Cloning Of Cattle IL-18, Construction And Expression Of Multi-epitope Recombinant Of O Type Of Foot-and-Mouth Disease Virus (FMDV) And Detection Of Immunitical Activity

Foot-and-mouth disease virus (FMDV) infection results in Foot-and-mouth disease (FMD), a widespread and highly contagious anthropozoonosis, which greatly threaten cloven-foot animals and human health. Asa kind of Picornaviridae ,FMDV can infect many animals, such as cattle,sheep, goats, buffalos, deer, antelope, and pigs. Spreading swiftly served asa character to the FMD endemic. Seven serotypes (A, O, C, Asia1, SAT1, 2,3) and several subtypes of FMDV were recognized worldwide. There iscross-antigen among the different serotypes. Vaccine inoculation is a safeand effective way to prevent FMDV. For the inactivated vaccine which waswidely used currently existed latent danger, researchers wanted to findsome new type vaccine to substitute the inactivated vaccine. As a safe andcheap new type vaccine, the DNA vaccine was the best choice. Howeverthe development of DNA vaccine was limited by the lower immunogenicity.In this study, a DNA vaccine was constructed by chasing the major antigenepitope gene (mVP1) from three domestic O-type strains. And theimmunological adjuvant was used to enhance the DNA vaccineimmunogenicity.The mVP1 was cloned and expressed in the prokaryotic cell. SDS-PAGE showed that the mVP1 could be expressed efficiently in the E.coli,and accounted 18% of total bacterium protein. In addition, the recombinantprotein was purified in this study, providing antigen for the diagnosis of O-type FMDV.In order to obtain the gene immunological adjuvant, was cloned thecattle IL-18 gene (bIL-18) into the pMD18-T vector. Then the insert DNAfragment was sequenced. The result showed that the bIL-18 gene wascloned successfully. According the gene of FMDV and bIL18, the primersof the two genes were designed, fragment of bIL18 and fragment of MFDVSEA were cloned by RT-PCR. And then they were insert into the pMD18-Tvector to construct two recombinant plasmids respectively, and theobjective fragments were 609bp and 719bp respectively. By restrictionenzymes digestion and sequence analysis, the results showed two genefragments were obtained successfully. The cloned bIL-18 sequence showed84.9%～99.5% nucleotide identity and 74.9%～99% amino acid identitywith the IL-18 sequences from boar, horse, sheep, goat, cattle, dog, rat andhuman. Polygenetic analysis revealed that the cloned bIL-18 gene was highhomology to IL-18 gene of the cattle (99.5% and 99%), which only onepoint amino acid mutation (S101F). while the recombinant plasmid wasconstructed by cloning super-antigen gene (SEA) into the pMD18-T vector.Then the bIL18 gene, sea gene and synthetic gene mVP1 wereinserted into the eukaryotic expression vector pVAX1, three recombinantplasmids,pVAX1-mVP1 ,pVAX1-SEA-mVP1, pIRES-IL18-mVP1 wereconstructed successfully. Then the recombinant plasmids were transfectedinto HeLa cells, and the transcription and expression of interest gene weredetected by RT-PCR, indirect immunofluorescent assay and Westernblotting. the results showed that interest genes could express correctly ineukaryotic cells.BALB/c mice were immunized by the recombinant plasmidsseparately .Another group BALB/c mice were mmunized by three plasmidat the same time, and their immuned level, including humoral mediatedimmunity and cell mediated immunity, were detected. The indexesmanifested that the specific CTL activity and the antibody against theserotype O of FMDV could be induced by nucleic acid vaccine plasmids .Inaddtion, the antibody response induced by the recombinant plasmids wasconsiderably higher than control group.