Immunogenicitv Of Chimeric Protein Constituting PCV2 Cap And FMDV VP1 Expressed By Baculovirus

Porcine circovirus type 2(PCV2)is a porcine disease that can damage the immune system of piglets from 5 to 12 weeks of age,causing them to die due to nutritional deficiencies or multiple complications.After infected with foot-and-mouth disease virus(FMDV)in the natural state,cloven-hoofed animals such as pigs often exhibit pathological conditions in which the skin of the lips and hoofs is eroded and a large amount of blister is generated,causing them to eat.Difficulties hinder their growth.At present,these two diseases have caused great harm to China's pig industry,and vaccine immunity is the main method to control its spread.However,the traditional vaccines against these two animal diseases still have many deficiencies and need to be improved.Therefore,it is still necessary to rely on genetic engineering and other methods to develop new animal vaccines to control the spread of these two diseases.PCV2 Cap protein is the major structural protein of PCV2 and mediates virus recognition and invasion,inducing the host to produce a specific immune response.FMDV VP1 protein is the main structural protein of FMDV and it is a key protein that affects its immunogenicity.It can bind to recognition receptors on the surface of host cells and stimulate the body to produce specific immune responses.Therefore,Cap protein and VP1 protein are rational target proteins for constructing genetically engineered vaccines against PCV2 and FMDV.So far,many studies have shown that the CD40 ligand(CD40L)can be used as a molecular adjuvant to enhance the immune effect of the vaccine.Therefore,the porcine CD40L can be used as a molecular adjuvant to enhance these two disease vaccines and improve their immunity.In this study,Cap and VP1 proteins were fused with porcine CD40L and expressed in baculovirus expression system.The fusion protein was displayed on the surface of baculovirus and three recombinant baculoviruses were constructed:rvAc-Cap,rvAc-VP1 and rvAc-Cap-VP1.The three recombinant baculovirus-infected sf9 cells were detected by SDS-PAGE and Western blotting to demonstrate the successful expression of the three fusion proteins,which were approximately 146kDa,138 kDa,and 165 kDa,respectively,in accordance with expected theoretical values.Indirect immunofluorescence and immunoelectron microscopy demonstrated that the fusion protein was successfully displayed on the surface of the infected cell membrane and the surface of the baculovirus envelope.BALB/c mice were subcutaneously immunized with purified recombinant baculoviral particles as a live vector vaccine at a dose of 1×10~8 pfu/mouse.Cap and VP1 antibody concentrations in mouse serum were detected by indirect ELISA to demonstrate that the recombination baculoviruses can stimulate mice to produce higher antibody levels,and there is no significant difference in antibody concentration of PCV2 and FMDV commercial vaccine immunized groups(P>0.05).The concentration of IL-4 in the serum of the mice was tested to show that the recombinant baculovirus can stimulate the increased level of IL-4 secretion.To further explore its immune effect,mice were immunized with rvAc-Cap-VP1 at a dose of 1×10~8 pfu/mouse by atomization.Indirect ELISA showed that the antibody concentration of Cap and VP1 in mice serum was significantly higher than that of PBS group(P<0.05),and the level of IL-4 secretion in rvAc-Cap-VP1 aerosolized group was increased.The experimental results show that the rvAc-Cap-VP1 recombinant baculovirus displaying Cap protein and VP1 protein can immunize mice with specific humoral immune responses against PCV2and FMDV and cellular immune response by subcutaneous injection and aerosolization.In this study,Cap and VP1 were displayed for the first time on the surface of baculovirus,and their immune effects in mouse were initially explored,laying a theoretical foundation for the further development of PCV2 and FMDV bivalent vaccine.