Enhancement of antigen-specific humoral immune responses and protein solubility through conjugation of bacterial flagellin, Vibrio vulnificus FlaB, to the N-terminus of porcine epidemic diarrhea virus surface protein antigen S0

Oh, Seo-ho; Kim Cho, Young-Saeng; Lee, Ho-Bin; Lee, Sang-Mok; Kim, Whee-Soo; Hong, Liang; Cho, Chong-Su; Choi, Yun-Jaie; Kang, Sang-Kee

Each expression plasmid (pS0, pS0-F, pF-S0 and pF) was transformed into E. coli BL21(DE3) (Invitrogen, USA). A seed culture was prepared with a single colony of recombinant E. coli Adjuvancy of flagellin conjugation to PED subunit vaccine antigen, S0 and grown overnight in LB broth containing 50 μg/mL kanamycin. One percent of the seed culture was inoculated into LB broth containing 50 μg/mL kanamycin and grown until the optical density at 600 nm (OD 600) reached 0.5. Cultures were then treated with 0.3 mM IPTG to induce expression of the protein of interest and grown at 25°C for 5 h with shaking set to 200 rpm. Cells were harvested and disrupted on ice by sonication (VCX750, SONICS, USA) using a preset program (45 cycles of 2 sec On/5 sec Off, amp 40%), followed by centrifugation at 20,000 × g for 15 min at 4°C. After centrifugation, supernatants were collected as a soluble fraction, while pellets were solubilized using 50 mM Tris-Cl, 2 M urea, pH 12.5 buffer and collected as an insoluble fraction. Equivalent volumes of solubilization buffer versus supernatants were used to prepare the insoluble fraction. Insoluble and soluble fractions of each recombinant protein were analyzed through SDS-PAGE followed by Coomassie Blue staining (Fig. 1A) . The insoluble fraction of S0 was subsequently refolded as described previously [8] .