N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine

Kim, Sae-Hae; Cho, Byeol-Hee; Lee, Kyung-Yeol; Jang, Yong-Suk

The mucin-binding activity of NTD 231-501 of the PEDV S1 protein was measured as described previously (17) . Briefly, for ELISA, bovine mucin (60 μg/mL in PBS) was coated in 96-well MaxiSorp plates (Nunc, Rochester, NY, USA). After blocking with 3% BSA in PBS, the wells were incubated with the indicated amounts of recombinant NTD 231-501 or control proteins at 37ºC for 2 h. Then, the plates were washed with PBS and incubated with an anti-GST Ab (Abcam, Cambridge, UK) followed by the HRP-conjugated secondary Ab. Finally, 3,3′,5,5′ tetramethylbenzidine substrate reagent was added, and enzyme activity was measured on a microplate reader (BMG Labtech, Ortenberg, Germany). isotype, at 4 wk after the last oral immunization, sera were prepared and analyzed by ISO-2 (Sigma-Aldrich) according to the manufacturer's instructions. Briefly, wells were coated with recombinant NTD 231-501 protein and incubated with sera. After washing, each indicated IgG isotype-specific Ab and alkaline phosphatase (AP)-conjugated secondary Ab were added sequentially to the plates. Finally, AP substrate was added, and enzyme activity was measured on a microplate reader (BMG Labtech).