Development Of DNA Vaccines With Multi-gene Of Foot-and-Mouth Disease Virus Serotype O

By use of site mutation strategy and PCR technology, we obtained the gene P12X3C that includes full length PI, 2A, 3C and a part of 2B and 3B and the gene P12X3C3D that includes full length PI, 2A, 3C, 3D and a part of 2B and 3B. After being digested by restriction enzyme respectively, the gene P12X3C and the gene P12X3C3D were cloned into the pcDNA3.1 (+) and pTARGET expression vector that were digested by the same enzyme. Recombinant plasmids were checked by restriction enzyme analysis and nucleic acid sequencing. Further more, recombinant plasmids were transfected into BHK-21 cells by using lipoid. The proteins of foot-and-mouth disease virus (FMDV), which were expressed in BHK-21 cells, were confirmed by sandwich-ELlSA and fluoroscopy, and the capsid of FMDV was tested by electron microscope. In order to evaluate enhanced immune response of guinea pigs against FMDV, DNA vaccines which were designed to produce viral capsids lacking infectious viral nucleic acid and contained the gene P12X3C and the gene P12X3C3D were injected respectively with FMDV 3D protein which was expressed in Pichia Pastoris Secreted expression System and purified or with pcDNA3.1/lFN which includes the gene IFN-a of cattle. Subsequently, Recombinant plasmids were injected to catties with or without pcDNA3.1/IFN. Anti-FMDV antibodies were detected by ELISA, and the T lymphocyte proliferation response was tested by MTT assay, neutralization antibodies liters were analyzed by micro-neutralization assay. The result shows:1) The gene P12X3C and P12X3C3D were cloned into eukaryotic expression plasmid correctly. The sequence of P12X3C and P12X3C3D contain 3042nt and 4452nt respectively. The percent identity of them to the same gene of FMDV China99 were 99.6% and 99.7% respectively.2) The recombinant eukaryotic expression plasmid pcDNA3.1/P12X3C, pcDNA3.1/P12X3C3D, pTARGET/P12X3C3D could express proteins of FMDV in BHK-21 cells, and the expressed proteins have immunocompetence and can produce the empty capsid of FMDV.3) Anti-FMDV antibodies were elicited and increased by plasmid pcDNA3.1/P12X3C in the second week after the first inoculation. Neutralization antibodies were induced with high level and the T lymphocyte proliferation response was enhanced after the second inoculation. In the challenge test, all guinea pigs immunized with pcDNA3.1/P12X3C were fully protected against FMDV challenge. But the result that obtained from the group injected with 3D together plasmid pcDNA3.1/P12X3C was not satisfied.4) Anti-FMDV antibodies were elicited and increased by plasmid pcDNA3.1/P12X3C, pcDNA3.1/P12X3C3D and pTAGRET/P12X3C3D in the second week after the first inoculation and increased further in the second inoculation. The T lymphocyte proliferation response was induced after the first inoculation and enhanced after the second inoculation. In the challenge test, only guinea pigs immunized with pcDNA3.1/P12X3C3D and pcDNA3.1/IFN were protected against FMDV challenge partially.5) Anti-FMDV antibodies and the T lymphocyte proliferation response of catties were elicited afterinoculation; The disease was delayed and symptoms were mild after challenge.In conclusion, the result encouraged further work towards the development of a DNA vaccine against FMDV and provided the basis of research for DNA vaccine.