Preliminary Study On The Immunogenicity Of BVDV Recombinant E2 Protein Conjugated To Mesoporous Silica Nanoparticles

Bovine viral diarrhea virus is the pathogeny of bovine viral diarrhea-mucosal disease and is widely prevalent worldwide,which has great harm to the development of cattle breeding and related biological products industry.In view of the current situation of dense cattle and high prevalence of BVDV in China,vaccination is the optimal strategy to prevent and control BVDV.At present,BVDV vaccines have some shortcomings,such as safety risks or insufficient immune protection,the development of new vaccines such as BVDV subunit vaccines has become a research hotspot.The BVDV genome encodes four structural proteins and eight non-structural proteins,among which the structural protein E2 has a variety of common epitopes and specific epitopes,which is a major candidate antigen for the development of subunit vaccines.With the rapid development of nanotechnology applications,mesoporous silica nanoparticles have significant advantages in targeted delivery and release of biomolecules,and become a new material for protein delivery carrier.Therefore,exploring the feasibility and immunogenicity of MSNs as a carrier of E2 protein lays a theoretical foundation and technical support for the development of new BVDV subunit vaccines.In this study,a cytopathic type of BVDV was successfully isolated by infecting MDBK cells with BVDV-positive bovine serum and re-identified by RT-PCR.After whole genome sequencing,the phylogenetic tree of whole genome and E2 gene was constructed by DNAStar software.The isolated BVDV was confirmed to be type BVDV-1,which was consistent with the epidemic situation in China,and its TCID₅₀was 10^5.5.The truncated E2 gene of BVDV was amplified by PCR,and the yE2 gene amplification product was cloned into the prokaryotic expression vector p ET-28a（+）and construct the p ET-28a-yE2 plasmid.Sequencing showed that yE2 gene was correctly inserted into the p ET-28a（+）vector.The recombinant plasmid p ET-28a-yE2was transformed into BL-21 competent expression vector,and the expression conditions were optimized.The yE2 protein was purified by nickel-affinity chromatography,and the yE2 protein showed good immunogenicity by Western Blot.The adsorption properties of NH₂-HMSNs and SBA-15 for yE2 protein were compared by SDS-PAGE and protein quantification.Mice were immunized with NH₂-HMSNs conjugated yE2 protein which has better adsorption effect,and yE2 protein emulsified with Freund’s adjuvant was used as control.The humoral and cellular immune responses were detected by virus neutralization test and mouse spleen lymphocyte proliferation test,and the results showed that NH₂-HMSNs as adjuvant elicited stronger humoral immune responses than Freund’s adjuvant.In summary,NH₂-HMSNs have the potential to be used as an animal subunit vaccine adjuvant.These results provide technical support for the development of novel BVDV subunit vaccines and based on this,a subunit vaccine platform rely on MSNs as antigen carriers may be constructed.