JIA-2019-11

2599 ZHANG Jun-qin et al. Journal of Integrative Agriculture 2019, 18(11): 2598–2604 cell-surface receptors (Bergelson et al . 1997; Tomko et al . 1997). Subsequently, penton binds to cell-surface integrins, promoting integrin-mediated endocytosis (Wickham et al . 1993; Mathias et al . 1994). Neutralizing antibody against penton base can also be detected in sera (Hong et al . 2003). Moreover, anti-fiber and anti-penton base antibodies exhibit synergetic effect on neutralization (Gahéry-Ségard et al . 1998). However, the immunogenicity of the FAdV-4 penton base protein expressed by baculovirus expression system has not been explored yet. In this study, two recombinant baculoviruses were constructed to express penton base protein and fiber-2. The protective efficiency of the recombinants was then investigated. Results indicate that immunization with penton base protein and fiber-2 alone or together confers complete protection against virulent FAdV-4. 2. Materials and methods 2.1. Viruses and cells The FAdV-4 virulent strain HB1502 (GenBank no. KX421401.2) was isolated from a natural case by our laboratory. The virus was purified and propagated in chicken hepatoma LMH cells (ATCC). Sf9 cells were maintained in Grace’s insect medium (Invitrogen, USA) with 10% fetal bovine serum at 27°C and used to propagate recombinant baculoviruses. 2.2. Construction of recombinant baculoviruses To obtain the recombinant plasmids, viral DNAwas extracted from HB1502-infected LMH cell supernatant. The coding sequences of the penton base (1578 bp) and fiber-2 (1440 bp) were amplified from the extracted DNA by using two pairs of primers (Table 1). The amplicons were separately inserted into pFastBac HTB donor plasmid (Invitrogen). The recombinant baculoviruses, namely, Ac-Penton and Ac-Fiber-2, were generated according to the manufacturer’s instructions. 2.3. Expression and purification of recombinant proteins For Western blot, Sf9 cells were seeded into six-well plates and infected with Ac-Penton and Ac-Fiber-2 at a series of multiplicity of infection (MOI) to determine the optimal incubation dose. The cell lysates were separated by 10% SDS-PAGE and electrotransferred onto nitrocellulose membranes. Mouse anti-GAPDH monoclonal antibody (Santa Cruz, USA) and rabbit anti-FAdV-4 polyclonal antibody (made in our laboratory) were used as primary antibodies. HRP-labeled goat anti-mouse IgGs and HRP- labeled goat anti-rabbit IgGs (BioPM, China) was used as a secondary antibody. For immunofluorescence assay (IFA), Sf9 cells were seeded into 24-well plates and infected with Ac-Penton and Ac-Fiber-2 at 1 MOI for 48 h. Rabbit anti-FAdV-4 polyclonal antibody was used as primary antibody. Cy3- conjugated goat anti-rabbit antibody (BioPM, China) was used as a secondary antibody. The nuclei were stained with 4´,6-diamidino-2-phenylindole (DAPI). The cells were observed under a laser scanning microscope (LSM 510, Zeiss, USA). Purified proteins were prepared by infecting Sf9 cells with Ac-Penton and Ac-Fiber-2. After 3–4 days of infection, cells were collected and washed with PBS. Then cells were disrupted on iced by ultrasound. The cell lysates were centrifuged at 12 000 r min –1 for 20 min at 4°C. The supernatant was loaded intoAffinity chromatography columns with Ni-NTA resin (Roche, USA) to obtain purified proteins. 2.4. Vaccination and challenge experiments Animal experiments protocols were approved and performed by the Biological Studies Animal Care and Use Committee in Hubei Province, China. The proteins for vaccination were prepared by mixing the antigens with Montanide TM ISA 71 VG adjuvant (Seppic, France) at a ratio of 30:70 (w/w). Fifty 14-day - old chickens with SPF (Vital Merial, Beijing, China) were evenly divided into five groups (A–E), with ten chickens in each group. The chickens were raised in separated negative-pressure isolators and vaccinated intramuscularly twice at 14- and 28-day of age with the same amount of proteins. Groups A to C were injected with 200 μL of penton (50 μg), fiber-2 (50 μg), and penton+fiber-2 (25 μg+25 μg), respectively. Group D (challenge control) was injected with 200 μL of non-infected insect cell supernatant mixed with the adjuvant. Group E (negative Table 1 Primers and restriction sites used in the construction of recombinant baculoviruses Gene Primer name Primer sequence (5´→3´) 1) Restriction site Amplicon size (bp) Penton Penton-F 5´-AGCGGATCCATGTGGGGGTTGCAGCCG-3´ Bam HI 1 578 Penton-R 5´-GGTAAGCTTCTACTGCAAGGTCGCGGAACTC-3´ Hin dIII Fiber-2 Fiber-2-F 5´-AGCTCTAGAATGCTCCGGGCCCCTAAA-3´ Xba I 1 440 Fiber-2-R 5´-GGTAAGCTTTTACGGGAGGGAGGCCG-3´ Hin dIII 1) The underlined nucleotides indicate restriction sites.