Evaluation of the Efficacy of a Recombinant Adenovirus Expressing the Spike Protein of Porcine Epidemic Diarrhea Virus in Pigs

Liu, Xinsheng; Zhao, Donghong; Zhou, Peng; Zhang, Yongguang; Wang, Yonglu

Blot. P3 rAd-PEDV-S was used to infect HEK293A cells, and the cells were harvested after 120 hours. e cells were lysed using the lysis buffer ( ermo Scientific, Waltham, MA) containing protease inhibitors (Invitrogen, USA), frozen/thawed 3 times, and then centrifuged and collected at 4°C and 12000 rpm for 10 minutes. en, the proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes under a constant voltage of 80 V for 2 hours. e blots were blocked with 5% skim milk at 37°C for 1 hour and then incubated with a primary mouse anti-His-tag monoclonal antibody (mAb) (1 : 1000 dilution; Abcam, USA) for 1 hour at 37°C. After washing 3 times for 5 minutes per wash with TBS-Tween 20 (50 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.6), the membrane was incubated with a secondary horseradish peroxidase-(HRP-) labeled goat antimouse IgG antibody (1 : 2000 dilution; Abcam, USA) for 1 hour at 37°C. Finally, the membrane was washed and visualized using an ECL chemiluminescent substrate reagent kit ( ermo Scientific, USA).