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http://dx.doi.org/10.4014/jmb.1408.08038

Bombyx mori Nucleopolyhedrovirus Bacmid Enabling Rapid Generation of Recombinant Virus by In Vitro Transposition  

Tao, Xue Ying (State Key Laboratory of Food Science and Technology, Nanchang University)
Choi, Jae Young (Research Institute for Agriculture and Life Sciences, Seoul National University)
Kim, Yang-Su (Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University)
Lee, Seok Hee (Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University)
An, Saes Byeol (Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University)
Pang, Ying (Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University)
Kim, Jong Hoon (Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University)
Kim, Woo Jin (Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University)
Je, Yeon Ho (Research Institute for Agriculture and Life Sciences, Seoul National University)
Publication Information
Journal of Microbiology and Biotechnology / v.25, no.3, 2015 , pp. 386-392 More about this Journal
Abstract
A novel recombinant bacmid, bEasyBm, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBm, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the nonrecombinant background. The bEasyBm bacmid could only replicate in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBm was transposed with pDualBac-EGFP, the resulting recombinant virus, EasyBm-EGFP, showed high levels of EGFP expression efficiency compared with that of non-purified recombinant virus BmGOZA-EGFP, which was constructed using the bBmGOZA system. In addition, nonrecombinant backgrounds were not detected in unpurified EasyBm-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established.
Keywords
Baculovirus expression system; EasyBm; in vitro transposition; barnase; high throughput;
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