Abstract

The activities of three insect cell-specific promoters were compared and analyzed here, including the cytoplasmic Actin 3 (A3) promoter from Bombox mori (silkworm), immediately early 2 (ie2) promoter from Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) and the very late polyhedrin (polh) promoter fromAutographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Results of transient expression assays showed the activity of ie2 promoter was approximately forty fold higher in Spodoptera frugiperda 9 (Sf9) cells and thirty fold higher in ovarian cell line of Bombyx mori (BmN) than that of A3 promoter. However, it showed reverse effect in baculovirus system that the activity of A3 promoter became approximately ten fold higher than that of ie2 promoter in the late stage of baculovirus infection. We constructed recombinant baculoviruses with enhanced green fluorescent protein gene(EGFP) reporter gene controlled by individual A3, ie2 and polh promoter. The analysis result of EGFP expression level showed the activity of polh promoter was nearly 50 fold higher than that of ie2, and 5 times higher than that of A3 at 72 h post infection in Sf9 cells. The systemic studies on the activities of the three promoters indicate they are ideal drivers for the construction of a time sequential baculovirus expression system.      

Key words: Promoter activity, immediately early promoter, very late promoter, transient expression, recombinant baculovirus.

Abbreviation

AcMNPV,     Autographa    californica   multicapsid nucleopolyhedro-virus; BmNPV, Bombyx mori nucleopolyhedrovirus; OpMNPV, Orgyia pseudotsugatamulticapsid nucleopolyhedrovirus; B. mori, Bombyx mori/silkworm; egfp, enhanced green fluorescent protein gene; A3, Actin 3; ie-2, immediately early 2; ie-1, immediately early; polh, polyhedron; hr, homologous region; E. coli, Escherichia coli;h.p.i, hours post infection; MOI, multiplicity of infection; PCR, polymerase chain reaction; PFU, plaque forming unit; Sf9, Spodoptera frugiperda 9; ONPG, o-Nitrophenyl beta-D-glucoside; PBS, phosphate buffered saline; TEN buffer, TrisEDTA Sodium chloride buffer; β-gal/lacZ, β-galactosidase.