One of the crucial factors for evaluation of an effective genetically engineered vaccine is whether susceptible animals are protected from virus challenge after vaccination. In this study, a recombinant pseudorabies virus (PRV-P12A3C) that expressed capsid precursor polypeptide P12A and nonstructural protein 3C of foot-and-mouth disease virus (FMDV) was used as a vaccine. The expression of P12A3C and its immunogenicity and protective efficacy against FMDV challenge were measured. Humoral and cellular immune responses were evaluated after each immunization. Subsequently, each piglet was challenged with 1000 ID50 (50% infection dose) FMDV serotype O, named OR/80, which is used to produce vaccine in China. PRV-P12A3C induced a high level of neutralizing antibody and FMDV-specific lymphocytes. Inactivated vaccine provided 100% protection, and the vector strain (TK−/gE−/gI−) showed no protection. PRV-P12A3C induced 60% protection, compared with piglets that were vaccinated with TK−/gE−/gI−. The severity of clinical signs for the remaining two piglets was lighter and the appearance of vesicles was delayed.

VP1 gene of foot-and-mouth disease virus(FMDV) is the major immunogenic gene as it contains T cell and B cell epitopes.In this study,we constructed a recombinant expressing plasmid(KG2VP1) including two VP1 genes(2VP1) of Asia 1 FMDV,and the 2VP1 gene was expressed in E.coli BL21.The fusional protein(GST2VP1) which was purified by Sephadex-G200 sieve chromatography demonstrated the reactionogenicity by Western blot.And the mice test indicated that the GST2VP1 can induce similar levels of ELISA and neutralization antibody like inactivated vaccine.We provide foundation for researching the immunogenicity of FMDV serotype Asia 1 and further application of GST2VP1 in future.

The primers was designed according to the sequences published in GenBank and genomic fragment containing P12A3C genes was amplied by PCR,then cloned into shuttle plasmid pShuttle-CMV.Subsequently the recombinant shuttle plamid(named pShuttle-PAC) was linearized and co-electroporated with the circular adenoviral genome plasmid pAdeasy-1 into competent BJ-5183 bacterial cells.The recombinant adenovirus plasmid(named pAd-PAC) was screened out via homologous recombination.After transfection of pAd-PAC into HEK293 cells,recombinant virus Ad-PAC was obtained and continuously cultured by ten cycles,bearing the titer of 105.5 TCID50/0.1 mL finally.The expression of the protein P12A3C was confirmed by indirect immunofluorescence.The genetic stability of Ad-PAC was examined by serial passages.Mice experiments showed Ad-PAC can induced high specific antiFMDV antibody,but the level of antibody was lower compared with inactivated vaccines.The results indicated the recombinant adenovirus Ad-PAC may provide a foundation of future research for the control strategy of FMD.

Monoclonal antibodies against FMDV vp2 protein were prepared and a competitive ELISA based on the monoclonal antibodies and vp2 protein was established. Balb/c mice were immunized with Escherichia coli expressed fusion protein. The splenocytes from immunized mice were fused with myeloma cells SP2/0. The hybridism cells were screened by indirect ELISA and limited dilution method. Two hybndoma cell Iines secreting mAbs against Asia I type foot-and-mouth disease were obtained. The titer and relative affinity of mAbs were determined by ELISA. Specificity of mAbs was analyzed by Western blotting. The ELISA titers of the ascites induced by the two hybridism cells were above 100 x 2(9).A competitive ELISA for the use of FMDV antibody detection was established using E. coli expressed fusion protein as coating antigen and HRP-labled mAb as detecting antibody. Clinical tests showed the method had 89.0 percent agreement with UBI Kit to detection of FMDV antibodies and 86.5 percent agreement with LPB- ELISA kit (Ceditest kit) for detection of antibodies against Foot-and-Mouth Disease Virus respectively.