[Improvement of baculovirus expression system and purification of IL-6 protein expressed in insect cells].

Abstract On the basis of site-specific transposition of an expression cassette into a baculovirus shuttle vector (Bacmid), which propagated in Escherichia coli , the Bac-to-Bac system provides a rapid and efficient method to generate recombinant baculoviruses and is widely used for high level expression of heterologous proteins. The efficiency of recombinant baculovirus infecting cells plays an important role in the protein expression. In this study, we have introduced an egfp expression cassette driven by polyhedrin promoter into the p74 locus of Bacmid by homologous recombination. The target Bacmid-egfp is then transformed into E. coli DH10B containing the transposition helper plasmid to construct a new transposition receipt strain E. coli DH10Bac-egfp. Because of the intact attTn7 sites in the lacZ α cassette, the target gene cloned in a pFastBac vector can be transposed into the Bacmid-egfp shutter vector to construct recombinant baculovirus, which would allow the tracking of the target protein expression and the recombinant Bacmid transfection and the recombinant baculoviral infection under fluorescence microscopes. Recombinant virus Bac-egfp-DsRed was constructed by transposing DsRed into the Bacmid-egfp in E. coli DH10Bac-egfp, and the Sf9 cells infected with the recombinant virus expressed DsRed and EGFP efficiently. Another protein IL-6 fused with 6×His tag was expressed and purified successfully from Sf9 cells infected with recombinant virus Bac-egfp-6×His-IL6 constructed by the improved Bac-to-Bac system.