Bombyx mori nucleopolyhedrovirus bacmid enabling rapid generation of recombinant virus by in vitro transposition

J Microbiol Biotechnol. 2015 Mar;25(3):386-92. doi: 10.4014/jmb.1408.08038.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins
  • Bombyx / virology*
  • Cell Line
  • Genetic Vectors
  • Genome, Viral*
  • Green Fluorescent Proteins
  • Insecta / microbiology
  • Nucleopolyhedroviruses / genetics*
  • Promoter Regions, Genetic
  • Recombinant Proteins
  • Recombination, Genetic*
  • Ribonucleases / genetics

Substances

  • Bacterial Proteins
  • Recombinant Proteins
  • Green Fluorescent Proteins
  • Ribonucleases
  • Bacillus amyloliquefaciens ribonuclease