Expression Of Type O FMDV Structure Protein VP1 Recombination Protein And Its Immunogenicity Research

Foot-and-mouth disease (FMD) is a highly contagious, acute vesicular disease of cloven-hoofed animals, and has been designed as type A by the Office International des Epizooties, World Organization Animal Health. FMD is a serious disease that spreads rapidly and requires responsible for large economic losses, both as a direct result of the disease, and because of the necessary restrictions imposed on animal products and trade for disease control. Many researchers in our country prevent and control this disease by inoculating inactivated whole-virus preparation that is formulated with adjuvant, which has integral immunogenicity and boost high protective antibody titer, but risk the escape of live virus from animal facilities or from improper vaccine preparations. Researchers have paid many attentions for several new type vaccines because of the safety and controllability along with the development of gene engineering technique, of which recombination protein vaccine is safety, protective , low cost because it only contains the effective virus structure, it is the best development direction of animal vaccine.The etiological agent of FMD is foot-and-mouth disease virus, which belongs to the genus Aphthovirus of the family Picornaviridae. There are seven distinguishable serological types, namely, O, A, C, Asia1, SAT1, SAT2, and SAT3, and several subtypes. The capsid of FMDV is constituted of 60 copies each of the four proteins VP1, VP2, VP3 and VP4, in which VP1, VP2 and VP3 form the capsomeres, VP4 is inside of virus particle. The VP1 capsid protein is the dominating antigen, in which the prominent G-H loop, spanning residues 134-158, has been identified as the major immunogenic site for neutralizing antibodies. This has formed the basis for the peptide approach to vaccination against FMD. The expression vector including four copies of tandem gene fragments of VP1 141-160aa-200-213aa, was constructed on the basis of type O FMDV strain TAW97. The ideal vaccine should include B cell epitopes but also T cell epitopes. The VP1 region itself lacks certain T cell epitopes. Thus, a fully protective VP1 vaccine needs additional T sites from a source outside VP1. The tandem fragments hepatitis B surface antigen and tetanus toxid was synthesized to insert into the N terminal of the fusion expression vector pET32a-4VP1, constructing the recombinant expression vectors pET32a-TCE-4VP1 and pET32a-TCE-2VP1. These three plasmids were transformed into the competent cells of the host cell E.coli BL21 (DE3). The expression of the target genes were induced with IPTG and were analyzed by SDS-PAGE and Western-blotting. Results showed that the recombinant vectors were successfully constructed and the relative molecular mass of the fusion proteins were measured to be 38.5kDa, 43.5kDa, 34.6kDa. The recombinant proteins were induced with 37℃, 0.1mmol/L IPTG for 4h, reached the maximum, expressed partly in the supernatant and partly in the inclusion body. The purified fusion proteins were obtained by Ni-sepharose affinity chromatography.ICR mice were immunized with the commercial inactivated vaccine to produce the positive and negative sera and 141-160aa of FMDV VP1 protein was commercially synthesized for developing an indirect ELISA for VP1 antibody detection. 96-well plates were coated with 5μg/mL commercially synthesized 20aa peptide , blocked with 10% FCS at 37℃for 2h, serum samples were diluted by 1:200 and incubated at 37℃for 60min, HRP-conjugated secondary antibodies were incubated at 37℃for 45min, and the chromogen, TMB was added and after 15min incubation at 37℃, the reaction was stopped by 2M H2SO4. The indirect ELISA was applied to detect the anti-recombinant protein antibody.16-22g weight ICR mice were inoculated with the three fusion proteins 4VP1, TCE-4VP1, TCE-2VP1 at a dose of 100μg per mice, whole bacteria expressing protein TCE-2VP1, inactivated vaccine and PBS were inoculated at the same time, respectively. Animals were boosted two weeks post the primary immunization. Blood samples were collected before immunization, 2, 4, 6weeks after first immunization. VP1 140-160aa specific antibodies in the sera were detected with indirect ELISA. 2 weeks after the third immunization the cytokine IFN-γand IL-4 were detected. The fusion protein TCE-4VP1 and whole bacteria expressing protein TCE-2VP1 could induce the highest level of antibodies. Higher mount of IFN-γwere induced by the fusion protein TCE-4VP1 and 4VP1. The three recombination proteins could increase the mount of IL-4, in which TCE-4VP1 is the best. It is concluded that the immunogenicity is increasing along with increasing the copies of 141-160aa-200-213aa fragments.