Study On Preparation Of A New Nano-Vaccine Of Porcine Circovirus Type 2 And Preliminary Evaluation Of Its Immune Effect On Mice

Porcine Circovirus Disease(PCVD),also known as Porcine Circovirus-Associated Disease(PCVAD),encompasses a variety of pig illnesses caused by Porcine Circovirus Type2.The primary symptoms include Post-weaning Multisystemic Wasting Syndrome(PMWS),Porcine Dermatitis and Nephropathy Syndrome(PDNS),swine respiratory diseases,enteritis,reproductive issues in sows,and congenital tremors in piglets.PMWS cases were first identified in Canada in 1991,with an outbreak occurring in 1997.Since then,numerous European and American countries have reported the disease and associated it with Porcine Circovirus Type 2.Presently,the disease has spread to pig-rearing countries and regions across the globe,resulting in significant economic losses for the worldwide pig industry.In2001,China reported its first cases of Porcine Circovirus Disease.Currently,the disease affects nearly all pig herds in the country,severely impacting the production and development of the Chinese pig industry and causing substantial economic losses.Porous nano-materials are essential in vaccine research and development.Nano-particles can transport vaccine antigens and proteins to target sites while protecting them from degradation in adverse conditions through mucosal administration.The uptake of nano-particles by antigen-presenting cells can enhance and stimulate a robust immune response.The slow-release properties of nano-materials enable the long-lasting effects of vaccine antigens,making them highly suitable for use in delivery systems.Among these,Covalent Organic Frameworks(COFs)and Metal-Organic Frameworks(MOFs)have emerged as innovative porous crystalline nano-materials.Their unique and exceptional features,such as customizable functionality,predictable structures,and adjustable pore sizes,combined with their high specific surface area and organized open nano-channels,guarantee a high loading capacity.This proves the viability of MOFs and COFs materials as universal antibody carriers.Employing these nano-materials as carriers for vaccine antigens has the potential to significantly enhance the immune response and protective efficacy induced by vaccines.In this study,we initially expressed the PCV2 Cap protein.The engineered Cap gene plasmids were individually introduced into competent cells of BL21(DE3),Rosetta,and BL21Star(DE3).After IPTG induction,the target protein was expressed.The recombinant protein was purified using Ni-column affinity chromatography,and its concentration was determined with the BCA method.SDS-PAGE analysis demonstrated the successful expression of a singular Cap protein band,exhibiting a molecular weight of 29 k Da.Utilizing the purified PCV2 Cap protein as a foundation,we developed an innovative porcine circovirus nano-vaccine by combining it with nano-porous crystalline materials,specifically COFs and MOFs,which served as carriers via a coating process.This approach aimed to enhance the thermal and chemical stability of the encapsulated PCV2 Cap protein while also increasing the level of immune antibodies.The BCA method was employed to assess the protein encapsulation efficiency of PCV2@MOFs and PCV2@COFs.The CCK-8cell toxicity assay evaluated the cytotoxicity of COFs,MOFs,PCV2@MOFs,and PCV2@COFs at concentrations ranging from 12.5 μg/m L to 200 μg/m L.The findings indicated that these materials exhibited no cytotoxicity towards RAW and PK-15 cells,suggesting that this novel nano-vaccine possesses favorable safety characteristics.Mice were immunized with PCV2@MOFs vaccine through subcutaneous injection to preliminarily assess the vaccine’s immunogenicity.The presence of PCV2 antibodies in the mice was detected using a porcine circovirus ELISA kit,with the secondary antibody replaced by a sheep anti-mouse antibody.The experimental results were analyzed under an enzyme-linked immunosorbent assay(ELISA)instrument at OD450.The results demonstrated that the groups injected with PCV2@MOFs 25 μg,PCV2@MOFs 50 μg,and PCV2@MOFs 100 μg all tested positive through the ELISA kit,indicating the production of PCV2 antibodies.In summary,this study,based on the prokaryotic expression of PCV2 Cap protein and its encapsulation with porous crystalline nano-materials,demonstrated a favorable immunological response for the novel porcine circovirus nano-vaccine.This research lays the foundation for further in-depth development of the new porcine circovirus nano-vaccine...