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Mol. Cells 2009; 28(1): 19-24

Published online July 31, 2009

https://doi.org/10.1007/s10059-009-0096-x

© The Korean Society for Molecular and Cellular Biology

Improved Baculovirus Vectors Expressing Barnase Using Promoters from Cotesia plutellae Bracovirus

Jae Young Choi, Yang-Su Kim, Yong Wang, Joong Nam Kang, Jong Yul Roh, Hee Jin Shim,Soo-Dong Woo, Byung Rae Jin, and Yeon Ho Je

Received: March 3, 2009; Revised: May 22, 2009; Accepted: May 25, 2009

Abstract

The goal of this study was to create a novel baculovirus expression system that does not require recombinant virus purification steps. Transfection of insect cells with transfer vectors containing barnase under control of the Cotesia plutellae bracovirus (CpBV) promoters ORF3004 or ORF3005 reduced cell growth. Co-transfection with bApGOZA DNA yielded no recombinant viruses and non-recombinant backgrounds. To further investigate the detrimental effects of barnase on insect cells, two recombinant bacmids harboring the barnase gene under control of the CpBV promoters, namely bAcFast-3004ProBarnase and bAcFast-3005ProBarnase, were constructed. While no viral replication was observed when only the recombinant bacmids were transfected, recombinant viruses were generated when the bacmids were co-transfected with the transfer vector, pAcUWPolh, through substitution of the barnase gene with the native polyhedrin gene by homologous recombination. Moreover, no non-recombinant backgrounds were detected from unpurified recombinant stocks using PCR analysis. These results indicate that CpBV promoters can be used to improve baculovirus expression vectors by means of lethal gene expression under the control of these promoters.

Keywords baculovirus, barnase, Cotesia plutellae bracovirus, early promoter, expression vector

Article

Research Article

Mol. Cells 2009; 28(1): 19-24

Published online July 31, 2009 https://doi.org/10.1007/s10059-009-0096-x

Copyright © The Korean Society for Molecular and Cellular Biology.

Improved Baculovirus Vectors Expressing Barnase Using Promoters from Cotesia plutellae Bracovirus

Jae Young Choi, Yang-Su Kim, Yong Wang, Joong Nam Kang, Jong Yul Roh, Hee Jin Shim,Soo-Dong Woo, Byung Rae Jin, and Yeon Ho Je

Received: March 3, 2009; Revised: May 22, 2009; Accepted: May 25, 2009

Abstract

The goal of this study was to create a novel baculovirus expression system that does not require recombinant virus purification steps. Transfection of insect cells with transfer vectors containing barnase under control of the Cotesia plutellae bracovirus (CpBV) promoters ORF3004 or ORF3005 reduced cell growth. Co-transfection with bApGOZA DNA yielded no recombinant viruses and non-recombinant backgrounds. To further investigate the detrimental effects of barnase on insect cells, two recombinant bacmids harboring the barnase gene under control of the CpBV promoters, namely bAcFast-3004ProBarnase and bAcFast-3005ProBarnase, were constructed. While no viral replication was observed when only the recombinant bacmids were transfected, recombinant viruses were generated when the bacmids were co-transfected with the transfer vector, pAcUWPolh, through substitution of the barnase gene with the native polyhedrin gene by homologous recombination. Moreover, no non-recombinant backgrounds were detected from unpurified recombinant stocks using PCR analysis. These results indicate that CpBV promoters can be used to improve baculovirus expression vectors by means of lethal gene expression under the control of these promoters.

Keywords: baculovirus, barnase, Cotesia plutellae bracovirus, early promoter, expression vector

Mol. Cells
Nov 30, 2022 Vol.45 No.11, pp. 763~867
COVER PICTURE
Naive (cyan) and axotomized (magenta) retinal ganglion cell axons in Xenopus tropicalis (Choi et al., pp. 846-854).

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