Development Of Nanobody Fusion Flagellin Adjuvant And Its Effect Evaluation In The Application Of PCV2 Virus-like Particles

Porcine circovirus type 2(PCV2)has caused severe damage to pig farms nationwide.At present,it is mainly used for vaccine immunization for prevention and control,and compared with traditional inactivated vaccines,commercial subunit vaccines have more development prospects.Cap protein is a key immunogenic protein for the prevention and control of PCV2 infection,and can spontaneously form virus-like particle seedlings(VLP),which is the first choice for subunit vaccines.However,due to its weak immunogenicity,the selection and correct use of adjuvants is particularly important.Aluminum adjuvants and Freund’s adjuvants are common traditional adjuvants,although they can effectively enhance the immune effect,but there are toxic side effects.Flagellin is a pathogenic immune promoting factor,which can effectively cause humoral and cellular immunity,not only has high immune activation but also high safety.Studies have shown that the fusion expression of PCV2 Cap protein and Salmonella flagellin can play a good role in immune promotion and protection,but because the antigen and adjuvant are fused expression,the optimal ratio cannot be optimized.Compared with traditional antibodies,nanobodies have the advantages of high antigen affinity,strong tissue penetration,and stable structure,and can be conjugated with flagellar protein as an adjuvant.In this study,PCV2 Cap protein and flagellin complex vaccine were prepared by using the E.coli expression system to fuse flagellin and Cap protein-specific nanobodies as adjuvants,and the immune efficacy of the vaccine was evaluated in vivo.The main findings are as follows:1.Construction of flagellin-Linker2-Cap Nb58-p Cold-SUMO(FLC-pcold-SUMO)with nanobody recombinant expression vector.The bivalent flagellin-linker2-foldon-p UC57 vector containing flagellin-linker2-foldon-p UC57 vector containing flagellin and the p Cold-SUMO vector and Cap Nb58-p Cold-SUMO plasmid containing Cap protein nanobody were constructed by digestion,ligation,transformation,etc.,and the PCR identification results showed that the flagellin-Linker2-Cap Nb58-p Cold-SUMO(FLC-pcold-SUMO)was successfully constructed recombinant expression vector.2.Expression and purification of flagellin-linker2-Cap Nb58(FLC)and flagellin proteinFLC-pcold-SUMO vector and flagellin-p ET-32 a vector were transferred to E.coli for expression,and the results showed that FLC protein and flagellin protein were solublely expressed in supernatant and the expression was high.FLC protein and flagellin protein were purified by nickel column affinity chromatography,and the purification effect of the two proteins was confirmed by SDS-PAGE and Western Blot.The results showed that a large number of FLC protein and flagellin protein were purified,and the protein purity was high.3.The protective effect induced by recombinant FLC protein in mice using recombinant FLC protein as an adjuvant.To evaluate the immunoprotective effect of FLC adjuvants,this study compared the immunogenicity of FLC protein adjuvant versus conventional flagellin protein adjuvant subunit vaccines,10 μg/Cap VLP vaccine + 4 μg/adjuvant dissolved in PBS,and 100μL/c immunizing BALB/c mice.Co-immunize mice twice by intramuscular injection of the legs.Mouse serum was collected for the detection of antibodies and cytokines,and FLC adjuvants were shown to induce higher levels of Cap protein antibodies,neutralizing antibody levels,and higher IFN-γ and TNF-α levels in mice.After 2 weeks of booster immunization,the mouse challenge was performed with PCV2 DBN-SX07 strain,and the virus content in the serum of mice was determined 3 weeks after challenge,and the results showed that the virus content in the serum of the FLC adjuvant immune group was the lowest.After that,the inguinal lymph nodes of the mice were stained with HE,and the results showed that the tissue damage of the mice in the FLC adjuvant immune group was the lightest.In summary,this study used the E.coli expression system to obtain flagellin protein and flagellin-nanobody(FLC)fusion protein,and confirmed that FLC adjuvants can effectively stimulate mice to produce stronger humoral immunity and cellular immune responses,and produce more effective immune protection through mouse immunity.Lay the foundation for the development of novel immune adjuvants for porcine circovirus vaccines.