Strategy Study Of Surface Disply And Immunogenicity For FMDV VP1 Protein GH Loop Epitope

Foot-and-mouth disease caused by Foot-and-mouth disease virus(FMDV),is a acute,fever and highly contagious viral vesicular disease.The virus mainly infects cattle,sheep and other cloven hoofed animal and can cause severe economic loss.At present,domestic FMDV vaccine are prepared with inactivated foot-and-mouth disease virus,emulsified with oil adjuvant,clinical adverse reaction very severe,immune protection remains to be further improved.Therefore,select effective surface display system to display the FMDV antigen epitope by gene engineering technology for the preparation of FMDV subunit vaccines has great practical value in enhancing the domestic vaccine quality and safety.VP1 protein is the main antigen of FMDV which contains a GH loop belonging to B cell epitopes:141-160aa and 200-213aa sequence.The epitopes can induce VP1 specific antibody and thereby induce protective immunity.Thus VP1 protein is an ideal antigen for the development of FMD vaccine.Synthetic peptide vaccine has been successfully listed domestic.PCV2 Cap protein surface display system is a new type of exogenous antigen epitope display system which has developed in recent years.The technique is to use exogenous peptide to replace Cap protein loop,expression of recombinant Cap protein,then 60 recombinant capsid protein can be self-assembly into virus like particles in vitro and then complete the presentation of exogenous antigen epitopes.Cholera toxin B subunit display system is to use exogenous antigen epitopes to replace cholera toxin B subunit KNG amino acid fragment,expression of recombinant CTB protein.Then five recombinant CTB protein aggregation in vitro as pentameric structure and then complete the display of exogenous antigen epitope.In the study,PCV2 Cap protein virus like particles and Cholera toxin B subunit were chosen as vector system to display FMDV epitope,successfully demonstrated FMDV VP1 GH loop epitope.On the basis of this,Preparation of the FMDV-PCV2 bivalent FMDV subunit vaccine,Preliminary evaluation of the effectiveness of the immune protection of the subunit vaccine.It could lay a good foundation for development of efficient subunit vaccine.The main contents are as follows:1.Construction of recombinant PCV2 Cap protein to display FMDV VP1 GH loop epitope by gene replacement methodIn this study,type O FMDV VP1 major B cell epitopes GH loop was used as exogenous genes.Four sites in the PCV2 internal Cap protein were identified to constructe new chimeric gene by gene replacement.The chimeric gene which were successfully constructed by connecting PQZ expression vector were highly expressed in Escherichia coli.SDS-PAGE revealed that the obtained recombinant protein was about 27 kDa and existed in soluble form;Western-blotting results showed that six recombinant PCV2 Cap protein could react with PCV2 or FMDV positive serum;TEM observation showed that recombinant Cap protein was able to assemble into virus-like particles in vitro which diameter was about 20 nm;After immunization of mice with the purified recombinant proteins 50 per mouse,all the homemade vaccine was capable of eliciting the body to produce specific cellular and humoral immune responses.Among them,the immune effect of Cap-loop4 homemade vaccine was the best.2.Construction of recombinant PCV2 Cap protein to display FMDV VP1 GH loop epitope by gene insert methodOn the basis of previous research,B-cell epitopes GH loop was used to insert PCV2 Cap protein internal selected four loop sites to constructe new chimeric gene by gene insertion.The chimeric gene which were successfully constructed by connecting PQZ expression vector were highly expressed in Escherichia coli.SDS-PAGE revealed that the obtained recombinant protein was about 35 kDa and existed in soluble form;Western-blotting results showed that four recombinant PCV2 Cap protein could react with PCV2 or FMDV positive serum;TEM observation showed that recombinant Cap protein was able to assemble into virus-like particles in vitro which diameter was about 20 nm;After immunization of mice with the purified recombinant proteins 50 ?g per mouse,all the homemade vaccine was capable of eliciting the body to produce specific cellular and humoral immune responses.Among them,the immune effect of Cap-iloop1 homemade vaccine was the best.3.The study of using cholera toxin B subunit as a carrier to display the FMDV VP1 GH epitopeThe object of this study is to explore whether CTB protein is suitable for the display GH loop epitope of type O FMDV.The GH loop epitope was used to replace the KNG sequence of CTB amino acids.The chimeric gene was constructed by gene replacement.then the chimeric gene which were successfully constructed by connecting PQZ expression vector was expressed in Escherichia coli.SDS-PAGE revealed that the obtained recombinant protein was highly expressed and existed in soluble form;Western-blotting results showed that the recombinant protein could react with FMDV positive serum;the antigen-coated plate coated with GM1 ganglioside identification results show that recombinant CTB protein capable of forming a pentamer structure;After immunization of pigs with the purified recombinant proteins 200 ?g per pig,test pigs can produce higher antibody levels and cellular immune responses.The above results showed that the recombinant protein has good immunogenicity for FMDV,this provides a new idea for the study of the subunit vaccine of foot and mouth disease.