Construction And Efficacy Of The Recombinant Protein Vaccine For Type O Foot And Mouth Disease Virus

Foot and mouth disease (FMD), an economically important disease of cattle, sheep, goats and pigs, is one of the most contagious diseases known to humanity. The agent of FMD is foot and mouth disease virus (FMDV). FMDV belongs to the aphthovirus genus of the Picornaviridae family. The particles are icosahedral symmetry which composed of 60 copies of each of four capsid proteins termed VP1, VP2, VP3 and VP4. The major immunodominant site on the virion surface resides on theββG-βH loop of VP1 (G-H loop) which includes a highly conserved Arg-Gly-Asp (RGD) sequence Its genome within the icosahedral symmetry is a single stranded RNA of about 8500 nucleotides of positive polarity.FMDV elicits a rapid humoral-specific response in infected animals. Neutralising antibodies can be detected soon after infection or vaccination with FMDV. Specific T-cell responses are also elicited by FMDV infection and vaccination. In addition, FMDV infection results in a rapid reduction of MHC class I expression on the surface of susceptible cells. At present, attenuated and killed viral vaccine are widespread utilized to prevent animal from being infected. But because of following risky factors such as the recovery of virulence and the incomplete inactivation of the virus and escape of live virus, new type of vaccine safer and more effective are under intensive study worldwide. Recombinant protein vaccine produced by host cells such as Ecoli carrying an encoding gene of the FMDV antigen epitopes only has the utility immunological strcture of FMDV, so it can stimulate the immune response and ensure the safety.To construct recombinant vaccine against FMDV, the epitopes of the FMDV was selected first of all. FMDV induce humoral immunity dependence of Th cell. So B cell epitopes and Th cell epitopes are selected to construct the recombinant vaccine. The second structure prediction method can reflect the conformation tendency of local sequence of the nature protein, so we predict the B cell epitopes of the VP1 of FMDV basing this method. The fineness strcture of T cell epitopes is also very important in the immunological recognition. Now, the T cell epitopes location the on the VP1 of FMDV have been exactly researched.In study, we have produced 3 recombinant vaccines against FMDV and tested the efficacy of one of the vaccines, composing of following parts:1. The prediction of B cell epitopes of VP1 of type O FMDV (XJ strain, GenBank Accession 830046)To predict B cell epitopes on VP1 protein of FMDV, the second structure, hydrophilicity, flexible regions, surface accessibility and antigenic index of VP1 sequences of FMDV type O–XJ strain were analyzed with DNA star. Upon the reserachment, above parameter are correlated with B cell epitopes. According above parameter, we have selected a B cell epitope on VP1 contain 21 amino acids.2. The design of recombinant proteinTo improve the immunogenicity of recombinant protein, the coding gene of T cell epitope and B cell epitope derived from VP1 of type O FMDV were connected to design the 4 kinds of fragment: H, A (Xianglan Jin, in our lab), B (Xianglan Jin, in our lab) and N (Ming Yang, in our lab).Then repeated these fragment several times to design the recombinant protein. In this way, 3 kinds of recombinant protein were designed: H6, H(AH)3 and (BBN)2, named them with OJ1, OJ2 and OJ3.3. The construction of encoding gene recombinant vaccinesThe coding genes of H fragment were synthesized after 2 round of PCR using specific primers and the products (165bp) of PCR were confirmed by agarose gel electrophorosis. The coding genes of H fragment were subsequently subcloned into pMD18T vector and the product of ligation reaction was transformed into JM109 host cell. After antibiotic screening, plasmids were extracted. The recombinant plasmid was digested by EcoRI, a gene fragment of 195 was released. After sequenced the insertion element, the gene was confirmed to be exactly the gene designed. Then the coding gene H6, H(AH)3 and (BBN)2 of OJ1, OJ2 and OJ3 were constructed via isocaudarner ligation reaction.4. The expression, identification and purification of OJ1, OJ2 and OJ3The recombinant plasmid pMD18-T-H6, pMD18-T-H(AH)3 and pMD18-T-(BBN)2 were digested by EcoRⅠand HindIII then the gene of H6, H(AH)3 and (BBN)2 were released and recovered from agarose gel. The genes were subcloned into pET28a vector at the site of EcoRⅠand HindIII. Recombinant clones were identified after agarose gel electrophoresis. After sequenced the insertion element of pET28a-H6, pET28a-H(AH)3 and pET28a-(BBN)2, the insertion element sequences were affirmed exactly what we have designed. Then recombinant plasmids pET28a-H6, pET28a-H(AH)3 and pET28a-(BBN)2 were transformed into L21 host cells and positive clones wree selected and cultured. After being induce in the presence of IPTG, the host cells were collected and lysyed. The lysates were characterized by SDS-PAGE. The exogenous proteins, presenting an additional band, were confirmed by SDS-PAGE with the predicted size. The proteins were further confirmed by Western blot analysis. At last recombinant proteins were purified through nickel affinity chromatography.5. The efficacy of OJ3 (recombinant protein vaccine for FMDV type O)To verify immunogenicity of OJ3, guinea pigs and calf were immunized with OJ3. Then the serum antibody of the guinea pigs and calf were detected with Elisa and suckling mouse protection test. In addition, the immuinized calves were challenged with FMDV virus in order to estimate whether OJ3 can induce protection of susceptible animals against FMDV.Indirect ELISA was carried out to test the specific antibody in sera collected from guinea pigs and calves. The result showed that: the immunization of guinea pigs with OJ3 can elicit antibody which recognized the FMDV, The titer of antibody on day 106 is nearly to the level on day 43 and much higher than the level of traditional killed vaccine on day 106; the immunizationof calves with OJ3 can elicit antibody which recognized the FMDV, the antibody titer elicit by immunization of OJ3 is much higher than the level of killed vaccine.To estimate the protection of suckling mouse attacked by FMDV with sera from guinea pigs and calves immunized with OJ3, sucking mouse protection test was also carried out. After being serial diluted, the serum of was mixed with 100 TCID50 of live FMD virus. After 1 hour of incubation, the mixture was injected at the back of suckling mouse. The mousese were observed for 72 hours and the number of survived mouse recorded. Based on judgment standard, if the serum at 1:32 dilution can protect all the test animals, the presence of neutralizing antibodies is believed. The result of guinea pigs sera suckling mouse protection test showed that: the immunization of OJ3 can elicit FMDV specific antibody in guinea pigs, on day 21 post first immunization, a total protection was abserved by using the sera from 50%guinea pigs at 1:32 dilution and from 25%guinea pigs at 1:64 dilution. On day 21 post second immunization, a total protection was abserved by using the sera from 50%guinea pigs at 1:32 dilution and from 25%guinea pigs at 1:256 dilution. The result of calves sera suckling mouse protection test showed that: the immunization of OJ3 can elicit FMDV specific antibody in calves sera. On day 21 post first immunization, a total protection was abserved by using the sera from 20%calves at 1:64 dilution. On day 14 post second immunization, a total protection was abserved by using the sera from 80%calves at 1:64 dilution and from 60%calves at 1:128. The immuinized calves were challenged with FMDV in order to estimate whether OJ3 can induce protection of susceptible animals against FMDV. The calves were inoculated with FMDV type O on day 21 post second immunization. After 10 days, 20% calves immunized with OJ3 develop symptom of FMD. The killed vaccine group calves and PBS group calves are 20% and 100%, respectively. The result of virus challenge test showed that OJ3 can induce protection of susceptible animals against FMDV.According this study, the OJ3 recombinant protein could be developed into an effective vaccine vaccine for prevention of type O FMD.