![]() doi: 10.1007/s0025-2ĭeschuyteneer M, Elouahabi A, Plainchamp D, Plisnier M, Soete D, Corazza Y et al. Applied microbiology and biotechnology 85: 459–470. Kato T, Kajikawa M, Maenaka K, Park EY (2010) Silkworm expression system as a platform technology in life science. Tani H, Abe T, Matsunaga TM, Moriishi K, Matsuura Y (2008) Baculovirus vector for gene delivery and vaccine development. ![]() Recombinant DNA Technology and Application New York: McGraw-Hill pp. Luckow VL (1991) Cloning and expression of heterologous genes in insect cells withbaculovirus vectors In: Prokop A, Bajpai RK, Ho CS, editors. * indicates significant difference at P = 0.05. Quantitative comparison of polyhedra production of Sf21 cells infected with either AcBacPolh-SV40UTR or AcBacPolh-PolhUTR. Comparison of polyhedra production of Sf21 cells infected with either AcBacPolh-SV40UTR or AcBacPolh-PolhUTR. A separate membrane identical to that for the EGFP detection was probed with an anti-VP39 antibody. The separated proteins from the gel were transferred onto a nitrocellulose membrane and probed with an anti-GFP antibody to detect the amounts of EGFP production from different viral infections. Equal amounts of total proteins from Sf21 cells infected with viruses were used for SDS-PAGE. Western blotting analysis of EGFP yields by viral constructs with or without the SV40 polyA signal. EGFP produced by the Sf21 cells infected by AcegtSV40 - were diluted and fluorescence emission values were plotted against the number of cells. Validation of the fluorescence measurement to ascertain fluorescence values falling in the linear range. of 10 p.f.u./cell and the cells were harvested at 48 h post infection for fluorescence emission measurement in three independent cell infections. Sf21 cells were infected by different viral constructs at an m. Quantitative analysis of EGFP yields to test the effects of SV40 polyA on EGFP expression in the baculovirus expression vector system. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications).Ī. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. ![]() In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS).
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