and purification of the recombinant Fiber-1 and Fiber-2 protein provided biological material for further development of FAdV-4 antibody detection method.2.Development of indirect ELISA for detection of FAdV-4 antibodyThe recombinant Fiber-1 and Fiber-2 protein were used as FAdV-4 specific coating antigen to develop Fiber-1 ELIS A and Fiber-2_ELISA,respectively.Based on checkboard titrations,we determined optimal condition of all required ELISA components including antigen coating concentration,sera dilution,working concentration of secondary antibody and reaction time,etc.,and the ELISA cut-off was set at 0.1（S/P value）.In this study,the specificity,sensitivity and repeatability of Fiber-1_ELISA and Fiber-2_ELISA were evaluated,respectively.Specificity analysis showed that these two ELISA have similar reaction profile,reacting only with the sera against FAdV-4,but not with the sera against other avian pathogens or other serotypes of FAdV tested;Sensitivity analysis demonstrated that these two ELISA were all sensitive than IFA;The inter-and intra-assay coefficient of variation（CV）of the ELISA were both lower than 10%.Comparison with different detection method revealed that the Fiber-1 and Fiber-2 antibodies detected by Fiber-1_ELIS A or Fiber-2_ELIS A not only had a linear correlation with neutralizing antibodies,but also had good compatibility with Biochek commercial ELISA and neutralization test in detecting the antibodies in chickens vaccinated with an inactivated FAdV-4 vaccine or infected with FAdV-4.Moreover,the Fiber-1_ELISA and Fiber-2_ELISA could be efficiently applied in detecting sera from clinical chicken flocks immuned with FAdV-4 inactivated vaccine or Fiber-2 subunit vaccine,and the sensitivity was higher than IFA accordingly.All these data demonstrated that the Fiber based indirect ELISA for FAdV-4 antibody detection have applaudable application prospect in monitoring the immunological state of the vaccination and diagnosing the clinical infection of FAdV-4.