Development Of Mycoplasma Suis Pneumonia Subunit Vaccine Based On Baculovirus Expression System And Selection Of Vaccine Adjuvant

Mycoplasma pneumonia of swine(MPS)is a chronic respiratory disease of pigs,and Mycoplasma hyopneumoniae(Mhp)is the main pathogen.Mhp infection seriously affects the daily gain and feed conversion rate of pig herds,and secondary infection with other pathogens can further lead to increased mortality and cause significant economic losses to the pig industry.Vaccination is still considered to be the most effective way to control Mhp infection.At present,MPS vaccines mainly include inactivated vaccines and live attenuated vaccines,but these vaccines still have many shortcomings.The exact protective mechanism required to prevent and control Mhp infection is not fully understood,and continuous efforts are still needed to develop new generation vaccines that can provide better protection.At present,the development of adhesin-based genetically engineered vaccines is a major direction.However,in general,the recombinant antigens in genetically engineered subunit vaccines have poor immunogenicity,and adjuvants need to be added to improve the immune response effect of the antigens.Molecular adjuvants refer to various immune-related molecules that can non-specifically change or enhance the body’s specific immune response to antigens and molecules that have immune-stimulating effects themselves.It can stimulate an effective immune response,has better safety,and is more suitable for improving the immune protection effect of genetically engineered subunit vaccines.The laboratory used prokaryotic expression system and insect baculovirus surface display system in the early stage to prove that the recombinant Mhp P97R1P46P42 chimeric antigen has good immunogenicity in mice and piglets.On this basis,the recombinant plasmid p FBD-P97R1P46P42 will be constructed in this study,and the recombinant protein r P97R1P46P42 will be prepared by the insect baculovirus expression system,which will be used as a candidate vaccine antigen after nickel column purification.BALB/c mice were immunized with r P97R1P46P42 mixed with commercial vaccine adjuvant,and the commercial adjuvant suitable for MPS genetic engineering subunit vaccine was screened by detecting the humoral and cellular immune effects of the immunized mice.At the same time,GM-CSF,CD40 L and ctx B genes with molecular adjuvant effect were screened out and embedded into the C-terminus of the P97R1P46P42 chimeric antigen protein gene,respectively.And recombinant proteins: r P97R1P46P42-GM-CSF,r P97R1P46P42-CD40 L and r P97R1P46P42-ctx B were prepared by insect baculovirus expression system.Finally,BALB/c mice were immunized with the commercial adjuvanted vaccine screened above and the vaccine carrying molecular adjuvant.And the immune effect was determined by specific Ig G antibody assay,spleen lymphocyte proliferation assay,and cytokine IL-4 and IFN-γ levels.SDS-PAGE and Western Blot identification confirmed that r P97R1P46P42 chimeric protein and r P97R1P46P42-GM-CSF,r P97R1P46P42-CD40 L and r P97R1P46P42-ctx B proteins were successfully prepared by insect baculovirus expression system.This provides the antigen for the eventual development of MPS subunit vaccine products based on the baculovirus expression system.Further,the detection of mouse immunization experiments showed that compared with PBS,commercial vaccine and r P97R1P46P42 group alone,the addition of commercial adjuvant and recombinant protein expressed by fusion with molecular adjuvant can enhance the induction of mice to produce specific Ig G antibodies(p < 0.0001)The recombinant protein adjuvanted with ISA 201 and the recombinant protein fused and expressed with molecular adjuvant were added to immunize mice.After immunization,the spleen lymphocytes of the immunized mice could be stimulated by the corresponding stimulator to produce a more significant proliferation effect(p < 0.0001).And also enhanced the induction of cytokines IL-4 and TNF-γ(p < 0.01).The above results show that commercial ISA 201 emulsion and GM-CSF,ctx B,CD40 L molecular adjuvants can enhance the recombinant Mhp protein r P97R1P46P42-induced humoral and cellular immunity in mice.Based on the results of mouse immunization experiments,this study confirmed that CD40 L is the best molecular adjuvant to enhance the immunogenicity of recombinant Mhp subunit vaccine antigens.This study provides data support for the final development of a baculovirus expression system-based Mycoplasma suis pneumonia subunit vaccine product and supporting adjuvants.