Prokaryotic Expression Of VP03、VP1、VP3Protein Of Foot-and-mouth Disease Virus Serotype O In Swine And Development Of Indirect ELISA

Foot-and-mouth disease (FMD) is a highly infectious and contagious disease of transboundaryimportance affecting cloven hoofed animals and has seriously endangered the development of animalindustry all over the world. Currently, it has been controlled by vaccine and diagnostic tests. One of themost effective and economical diagnostic methods is enzyme-linked immunosorbnent assay (ELISA).Most of ELISA methods use whole virus as an antigen to detect serum antibodies, which entails the riskof spreading the virus. Therefore, a safer and more effective way in preparation of foot-and-mouthdisease virus (FMDV) antigen is to express the viral structural protein gene in vitro and use the purifiedexpress protein in the ELISA. In addition, immunized animals with purified proteins expressed in vitrodeveloped immune response, and the expressed proteins can serve as the foundation of FMD subunitvaccines.In this study, P1gene coding for a structural protein precursor of FMDV was synthetized accordingto the full sequence of FDMV serotype O from GenBank (JN998085.1). VP0, VP1and VP3genes wereamplified by using pUC57-P1plasmid (Shanghai Biological Engineering Co., Ltd) as a template. Therecombinant proteins were refolded and purified. In the process of SUMO-VP3purification, The VP3protein was obtained from SUMO-VP3by the SUMO fusion tags digested with SUMO Protease1.An indirect ELISA method was established using the renatured and purified SUMO-VP0,SUMO-VP1, SUMO-VP3and VP3proteins which showed good antigenicity. The results showed thatthe proteins can be used as candidates for an indirect ELISA method to detect anti-FMD antibody fromserum samples. Compared with SUMO-VP0-ELISA and SUMO-VP3-ELISA, a significant differencebetween negative and positive serum OD450value was detected in the SUMO-VP1-ELISA, while P/Nratio was higher. In addition, comparing to SUMO-VP3-ELISA, difference between negative andpositive serum OD450value and P/N ratio were more significant, while it was in VP3–ELISA. The totalnumber of positive samples detected of SUMO-VP0-ELISA matchs with O-LPBE and Asia1-LPBE.The results indicated that SUMO-VP0proteins can be used in an ELISA to detect antibody againstFMDV serotype and Asia1.The high antibody titer was detected in the Guinea pigs immunized with refolded recombinantproteins SUMO-VP0, SUMO-VP1or SUMO-VP3. The highest antibody level existed in the animalswhich were immunized with a protein cocktail of them. It was suggested that SUMO-VP0, SUMO-VP1and SUMO-VP3have good immunogenicity and are able to induce specific antibodies in vivo.In summary, SUMO-VP0, SUMO-VP1, SUMO-VP3and VP3proteins produced in the prokaryoticexpression system are reactogenicity and immunogenic. Those proteins can be used as good candidatesfor serotype O FMD diagnostic reagents, and development of genetic engineering subunit vaccinesagainst FMD.