title: Bombyx mori nucleopolyhedrovirus bacmid enabling rapid generation of recombinant virus by in vitro transposition.
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.
authors.affiliation: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P.R. China, Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, P.R. China.
authors.forename: Xue Ying
authors.initials: XY
authors.lastname: Tao
authors.forename: Jae Young
authors.initials: JY
authors.lastname: Choi
authors.forename: Yang-Su
authors.initials: YS
authors.lastname: Kim
authors.forename: Seok Hee
authors.initials: SH
authors.lastname: Lee
authors.forename: Saes Byeol
authors.initials: SB
authors.lastname: An
authors.forename: Ying
authors.initials: Y
authors.lastname: Pang
authors.forename: Jong Hoon
authors.initials: JH
authors.lastname: Kim
authors.forename: Woo Jin
authors.initials: WJ
authors.lastname: Kim
authors.forename: Yeon Ho
authors.initials: YH
authors.lastname: Je
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D012831:Bombyx / Q000821:virology*; D002460:Cell Line; D005822:Genetic Vectors; D016679:Genome, Viral; D049452:Green Fluorescent Proteins; D007313:Insecta / Q000382:microbiology; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins; D011995:Recombination, Genetic; D012260:Ribonucleases / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease