Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Improved Baculovirus Vectors Expressing Barnase Using Promoters from Cotesia plutellae Bracovirus

  • Choi, Jae Young (Research Institute for Agriculture and Life Sciences, Seoul National University) ;
  • Kim, Yang-Su (Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University) ;
  • Wang, Yong (Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University) ;
  • Kang, Joong Nam (Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University) ;
  • Roh, Jong Yul (Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University) ;
  • Shim, Hee Jin (Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University) ;
  • Woo, Soo-Dong (Department of Plant Medicine, College of Agriculture, Life and Environment Sciences, Chungbuk National University) ;
  • Jin, Byung Rae (College of Natural Resources and Life Science, Dong-A University) ;
  • Je, Yeon Ho (Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University)
  • Received : 2009.03.03
  • Accepted : 2009.05.25
  • Published : 2009.07.31
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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 nonrecombinant 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

Acknowledgement

Supported by : Rural Development Administration

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