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

To express each recombinant protein in the presence of the chaperone tig, we transformed each expression plasmid (pS0, pS0-F and pF-S0) into E. coli BL21(DE3)/pTf16 (Takara, Japan), which can induce the expression of trigger factor chaperone tig. A seed culture was prepared by inoculating a single colony in LB broth containing 50 μg/mL kanamycin and 20 μg/mL chloramphenicol overnight at 37°C. tig was expressed before the induction of each recombinant protein. One percent of the seed culture was inoculated into LB broth supplemented with 0.5 mg/ml L-arabinose, 20 μg/mL chloramphenicol and 50 μg/mL kanamycin until the OD 600 reached 0.35. At this point, cells were cooled to 4°C for 15 min and subsequently induced with 0.1 mM IPTG at 15°C for 24 h. The cell disruption process was as described above. Insoluble and soluble fractions of each recombinant protein were analyzed by SDS-PAGE followed by Coomassie Blue staining (Fig. 1B) . The band densities of each recombinant protein were measured using Image Lab software (Bio-Rad Laboratories) ( Fig. 1C and D) .