Expression, Purification and Polyclonal Antibody Preparation of Fowl adenovirus Serotype 4 Fiber2 Protein
GUO Hao-Ran1,2, JIA Yan-E2, JI Yan-Hong2,*, LI Yu1,*
1 College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China; 2 State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
Abstract：Since 2015, the number of cases of inclusion body hepatitis and pericardial effusion in domestic chickens (Gallus domesticus) with Fowl adenovirus serotype 4 (FAdV-4) infection has been increasing. In order to prepare a serological diagnostic antigen of FAdV-4 with good reactiongenicity, the FAdV-4 Fiber2 whole gene synthesis sequence after codon optimization was amplified and cloned into the pCold expression vector to construct the prokaryotic expression vector pCold-Fiber2. Then pCold-Fiber2 was transformed into PG-Tf2.The supernatant induced by IPTG (isopropyl β-D-thiogalac toside) was purified and immunized rabbits (Oryctolagus cuniculus). The polyclonal antibodies were successfully prepared. The results of Western blot and indirect immunofluorescence assay (IFA) showed that the purified polyclonal antibodies could react specifically with Fiber2 protein and FAdV-4. It was proved that Fiber2 protein demonstrated good immunogenicity. The indirect ELISA method for FAdV-4 antibody showed that the sensitivity and specificity of the method were 96% and 100%. Polyclonal antibodies with good Western blot and IFA reactivity were successfully prepared. An indirect ELISA method for detection of FAdV-4 was established with good reaction specificity, high sensitivity, simple operation and stable and reliable reaction results. This method would be suitable for monitoring, evaluating and detecting early latent infection of chicken group antibody level. It provides basic data for the development of serological diagnosis technology and prevention and control of the disease.
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