Prokaryotic Expression of VP7 Gene and Subunit Vaccine Preparation of Bovine Rotavirus

Abstract The aims was to construct a prokaryotic expression plasmid and develop a subunit vaccine for bovine rotavirus (BRV), also to investigate the efficacy of this vaccine. BRV total RNA was extracted from M-A104 cell infected by the strain GSB01 of bovine rotavirus. Total RNA as template, BRV VP7 gene was amplified with real time fluorescence quantitative PCR (qPCR), PCR product was cloned into pEASY-T3 vector. The pEASY-T3-VP7 plasmid was recombined into the prokaryotic expression vector pET32a (+) after it was digested using double enzymes. The pET32a-VP7 and VP7-LTB used for prokaryotic expression were constructed, which were converted into Escherichia coli BL21 (DE3) competent cells. After IPTG induction and SDS-PAGE analysis were performed, the fusion proteins of pEASY-T3-VP7 and VP7-LTB with molecular weights of 42.2kD and 53.2kD were acquired respectively. Such a gene engineering bacteria for expressing VP7 protein within BL21(DE3) cells was constructed successfully. The mice immunized using both fusion proteins of pEASYT3- VP7 and VP7-LTB could promote VP7 IgG antibody production (8.33 vs 17.3). The immunization protection ratio of both fusion proteins in neonatal mice was 86.4% and 91.7%, respectively. These findings laid a foundation for further developing a high-efficiency subunit vaccine of BRV VP7 gene