Expression of FMDV RNA polymerase 3D gene in Pichia pastoris

Pichia expression system was used to express recombinant 3D gene in vitro. According to the codon preference of Pichia pastoris, the 3D gene got by digesting the pGEM-3D vector (using) restriction endonuclease,was modified artificially and cloned into secreted expression vector pPIC9K and transformed into E.coli. Positive recombinants were selected, sequenced and named pPIC9K/3D. The DNA linearized by SacⅠ was transformed into Pichia pastoris SMD1168 by electroporation. The expression vector pPIC9K/3D was integrated into SMD1168 via homologous recombination between the transforming DNA and regions of homology within the genome. The in vivo method utilizes (hyper) resistance against G418 to screen for possible multicopy inserts. The multicopy recombinant was named SMD1168/pPIC9K/3DHIS~(+)MUT~(+). The multicopy recombinant was induced expression with methanol. The expression products were tested by SDS-PAGE and Western blotting. The results showed that the 3D gene had been expressed successfully in Pichia pastoris. The expression product is a 52 ku protein and can react with the positive bovine serum of FMDV. The ratio of the protein is over 36% to the total protein secreted.