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 partial NTD gene (encoding amino acids 231-501) was amplified from the S1 protein gene of the PEDV BM1 strain, which was synthesized by GeneScript (Piscataway, NJ, USA) using a specific primer set (F: 5′-CGC GGA TCC ACA GCT AAT TGC ATT-3′, R: 5′-CCG CTC GAG TCA AAA AGA AAT TGG CTG-3′). The amplified NTD 231-501 gene was cloned into the pGEX 4T-1 expression vector (GE Healthcare Life Sciences, Buckinghamshire, UK) or the pET SUMO vector (Thermo Fisher Scientific, Waltham, VA, USA). The recombinant NTD 231-501 protein expressed in the Escherichia coli (E. coli) BL21 (DE3) strain was purified using a glutathione-or Ni-NTA-based affinity purification system (GE Healthcare Life Sciences and Thermo Fischer Scientific, respectively) according to the manufacturers' instructions. The recombinant NTD 231-501 protein containing a glutathione S-transferase (GST) tag was expressed from the pGEX 4T-1 vector, and the recombinant NTD 231-501 protein without a GST tag was expressed from the pSUMO vector; these constructs were used as the immunization and coating Ags, respectively, in subsequent experiments.