Expression, purification and antigenic evaluation of toxin-coregulated pilus B protein of Vibrio cholera

Abstract

The toxin co-regulated pilus B (TcpB) as well as tcpA has been verified as a critical colonization factor for Vibrio cholera O1. TcpB is a candidate for making subunit vaccine against cholera; this study aims to produce an oral vaccine by expressing recombinant toxin co-regulated pilus B in Escherichia coli (E. coli). The toxin co-regulated pilus B (tcpB) gene was amplified by polymerase chain reaction (PCR) method. PCR product was sub-cloned to prokaryotic expression vector PET32a, was transformed in E. coliBL21 (DE3) and was induced by Isopropyl_β-D-1-thiogalactopyranoside (IPTG). Recombinant protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and purified by Ni-NTA resin. The immune response to TcpB in animal model was studied. PCR product of tcpB gene has 1297 bp and it was confirmed by sequencing. In SDS-PAGE analysis a band with 65 kDa was seen. In patient recovering from cholera and animal model such as Mice, rabbit with oral inoculation, high titer of antibody in serum was detected. These results demonstrated that the recombinant TcpA is antigenic and can be used in a carrier host as an oral vaccine against cholera. Key words: Antigenicity, Escherichia coli, recombinant protein, toxin co-regulated pilus B.