Expression of human granulocyte‐macrophage colony‐stimulating factor gene in insect cells by a baculovirus vector

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Abstract
A plasmid pAc373GM‐CSF was constructed and co‐transfected into Spodoptera frugiperda (Sf9) cells with wild‐type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. The recombinant virus vAc373GM‐CSF was identified and purified by several rounds of plaque hybridization. By assaying the culture medium, we demonstrated recombinant virus infected Sf9 cells expressing hGM‐CSF. Recombinant hGM‐CSFs with apparent molecular masses of 14.5, 15.5 and 16.5 kDa were detected by the Western blot method. All 3 forms have biological activity of hGM‐CSF. Following N‐glycanase treatment, a single band of 14.5–15.5 kDa appeared in SDS‐PAGE. Western blot analysis of expression in Sf9 cells treated with tunicamycin revealed only the presence of the 14.5 kDa species. Thus, the signal sequence of recombinant hGM‐CSF could be recognized and cleaved by infected insect cell and the resultant molecule secreted into the media.