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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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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)
  • Published : 2005.01.31
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Abstract

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.

Keywords

References

  1. Buckle, A. M., Schreiber, G. and Fersht, A. R. (1994) Proteinprotein recognition: Crystal structural analysis of a barnasebarstar complex at 2.0-angstrom resolution. Biochemistry 33, 8878-8889 https://doi.org/10.1021/bi00196a004
  2. Chakraborty, S., Greenfield, P. and Reid, S. (1996) In vitro production studies with a wild-type Helicoverpa baculovirus. Cytotechnology 22, 217-224 https://doi.org/10.1007/BF00353942
  3. Cuyno, L. C. M., Norton, G. W. and Rola, A. (2001) Economic analysis of environmental benefits of integrated pest management: a Philippine case study. Agric. Econ. Res. 25, 227-233 https://doi.org/10.1111/j.1574-0862.2001.tb00203.x
  4. Deyev, S. M., Yazynin, S. A., Kuznetsov, D. A., Jukovich, M. and Hartley, R. W. (1998) Ribonuclease-charged vector for facile direct cloning with positive selection. Mol. Gen Genet. 259, 379-382 https://doi.org/10.1007/s004380050825
  5. Hartley, R. W. and Rogerson, D. L. (1972) Production and purification of the extracellular ribonuclease of Bacillus amyloliquefaciens (barnase) and its intracellular inhibitor (barstar), I Barnase. Prep. Biochem. 2, 229-242 https://doi.org/10.1080/00327487208061473
  6. Hartley, R. W., Rogerson, D. L. and Smeaton, J. R. (1972) Production and purification of the extracellular ribonuclease of Bacillus amyloliquefaciens (barnase) and its intracellular inhibitor (barstar), II. Barstar. Prep. Biochem. 2, 43-250
  7. Hartley, R. W. and Smeaton, J. R. (1973) On the reaction between the extracellular ribonuclease of Bacillus amyloliquefaciens (barnase) and its intracellular inhibitor (barstar). J. Biol. Chem. 248, 5624-5626
  8. Hartley, R. W. (1989) Barnase and Barstar: two small proteins that fold and fit together. Trends. Biochem. Sci. 14, 450-454 https://doi.org/10.1016/0968-0004(89)90104-7
  9. Jagannath, A., Bandyopadhyay, P., Arumugam, N., Gupta, V., Burma, P. K. and Pental, D. (2001) The use of a Spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high-frequency generation of transgenic male sterile lines in Brassica juncea L. Mol. Breed. 8, 11-23 https://doi.org/10.1023/A:1011916216191
  10. Javis, D. L., Fleming, J. O., Koace, C. R., Summers, M. D. and Guarino, L. A. (1990) Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells. Biotechnology 8, 950-955 https://doi.org/10.1038/nbt1090-950
  11. Kitts, P. A., Ayers, M. D. and Possee, R. D. (1990) Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vector. Nucleic. Acids. Res. 18, 5667-5672 https://doi.org/10.1093/nar/18.19.5667
  12. Kitts, P. A. and Possee, R. D. (1993) A method for producing recombinant baculovirus vectors at high frequency. Bio-Techniques 14, 810-817
  13. Kuvshinov, V., Koivu, K., Kanerva, A. and Pehu, E. (2001) Molecular control of transgene escape from genetically modified plants. Plant Sci. 160, 517-522 https://doi.org/10.1016/S0168-9452(00)00414-3
  14. Leuchtenberger, T. H., Dunfield, P. F. and Liesack, W. (2001) Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells. Nucleic. Acids. Res. 29, 16-76 https://doi.org/10.1093/nar/29.4.e16
  15. Lu, S.-Y., Qi, Y.-P. and Ge, G.-Q. (2002) Interaction of Heliothis armigera nuclear polyhedrosis viral capsid protein with its host actin. J. Biochem. Mol. Biol. 35, 562-567 https://doi.org/10.5483/BMBRep.2002.35.6.562
  16. Luckow, V. A., Lee, S. C. and Barry, G. F. (1993) Efficient generation of infectious recombinant baculovirus by sitespecific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J. Virol. 67, 4566-4579
  17. Maugen, Y., Hartley, R. W., Dodson, E. J., Dodson, G. G., Bricogne, G., Chothia, C. and Jack, A. (1982) Molecular structure of a new family of ribonucleases. Nature 297, 162- 164 https://doi.org/10.1038/297162a0
  18. Maruniak, J. W. and Summers, M.D. (1981) Autographa californica nuclear polyhedrosis virus phosphoproteins and synthesis of intracellular proteins after virus infection. Virology 109, 25-34 https://doi.org/10.1016/0042-6822(81)90468-2
  19. Mishra, S. (1998) Baculoviruses as biopesticides. Curr. Sci. 75, 1015-1022
  20. Paddon, C. J. and Hartley, R. W. (1986) Cloning, sequencing and transcription of an inactivated copy of Bacillus amyloliquefaciens extra-cellular ribonuclease (barnase). Gene 40, 231-239 https://doi.org/10.1016/0378-1119(85)90045-9
  21. Paddon, C. J. and Hartley, R. W. (1987) Expression of Bacillus amyloliquefaciens extracellular ribonuclease (barnase) in Escherichia coli following an inactivated mutation. Gene 53, 11-19 https://doi.org/10.1016/0378-1119(87)90088-6
  22. Patel, G., Nasmyth, K. and Jones, N. (1992) A new method for the isolation of recombinant baculovirus. Nucleic. Acids. Res. 20, 97104
  23. Peakman, T. C., Harris, R. A. and Gewert, D. R. (1992) Highly efficient generation of recombinant baculoviruses by enzymatically mediated site-specific in vitro recombination. Nucleic. Acids. Res. 20, 495-500 https://doi.org/10.1093/nar/20.3.495
  24. Possee, D. R. (1997) Baculovirus as expression vectors. Curr. Opin. Biotechnol. 8, 569-572 https://doi.org/10.1016/S0958-1669(97)80030-4
  25. Raghunathan, V., Khurana, S., Gupta, V., Khurana, R., Udgaonkar, J. B. and Salunke, D. M. (1994) Crystallization and molecular packing analysis of barstar crystals. J. Mol. Biol. 243, 533-536 https://doi.org/10.1006/jmbi.1994.1679
  26. Smith, G. E., Summers, M. D. and Fraser, M. J. (1983) Production of human $\beta$-interferon with a baculovirus expression vector. Mol. Cell. Biol. 3, 2156-2165
  27. Zhu, Y., Qi, Y., Liu, D., Mallam, N. J. and Wang, Y. (1998a) A novel expression system based on host-range expansion of baculovirus. J. Virol. Methods. 76, 101-108 https://doi.org/10.1016/S0166-0934(98)00128-1
  28. Zhu, Y., Qi, Y. and Liu, Z. (1998b) Baculovirus-mediated gene transfer into mammalian cells. Sci. China. 41, 473-478 https://doi.org/10.1007/BF02877738

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