Analyses Of The Immunogenicity Of The Multi-epitope DNA Vaccine Against Food-and-mouth Disease Virus Type Asia Ⅰ

To construct the eukaryotic expression plasmid encoding the multi-epitope of Food-and-mouthDisease Virus(FMDV) type AsiaⅠ, a genetic fragment F comprising two segments of B cell epitope(VP1135-159aa、VP1194-211aa) and two segments of T cell epitope (3A21-35aa、3D795-803aa) was synthesized.Then the recombinant plasmid pc-F was constructed by cloned the genetic fragment F into theeukaryotic expression vector pcDNA3.1(+) using the restriction enzyme HindⅢ and XbaⅠ. Afterconfirmed by enzyme digestion and sequence analysis, the recombinant plasmids pc-F were transfectedinto BHK-21cells by using LipofectamineTM2000. The western blot and IFA were used to confired thatif the recombant plasmids pc-F could correctly espressed in BHK-21cells.The gene of bovine IL-4was obtained by RT-PCR from peripheral blood lymphocytes and wascloned into the vector pMD-18T. And The gene of bovine IL-4without the sequences of the signalpeptide, named SIL4, was obtained by PCR and was cloned into the the eukaryotic expression vectorpcDNA3.1(+) using the restriction enzyme NheⅠand KpnⅠto construct the recombant plasmidpc-SIL4. After confirmed by enzyme digestion and sequence analysis, the recombinant plasmidspc-SIL4were transfected into BHK-21cells by using LipofectamineTM2000. Then the IFA was used toidentify the protein expressed by the recombinant plasmids pc-SIL4within the BHK-21cells.The BALB/C mouses were divided into five groups, and were vaccinated with PBS、pcDNA3.1(+)、pc-F、pc-F+pc-SIL4and inactive vaccine to value the potence of the DNA vaccine pc-F.The results showed:1) The genetic fragment F was synthesized and had759nt. And the genetic fragment F was clonedinto the pcDNA3.1(+) successfully. The percent identity of it to the same gene provided by thereport was100%.2) Bovine IL-4gene had576nt. And The percent identity of it to the same genes of bovine IL-4genewas99.7%. Bovine IL-4gene without the sequences of the signal peptide, named SIL4, wasamplified by PCR and was cloned into the pcDNA3.1(+) successfully. And this genetic fragmentSIL4had342nt.The percent identity of it to the same genes of bovine IL-4gene was100%.3) The results of IFA test showed that the proteins expressed by the recombant plasmids pc-SIL4andpc-F had good reactionogenicity. And the expressed protein of the recombant plasmid pc-F was25.4Ku.4) Compared with the control groups, the humoral and cell-mediated immune responses were seen inthe pc-F+the pc-SIL4coinoculated group、pc-F inoculated group and inactivated virus vaccineinoculated group.5) Compared to the group immunized with pc-F alone, the co-inoculation with pc-SIL4induced ahigher levels of IgG and expression of IL-4, but the lower level of expression of IFN-γ.