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Research article

References

  1. Airenne KJ, Peltomaa E, Hytonen VP, Laitinen OH, YlaHerttuala S. 2003. Improved generation of recombinant baculovirus genomes in Escherichia coli. Nucleic Acids Res. 31:e101.
    Pubmed PMC CrossRef
  2. Choi JY, Kim YS, Wang Y, Tao XY, Liu Q, Roh JY, et al. 2012. Fast and efficient generation of recombinant baculoviruses by in vitro transposition. Appl. Microbiol. Biotechnol. 96: 1353-1360.
    Pubmed CrossRef
  3. Choi JY, Kwon SJ, Roh JY, Yang TJ, Li MS, Park BS, et al. 2009. Analysis of promoter activity of selected Cotesia plutellae bracovirus genes. J. Gen. Virol. 90: 1262-1269.
    Pubmed CrossRef
  4. Choi JY, Woo SD, Lee HK, Hong HK, Je YH, Park JH, et al. 2000. High-level expression of canine parvovirus VP2 using Bombyx mori nucleopolyhedrovirus vector. Arch. Virol. 145:171-177.
    Pubmed CrossRef
  5. Hartley RW. 1989. Barnase and barstar - 2 small proteins to fold and fit Together. Trends Biochem. Sci. 14: 450-454.
    CrossRef
  6. Hong HK, Woo SD, Choi JY, Lee HK, Kim MH, Je YH, Kang SK. 2001. Comparison of promoter activity of the p10 gene between Bombyx mori nucleopolyhedrovirus variants. J. Microbiol. Biotechnol. 11: 585-591.
  7. Je YH, Chang JH, Choi JY, Roh JY, Jin BR, O’Reilly DR, Kang SK. 2001. A defective viral genome maintained in Escherichia coli for the generation of baculovirus expression vectors. Biotechnol. Lett. 23: 575-582.
    CrossRef
  8. Je YH, Chang JH, Kim MH, Roh JY, Jin BR, O’Reilly DR. 2001. The use of defective Bombyx mori nucleopolyhedrovirus genomes maintained in Escherichia coli for the rapid generation of occlusion-positive and occlusion-negative expression vectors. Biotechnol. Lett. 23: 1809-1817.
    CrossRef
  9. Kawakami N, Lee JM, Mon H, Kubo Y, Banno Y, Kawaguchi Y, et al. 2008. Efficient protein expression in Bombyx mori larvae of the strain d17 highly sensitive to B. mori nucleopolyhedrovirus. Mol. Biotechnol. 40: 180-185.
    Pubmed CrossRef
  10. Lee KS, Kim BY, Je YH, Woo SD, Sohn HD, Jin BR. 2007. A new technique for producing recombinant baculovirus directly in silkworm larvae. Biotechnol. Lett. 29: 175-180.
    Pubmed CrossRef
  11. Maeda S. 1989. Expression of foreign genes in insects using baculovirus vectors. Annu. Rev. Entomol. 34: 351-372.
    Pubmed CrossRef
  12. Motohashi T, Shimojima T, Fukagawa T, Maenaka K, Park EY. 2005. Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system. Biochem. Biophys. Res. Commun. 326:564-569.
    Pubmed CrossRef
  13. O’Reilly DR. 1997. Use of baculovirus expression vectors. Methods Mol. Biol. 62: 235-246.
    CrossRef
  14. Possee RD, Hitchman RB, Richards KS, Mann SG, Siaterli E, Nixon CP, et al. 2008. Generation of baculovirus v ectors for the high-throughput production of proteins in insect cells. Biotechnol. Bioeng. 101: 1115-1122.
    Pubmed CrossRef
  15. Qin Q, Liu YL, Zhu Y, Li SY, Qi YP. 2005. Construction of a transposon-mediated baculovirus vector Hanpvid and a new cell line for expressing barnase. J. Biochem. Mol. Biol. 38: 41-48.
    Pubmed CrossRef
  16. Reis U, Blum B, von Specht BU, Domdey H, Collins J. 1992. Antibody production in silkworm cells and silkworm larvae infected with a dual recombinant Bombyx mori nuclear polyhedrosis virus. Biotechnology (NY) 10: 910-912.
    CrossRef
  17. Schlaeppi JM, Henke M, Mahnke M, Hartmann S, Schmitz R, Pouliquen Y, et al. 2006. A semi-automated large-scale process for the production of recombinant tagged proteins in the Baculovirus expression system. Protein Expr. Purif. 50:185-195.
    Pubmed CrossRef
  18. Yao LG, Liu ZC, Zhang XM, Kan YC, Zhou JJ. 2007. A highly efficient method for the generation of a recombinant Bombyx mori nuclear-polyhedrosis-virus Bacmid and largescale expression of foreign proteins in silkworm (B. mori) larvae. Biotechnol. Appl. Biochem. 48: 45-53.
    Pubmed CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2015; 25(3): 386-392

Published online March 28, 2015 https://doi.org/10.4014/jmb.1408.08038

Copyright © The Korean Society for Microbiology and Biotechnology.

Bombyx mori Nucleopolyhedrovirus Bacmid Enabling Rapid Generation of Recombinant Virus by In Vitro Transposition

Xue Ying Tao 1, 2, Jae Young Choi 3, Yang-Su Kim 4, Seok Hee Lee 4, Saes Byeol An 4, Ying Pang 4, Jong Hoon Kim 4, Woo Jin Kim 4 and Yeon Ho Je 3, 4*

1State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P.R. China, 2Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, P.R. China, 3Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Republic of Korea, 4Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University, Seoul 151-742, Republic of Korea

Received: August 18, 2014; Accepted: October 15, 2014

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.

Keywords: baculovirus expression system, EasyBm, in vitro transposition, barnase, high-throughput

References

  1. Airenne KJ, Peltomaa E, Hytonen VP, Laitinen OH, YlaHerttuala S. 2003. Improved generation of recombinant baculovirus genomes in Escherichia coli. Nucleic Acids Res. 31:e101.
    Pubmed KoreaMed CrossRef
  2. Choi JY, Kim YS, Wang Y, Tao XY, Liu Q, Roh JY, et al. 2012. Fast and efficient generation of recombinant baculoviruses by in vitro transposition. Appl. Microbiol. Biotechnol. 96: 1353-1360.
    Pubmed CrossRef
  3. Choi JY, Kwon SJ, Roh JY, Yang TJ, Li MS, Park BS, et al. 2009. Analysis of promoter activity of selected Cotesia plutellae bracovirus genes. J. Gen. Virol. 90: 1262-1269.
    Pubmed CrossRef
  4. Choi JY, Woo SD, Lee HK, Hong HK, Je YH, Park JH, et al. 2000. High-level expression of canine parvovirus VP2 using Bombyx mori nucleopolyhedrovirus vector. Arch. Virol. 145:171-177.
    Pubmed CrossRef
  5. Hartley RW. 1989. Barnase and barstar - 2 small proteins to fold and fit Together. Trends Biochem. Sci. 14: 450-454.
    CrossRef
  6. Hong HK, Woo SD, Choi JY, Lee HK, Kim MH, Je YH, Kang SK. 2001. Comparison of promoter activity of the p10 gene between Bombyx mori nucleopolyhedrovirus variants. J. Microbiol. Biotechnol. 11: 585-591.
  7. Je YH, Chang JH, Choi JY, Roh JY, Jin BR, O’Reilly DR, Kang SK. 2001. A defective viral genome maintained in Escherichia coli for the generation of baculovirus expression vectors. Biotechnol. Lett. 23: 575-582.
    CrossRef
  8. Je YH, Chang JH, Kim MH, Roh JY, Jin BR, O’Reilly DR. 2001. The use of defective Bombyx mori nucleopolyhedrovirus genomes maintained in Escherichia coli for the rapid generation of occlusion-positive and occlusion-negative expression vectors. Biotechnol. Lett. 23: 1809-1817.
    CrossRef
  9. Kawakami N, Lee JM, Mon H, Kubo Y, Banno Y, Kawaguchi Y, et al. 2008. Efficient protein expression in Bombyx mori larvae of the strain d17 highly sensitive to B. mori nucleopolyhedrovirus. Mol. Biotechnol. 40: 180-185.
    Pubmed CrossRef
  10. Lee KS, Kim BY, Je YH, Woo SD, Sohn HD, Jin BR. 2007. A new technique for producing recombinant baculovirus directly in silkworm larvae. Biotechnol. Lett. 29: 175-180.
    Pubmed CrossRef
  11. Maeda S. 1989. Expression of foreign genes in insects using baculovirus vectors. Annu. Rev. Entomol. 34: 351-372.
    Pubmed CrossRef
  12. Motohashi T, Shimojima T, Fukagawa T, Maenaka K, Park EY. 2005. Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system. Biochem. Biophys. Res. Commun. 326:564-569.
    Pubmed CrossRef
  13. O’Reilly DR. 1997. Use of baculovirus expression vectors. Methods Mol. Biol. 62: 235-246.
    CrossRef
  14. Possee RD, Hitchman RB, Richards KS, Mann SG, Siaterli E, Nixon CP, et al. 2008. Generation of baculovirus v ectors for the high-throughput production of proteins in insect cells. Biotechnol. Bioeng. 101: 1115-1122.
    Pubmed CrossRef
  15. Qin Q, Liu YL, Zhu Y, Li SY, Qi YP. 2005. Construction of a transposon-mediated baculovirus vector Hanpvid and a new cell line for expressing barnase. J. Biochem. Mol. Biol. 38: 41-48.
    Pubmed CrossRef
  16. Reis U, Blum B, von Specht BU, Domdey H, Collins J. 1992. Antibody production in silkworm cells and silkworm larvae infected with a dual recombinant Bombyx mori nuclear polyhedrosis virus. Biotechnology (NY) 10: 910-912.
    CrossRef
  17. Schlaeppi JM, Henke M, Mahnke M, Hartmann S, Schmitz R, Pouliquen Y, et al. 2006. A semi-automated large-scale process for the production of recombinant tagged proteins in the Baculovirus expression system. Protein Expr. Purif. 50:185-195.
    Pubmed CrossRef
  18. Yao LG, Liu ZC, Zhang XM, Kan YC, Zhou JJ. 2007. A highly efficient method for the generation of a recombinant Bombyx mori nuclear-polyhedrosis-virus Bacmid and largescale expression of foreign proteins in silkworm (B. mori) larvae. Biotechnol. Appl. Biochem. 48: 45-53.
    Pubmed CrossRef