Construction, Expression, Purification And Structural Optimization Of Recombinant Protein Vaccine For Type O Bovine Foot And Mouth Disease

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals such as cattle and pig. The disease causes explosive epidemics and heavy economic losses in the agriculture worldwide. As the causative agent, FMD virus is a single stranded positive sense RNA virus of 8500 bp and is enclosed by an icosahedral capsid. The capsid contains four structural proteins including, VP1, VP2, VP3 and VP4. FMDV shows a high genetic and antigenic variability, and has seven serotypes of O, A, C, AsiaI, SAT1, SAT2 and SAT3. No cross-protective immunity has been demonstrated among these serotypes.?Currently, using chemically inactivated FMDV vaccine is major strategy for controlling FMD. However, there still exist some dangerously potent factors including: limited cross protection between virus types; slow onset of immunity; persistent infection in vaccinated ruminants (carrier state) and an inability to serologically differentiate infected from vaccinated animals. To avoid using live FMD, efforts have been made to develop various other vaccines such as DNA vaccines, subunit vaccines, recombinant viral vector vaccines, transgenic plant vaccines, peptide-based vaccines and recombinant protein vaccines. Epitope vaccines have been explored and have key advantages. Due to consisting of only a small part of the FMDV, the epitope vaccine induces immune responses that can be distinguished from that induced by infection with FMDV and inoculation with FMDV inactivated vaccine. In recent years, recombinant FMDV epitope vaccine has been extensively studied, consisting of epitopes of VP1 in which a fragment (141—160aa) has been found to elicit protective neutralizing antibody in several species. Precisely, one of the major antigenic sites locates in the G-H loop of VP1. Using synthetic peptide-competition studies, this loop includes a highly conserved Arg-Gly-Asp (RGD) motif that has been identified as a cell binding site.?In the capsid protein of VP1, there are also two T-cell epitopes (positions 20-40aa and 200-213aa) capable of inducing T helper cell responses that are crucial for the production of FMDV neutralizing antibodies. Ideally, an effective FMD vaccine should include both B cell epitope and T cell epitope for stimulating higher titer neutralizing antibody which can protect animals against FMDV. ?In previous work in our lab, three bovine O type of FMD recombinant vaccines, FMD-J4, FMD-J5 and FMD-J8, were constructed by linking B-cell epitope and T-cell epitopes. However, the vaccine failed to induce the neutralizing antibody that protects the suckling mice from attacking by FMDV. So, we need to construct efficient recombinant protein vaccines for controlling type O bovine foot and mouth disease.In this study, we designed a series of encoding genes for recombinant proteins by using repeated B cell epitope (140-160aa) and two T cell epiopes (positions 20-40aa and 200-213aa) in different ways. Then, we predicted the 3D structure of these constructs by Gene 3D. According to the prediction, we chose one of the proteins, FMD-J11 whose 3-D structure of the exposed B-cell epitope was similar to one of the natural VP1 protein. Consequently, we constructed the encoding gene which was then subcloned into prokaryotic expression system pET28a. After being expressed in the host cell BL21(DE3), the stabilized and high yield genetic engineering bacteria was selected as seed cell for the fermentation. FMD-J11 protein was expressed in medium density fermentation successfully. Subsequently, FMD-J11 was purified through nickel affinity column. Guinea pigs were immunized with this protein. The result of ELISA and sera suckling mouse protection test showed that: The immunization of FMD-J11 can elicit FMDV specific antibody. Finally, in order to solve the purification problem derived from Cys residue of FMD-J11, the Cys of FMD-J11 was substituted with Ser, Gly and Ala by means of site-directed mutagenesis. Then we expressed, purified and identified these mutants.This study was composed of following parts1. Predict the 3D structure of proteinWith B cell epitope and T cell epitope derived from VP1 capsid protein of FMDV, we designed nine proteins with different structures predicted by Gene3D. In these proteins, it was found that FMD-J11 was a global protein which has one B-cell epitope located on the surface of the protein and the RGD sequence in the epitope was exposed. The 3-D structure of the exposed B-cell epitope was similar to that of the natural VP1 protein. Because of these features in the structure, we chose FMD-J11 as a candidate for further study.2. Construction, expression and identification of FMD-J11For constructing FMD-J11 encoding gene, we synthesized three different gene fragments (P, D and E) which were composed of B cell epitope and T cell epitopes by 2 round of PCR. The PCR products were analyzed by agarose gel electrophoresis. Then, P, D and E gene fragments were separately cloned into pMD18T vector, constructing the recombinant plasmids PMD18-T-P, PMD18-T-D and PMD18-T-E. The recombinant plasmid pMD18-T-E was digested by BglII and BamHⅠand E fragment was released. The E fragment was subcloned into pMD18-T-E at the site of BglII for constructing pMD18-T-EE. In similar way, pMD18-T-PDPDPEE recombinant plasmid was constructed. The PDPDPEE fragment was released from pMD18-T-PDPDPEE and then subcloned into the expression vector pET28a for constructing pET28a-PDPDPEE which was confirmed by DNA sequencing. pET28a-PDPDPEE was transformed into BL21(DE3) host cells. The recombinant protein FMD-J11 was expressed in the transformed BL21 cells by inducing with IPTG for 3 hours. The FMD-J11 was identified by SDS-PAGE and western blot. The result showed that FMD-J11 protein was successfully expressed but formed monomer, dimer and tetramer.3. The expression of FMD-J11 in medium density fermentationTo increase the expression yield of recombinant FMD-J11, we screened engineering bacteria as seed cell and optimized the culture conditions of fermentation. In the fermentation, a 10-L fermentor with a 5 L initial working volume was used in fed-batch mode. The temperature was 37°C and the pH was maintained at 7.0. The air was supplied at a rate of 1L/min and the agitation rate was controlled between 200 and 600 rpm to maintain dissolved oxygen (DO2) concentration at 50% air saturation. To control the specific growth rate and limit the production of metabolite by-products, we feed carbon source in DO2-State feeding strategy in the process of the fermentation. The bacteria grown continuesly until A600 value of the cell density was 5. Then the bacteria were induced with 1mM IPTG for 3 hours. The expression level of FMD-J11 was 30% of total bacterium protein analyzed by SDS–PAGE. The yield of the FMD-J11 in the medium density fermentation reached 30g/L.4. The purification of FMD-J11FMD-J11 was purified using nickel affinity chromatography under denaturing conditions and refolded to its native conformation. The result of SDS-PAGE showed that the expressed recombinant FMD-J11 was accumulated in inclusion body forms. The inclusion bodies were solubilized in a buffer containing 8M urea at pH 9.5. Then the bacteria suspension was centifugated to remove the cellular debris. The upernatant containing the recombinant FMD-J11 was applied onto the nickel affinity column. Non-specific binding protein was removed in a buffer containing 3M urea at pH 7.8 and. The denatured protein was refolded during the process of flowing buffer containing 1M urea. Finally, the His-tagged recombinant FMD-J11 was eluted in a buffer containing 1M urea at pH 7.8. The eluted fractions from affinity chromatography were applied gel filtration G-25 to remove the small molecular salt. The sample containing recombinant FMD–J11 was analyzed by 12% SDS–PAGE and confirmed by Western blot . SDS-PAGE analysis revealed that most impurities were FMD-J11 proteins which bind strongly to the affinity column. The purity of the recombinant FMD-J11 was approximately 98% as analyzed by SDS–PAGE, indicating that was an efficient purification process.5. The efficacy of FMD‐J11To determine that the recombinant protein of FMD-J11 induce a specific antibody response against serotype O of FMDV. The guinea pigs were inoculated with 200μg of FMD-J11 protein. As a comparison, we also inoculated guinea pigs with inactivated vaccines and FMD-J8 protein. Then the serum antibody of the guinea pigs was detected with Elisa and suckling mouse protection test. ELISA experiment revealed that the guinea pigs inoculated with FMD-J11 were able to elicit specific antibody which recognized the FMDV. The antibody titer was similar to the level of inactivated vaccine much higher than the level of FMD-J8. In addition, Suckling mice protection test was also performed. After being serial diluted, the serum of different concentrations was mixed with 100 TCID50 of live FMD virus. After 1 hour of incubation, the mixture was inoculated at the back of suckling mice. The mice were observed for 48 hours and the number of survived mice was recorded. By calculating formula, the titer of the antibody was nearly 1:125, which was similar to the level of inactivated vaccine and higher than the level of FMD-J8.6. The construction, expression and antigenic study of FMD-J11 mutantsTo solve the polymerization problem in the process of purification derived from cys residue in primary structure of recombinant protein FMD-J11, the Cys of FMD-J11 was substituted with Ser, Gly and Ala by means of site-directed mutagenesis. Codon cys was mutated to Ser, Gly and Ala through PCR. Then, mutation genes have been successfully achieved, as verified by sequencing. Using pET- 28a as vector, the mutants (FMD-J11-A, FMD-J11-S and FMD-J11-G) encoding gene were cloned and then transferred into BL21 (DE3). After induction by IPTG, the mutant proteins were obtained and analyzed by SDS - PAGE. The express level is about 30%. The mutant protein was purified successfully by nickel affinity column without formatation of polymers. By ELISA assay, the mutant FMD-J11-A showed higher antigenic than FMD-J11. So, this mutant FMD-J11-A represented a desirable substitute for native FMD-J11 to develop recombinant protein vaccine for type O bovine foot and mouth disease.Generally, we successfully constructed and expressed recombinant O type FMDV protein vaccine. Elisa and suckling mice protection test concluded that high titers of protective neutralizing antibody were produced after immunization of FMD-J11. To solve the problem of purification, we designed three mutants, experiment revealed that the mutant FMD-J11-A showed higher antigenic than FMD-J11. So, this mutant FMD-J11-A represented a desirable substitute for native FMD-J11 to develop recombinant protein vaccine for type O bovine foot and mouth disease.