Expression of truncated fowl adenovirus serotype 4 Fiber-2 protein and the preparation of Anti-FAdV-4 monoclonal antibodies

【Objective】 To obtain the truncated recombinant fowl adenovirus serotype 4（FAdV-4）Fiber-2 protein and its monoclonal antibodies，thereby providing the basic materials for the rapid and sensitive detection of fowl adenoviruses.【Method】 Codon optimization was performed on the Fiber-2 gene sequence，and a sequence fragment encoding a truncated Fiber-2 protein with strong antigenicity was amplified by PCR and cloned into pET-32a（+）to construct the vector for its prokaryotic expression.The recombinant protein was purified，and its expression in Escherichia coli was validated by SDS-PAGE and Western blotting with anti-FAdV-4 antibodies.Following immunization of BALB/c mice with the recombinant protein，splenocytes were harvested and fused with SP2/0 cells，from which the positive cell lines were identified by colchicine lysis and screened using indirect enzyme-linked immunosorbent assay（ELISA）and indirect immunofluorescence assay（IFA）.【Result】 The prokaryotic expression system of the truncated fragment of Fiber-2 gene was successfully constructed，and the truncated Fiber-2 protein of about 67 kD was obtained in the form of inclusion bodies.After purification，a single specific band was obtained in SDS-PAGE.Western blotting results confirmed this single band could bind to FAdV-4 positive serum.5 hybridoma cell lines（2C2，4F6，5A5，6G10 and 10B5）characterized by good stability and specificity in antibody secretion were obtained after four times of subcloning.The antibody titers of the resulting cell supernatant and ascites were 1:1600-1:6400，1:320000-1:1280000，respectively，and the number of chromosomes detected in colchicine lysis was 94-110.In addition，the heavy chain of antibodies secreted by 2C2，4F6 and 10B5 cells was the IgG2b heavy chain，while the light chain was Kappa chain.Likewise，the heavy chain of antibodies secreted by 5A5 and 6G10 was the IgG1 heavy chain，whereas the light chain was Kappa chain.IFA was performed on the 5 hybridoma cell lines and chicken hepatoma cell lines（LMH）infected with FAdV-4，and the results were all positive.【Conclusion】 The truncated recombinant Fiber-2 protein prepared shows good immunogenicity and the monoclonal antibodies prepared can be used in research and the development of a FAdV-4 virus detection kit against fowl.