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KMID : 0545120150250030386
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Journal of Microbiology and Biotechnology
2015 Volume.25 No. 3 p.386 ~ p.392
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Bombyx mori Nucleopolyhedrovirus Bacmid Enabling Rapid Generation of Recombinant Virus by In Vitro Transposition
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Xue Ying Tao
Choi Jae-Young
Kim Yang-Su
Lee Seok-Hee
An Saes-Byeol
Ying Pang
Kim Jong-Hoon
Kim Woo-jin
Je Yeon-Ho
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Abstract
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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.
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KEYWORD
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baculovirus expression system, EasyBm, in vitro transposition, barnase, high throughput
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