| Brovine Rotavirus(BRV)is a member of the Rotavirus genus of the Reoviridae family.The virus is one of the main pathogens causing viral diarrhea in calves.It is widely prevalent in the world and seriously affects the development of cattle industry.Recent studies have shown that the VP6 gene is a commonly used molecular detection target for BRV,and the VP6 protein encoded by it is also a commonly used serological detection target protein and candidate antigen for subunit vaccines.Therefore,the cloning and expression of the VP6 gene in this study lays the foundation for the further establishment of serological detection methods and the study of subunit vaccines.And use HBc as a vector to prepare virus-like particles containing BRV VP6 gene,at the same time,the recombinant pcDNA3.1-HBcAg-VP was constructed,and its immune effect was analyzed,which provided a certain data basis for the research of BRV vaccine.In this study,HBcAg(encoding 1-74aa),HBcAg(encoding 83-144aa)and truncated BRV VP6 genes were obtained through PCR amplification,and construct the recombinant plasmid pET32a-HBcAg-VP6.The recombinant protein HBc-VP6 with a size of about 57 kDa was obtained after induction and purification.The results of Western blot showed that the recombinant protein HBc-VP6 had good reactogenicity.After dialysis and renaturation of the obtained recombinant protein and concentration by ultrafiltration,it can be observed through transmission electron microscope that the recombinant protein HBc-VP6 can autonomously assemble into virus-like particles.In this study,the BRV VP6 sequence in GenBank was used to obtain a truncated VP6 gene with a length of 597 bp through PCR amplification.The recombinant expression plasmid pET32a-VP6 was constructed,and a large amount of protein with a size of about 42kDa was induced by the inducer IPTG.Wesstern blot test showed that the recombinant VP6 protein has good immunogenicity to BRV positive serum.In this study,two pairs of specific expression primers were designed according to the gene sequence of the recombinant plasmid pET32a-HBcAg-VP6,and the fusion gene HBcAg(coding 1-74aa)-VP6 with restriction sites Hind Ⅲ and EcoR1 was obtained by PCR And HBcAg(encoding 83-144aa)gene with restriction sites EcoR I and Xho I.The amplified genes were introduced into the expression plasmid pcDNA3.1 to obtain the recombinant plasmid pcDNA3.1-HBcAg-VP6.Recombinant protein HBc-VP6,recombinant protein VP6 and recombinant plasmid pcDNA3.1-HBcAg-VP6 were respectively immunized to mice,and the mouse serum related antibodies,related cytokines and the protective effects of challenge were determined,and whether the recombinant protein caused The activation of mast cells in mice has been preliminarily studied.The results showed that VP6 protein,recombinant protein HBc-VP6 and recombinant plasmid pcDNA3.1-HBcAg-VP6 all have good immunogenicity,and recombinant protein HBc-VP6 and recombinant plasmid pcDNA3.1-HBcAg-VP6 have immunogenicity Better than VP6 protein.After the mice were stimulated by the recombinant protein,the mast cells of the mice in the immunized group were degranulated,while the mast cells of the mice in the PBS group hardly appeared degranulated.In summary,the recombinant protein HBc-VP6,recombinant protein VP6 and recombinant plasmid pcDNA3.1-HBcAg-VP6 prepared in this research all have good immunogenicity and protection against challenge,and can stimulate the activation of mast cells.However,the recombinant protein HBc-VP6 and the recombinant plasmid pcDNA3.1-HBcAg-VP6 prepared with HBc as the carrier are superior to the recombinant protein VP6 in terms of immunogenicity and protective effect after challenge. |
