Co-expression And Identified Bioactivity Of VP1 Gen Of FMDV Type O And Goat IL-18 Gene In Sf9 Cells

Foot and Mouth Disease (FMD) is a highly contagious viral disease for cattle, swine, sheep, goat and other cloven-hoofed animals and the human, and was on the A list infectious disease of the Office International Epizooties, World Organization Animal Health (OIE). UP to date, the vaccination is a major means to prevent and control FMD. Many studies show that the structural protein VP1 is the dominating antigen and carries critical epitopes responsible for the induction of neutralizing antibodies, which has been paid many attentions for immunoprophylaxis of FMD and has significance in development of genetic engineering vaccine. Interleukin-18 has various bioactivities in immunoregulation, anti-infection and chronic inflammation. So IL-18 which was as a kind of efficient immune adjuvant for vaccine, can coexpress with some protective gene of pathogenic microorganism, thereby enhance and improve the immune effective of vaccine. The gene encoding goat IL-18 (gIL-18) and VP1 were coexpressed in insect baculovirus in our laboratory to search for gIL-18's immunoloregulation in FMD, and provides theoretical base for the prevention of FMD.According to the sequence published on GenBank, VP1 gene of FMDV type O and goat IL-18 gene were PCR amplified from recombinant plasmid pMD18-T-VP1 and pET32a-gIL18 by two pairs primers, respectively. Then the VP1 gene and gIL-18 gene were inserted into the downstreams of P10 promoter and PH promoter of baculovirus expression plasmid pFastBac~TM Dual, respectively. Finally, the constructing eukaryotic expression plasmid pFastBac~TM Dual-gIL18-VP1 was transformed into DH10Bac competent cells, and screened by three antibiotics (kanamycin, gentamycin and tetracycline) and blue-white patch. The recombinant bacmid DNA is extracted, on the basis of M13 universal primers(The bacmid contains M13 Forward and M13 Reverse priming sites flanking the mini-attTn7 site within the lacZα-complementation region to facilitate PCR analysis), we analyze the recombinant bacmid-gIL18-VP1 DNA by PCR. The recombinant Bacmid- gIL18-VP1 was transfected into sf9 cells with the Cellfectin regent. Then the recombinant baculovirus was infected sf9 cells. Harvested the cells in different times, the infected sf9 cells and its culture supernatant were collected and assayed by SDS-PAGE and Western blot. The recombinant baculovirus Bacmid-gIL18-VP1 can be successfully constructed by insect baculovrius expression system, and had high expression in the lysate supernatant of sf9 cells, the protein were dissolvable and had good immunogenicity, the intention banding were 23.5KD and 18.3KD by assay of SDS-PAGE and Western blot, respectively. The coexpression of gIL-18 and VP1 protein in sf9 cells were identified by IFA. Then identified bioactivity of the coexpressed production, the result showed that the recombinant protein could stimulate the proliferation of lymphocyte and inhibit the growth of VSV in MDBK by inducing IFN-γproduction in goat spleen lymphocyte. The study laid a foundation for gIL-18's immunoloregulation in FMD.