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

The nucleotide sequence of S0 was amplified by PCR using the primers NS0-F/XS0-R and cloned into the vector pET28a(+) (Novagen, USA) using NheI and XhoI restriction enzyme sites (pS0). The FlaB DNA fragment was amplified by PCR using BFlaB-F/XFlaB-R primers and was cloned into pET28a(+). The resulting construct was digested using NheI and BamHI restriction enzymes and ligated with the S0 DNA fragment that was previously PCR-amplified using NS0-F/BS0-R primers (pS0-F). The FlaB DNA fragment, which was PCR-amplified using SFlaB-F/NFlaB-R primers, was cloned into pET28a(+) pretreated with rSAP. The resulting construct, which contained the correct FlaB direction, was digested using NheI restriction enzymes and ligated with the PCR-amplified S0 DNA fragment using NS0-F/XS0-R primers (pF-S0). To express FlaB alone, the FlaB DNA fragment PCR-amplified using NFlaB-F/ XFlaB-R primers was cloned into pET28a(+) using NheI and XhoI restriction enzyme sites (pF). As pET28a(+) supports 6His-tag nucleotide sequences on both ends of the multiple cloning site, the resulting recombinant proteins contained an additional 6His-tag on both the C-and N-terminal regions. DNA sequences of the resulting expression vectors were verified by the dideoxy-chain termination method.