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생화학분자생물학회 (Korean Society for Biochemistry and Molecular Biology)
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Construction of a Transposon-mediated Baculovirus Vector Hanpvid and a New Cell Line for Expressing Barnase

  • Qin, Qin (School of Life Science, Hubei University) ;
  • Liu, Ying-Le (School of Life Science, Wuhan University) ;
  • Zhu, Ying (School of Life Science, Wuhan University) ;
  • Li, Shun-Yi (School of Life Science, Hubei University) ;
  • Qi, Yi-Peng (School of Life Science, Wuhan University)
  • 발행 : 2005.01.31
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초록

In this study we developed the transposon-mediated shuttle vector 'Hanpvid', which composed of HaNPV (Heliothis armigera nuclear polyhedrosis virus) genomic DNA and a transposon cassette from Bacmid of Bac-to-Bac system. Hanpvid replicates in E. coli in the same way as Bacmid and retains infective function in cotton bollworm cells (Hz-AM1). Using Hanpvid we constructed a recombinant virus, which could infect Hz-AM1 cells and generate recombinant HaNPV (rHa-Bar) containing the barnase gene, a ribonuclease gene from Bacillus amyloliquefaciens. Since the expression vector carrying barnase gene cannot replicate in the absence of barstar, a specific inhibitor of barnase, we constructed a new cotton bollworm cell line (AM1-NB) using the marker rescue method. In AM1-NB barstar was integrated into the cellular chromosome to sustain the replication of rHa-Bar. To screen out recombinant HaNPV for potential use as biopesticide, Hz-AM1 and AM1-NB cell lines were infected with rHa-Bar, respectively. The results obtained indicate that Viral progenies in AM1-NB were 23 and 160 times greater than those in Hz-AM1 48 h and 72 h after infection, respectively. With additional insertion of the polyhedron gene from AcNPV (Autographa californica nuclear polyhedrosis virus) into the Hanpvid genome, rHa-Bar regained the polyhedron phenotype and its pest-killing rate greatly improved. Toxic analysis showed that the lethal dosages ($LD_{50}$) and the lethal time(s) ($LT_{50}$) of rHa-Bar were reduced by 20% and 30%, respectively, compared to wt-HaNPV in the third instar larvae of cotton bollworm. This study shows that in AM1-NB barnase can be effectively produced and used as pest-killing agent for the biological control of cotton pests.

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참고문헌

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피인용 문헌

  1. Improved baculovirus vectors expressing barnase using promoters from Cotesia plutellae bracovirus vol.28, pp.1, 2009, https://doi.org/10.1007/s10059-009-0096-x
  2. Fast and efficient generation of recombinant baculoviruses by in vitro transposition vol.96, pp.5, 2012, https://doi.org/10.1007/s00253-012-4468-3