B subunit of cholera toxin fused with VP7 from GCRV (grass carp reovirus) was expressed in E. coli and folds into an active protein.

Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idella) production and results in high mortality in China. To obtain a genetically engineered oral vaccine against GCRV, the cholera toxin B subunit (CTB) of Vibrio cholerae was fused to VP7 (CTB-VP7) and transformed into BL21(DE3) for expression. SDS-PAGE and Western blotting showed that the purified rCTB-VP7 fusion protein (rCTB-VP7) was approximately 49.0 kDa. The monomeric nature of rCTB-VP7 through multistage purification showed a binding affinity for GM1, a receptor for biologically active CTB. rCTB-VP7 is not vulnerable to disassembly by SDS but is vulnerable to disassembly by 2-mercaptoethanol. rCTB-VP7 is stable and highly active at room temperature. The binding affinity experiment between rCTB-VP7 and GM1 also confirms the effects of acid and alkalinity in solution on the structure of rCTB-VP7. rCTB-VP7 bound to GM1 with different affinities under different temperatures and pH values. Prokaryotic expression of rCTB-VP7 was characterized by high expression and easy purification and had a strong binding force with GM1 at 37 °C and pH 7.4. Our results suggest that rCTB-VP7 has the potential as an oral vaccine for protection against GCRV in aquaculture.