JIA-2019-11

2600 ZHANG Jun-qin et al. Journal of Integrative Agriculture 2019, 18(11): 2598–2604 control) was injected with sterile PBS. Blood samples were collected weekly for four weeks since the first vaccination. Two weeks after secondary immunization, chickens in groups A–D were intramuscularly challenged with 200 μL of 10 7 TCID 50 mL –1 FAdV-4 virus strain HB1502. All surviving birds were humanely euthanatized two weeks after the challenge. 2.5. Detection of antibody Serum antibody was detected using commercial Fowl Adenovirus Group 1Antibody Test Kit (Biochek, Netherlands) according to the manufacturer’s instructions. 2.6. Necropsy and histopathology All birds were euthanized and necropsied. Heart and liver samples were dipped in 4% paraformaldehyde, embedded in paraffin and stained with hematoxylin and eosin (HE) for examination. 2.7. Statistical analysis Data were analyzed using GraphPad Prism Version 5 (GraphPad Software, USA). Differences were considered statistically significant when P <0.05. 3. Results 3.1. Confirmation of recombinant plasmids The recombinant plasmids were confirmed by restriction enzymes digestion (data not shown). The nucleotides of the penton base gene (1 578 bp) and the fiber-2 gene (1440 bp) were verified by sequencing. 3.2. Expression of recombinant proteins The expression of the recombinant proteins was evaluated by Western blot analysis (Fig. 1-Aand B) and IFA (Fig. 1-C). The Western blot showed that the optimal incubation doses were at least 1 MOI for Ac-Penton and 0.5 MOI for Ac-Fiber-2 (Fig. 1-Aand B). The molecular weights of the recombinant penton base protein and fiber-2 were approximately 65 and 56 kDa, respectively. These two proteins were expressed in the supernatant in a soluble form. No protein was detected in normal cells (negative control). To further determine the protein expression, IFA was DAPI Sf9 cells Ac-Penton Ac-Fiber-2 Anti-FAdV-4 Merge Ac-Fiber-2 GAPDH Ac-Penton 65 kDa 1 A C B 2 3 4 5 6 7 1 2 3 4 5 6 7 56 kDa GAPDH Fig. 1 Western blot (A and B) and immunofluorescence assay (C) of the expression of recombinant penton and fiber-2 proteins. Sf9 cells were infected with Ac-Penton and Ac-Fiber at a series of multiplicity of infection for 72 h. A, lane 1, cell lysates of uninfected Sf9 cells (negative control); lanes 2–7, cell lysates of cells infected with 0.1, 0.5, 1, 2, 5, and 10 multiplicity of infection (MOI) Ac-Penton, respectively. B, lane 1, negative control; lanes 2–7, cell lysates of cells infected with 0.1, 0.5, 1, 2, 5, and 10MOI Ac-Fiber-2, respectively. C, confirmation of the expression of the recombinant penton and fiber-2 proteins in Sf9 cells by immunofluorescence assay.