Production of the BmCecB1 antimicrobial peptide in transgenic silkworm

Kim, Seong Wan;Kim, Seong Ryul;Park, Seung Won;Choi, Kwang Ho;Goo, Tae Won

  • Received : 2015.11.06
  • Accepted : 2015.11.09
  • Published : 2015.12.31


This peptide has antibacterial activity against several Gram-positive and Gram-negative bacteria. Bombyx mori cecropinB1(BmCecB1) is antimicrobial peptides from Bombyx mori and belongs to cecropin family. Antimicrobial peptides are important components of the innate immune systems in all living organism. To produce the BmCecB1 antimicrobial peptide, we constructed transgenic silkworm that expressed BmCecB1 gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 600 eggs of bivoltin silkworms, Baegokjam. In total, 49 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 22 broods, and we selected 2 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 9%. We identified 9 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a BmCecB1 as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that BmCecB1 possesses high antibacterial activities against Gram-negative bacteria.


Cecropin;Transgenic silkworm;piggyback;EGFP;Bombyx mori


  1. Steiner H, Hultmark D, Engstrom A, Bennich H and Boman HG (1981) Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292(5820), 246-248.
  2. Sugiyama M, Kuniyoshi H, Kotani E, Taniai K, Kadono-Okuda K, Kato Y, Yamamoto M, Shimabukuro M, Chowdhury S and Xu J (1995) Characterization of a Bombyx mori cDNA encoding a novel member of the attacin family of insect antibacterial proteins. Insect biochemistry and molecular biology 25(3), 385-392.
  3. Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC and Couble P (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nature biotechnology 18(1), 81-84.
  4. Tanaka H, Ishibashi J, Fujita K, Nakajima Y, Sagisaka A, Tomimoto K, Suzuki N, Yoshiyama M, Kaneko Y, Iwasaki T, Sunagawa T, Yamaji K, Asaoka A, Mita K and Yamakawa M (2008) A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori. Insect biochemistry and molecular biology 38(12), 1087-1110 .
  5. Tanaka H and Yamakawa M (2011) Regulation of the innate immune responses in the silkworm, Bombyx mori. Isj-Invert Surviv J 8(1), 59-69.
  6. Tomotake H, Katagiri M and Yamato M (2010) Silkworm pupae (Bombyx mori) are new sources of high quality protein and lipid. Journal of nutritional science and vitaminology 56(6), 446-448.
  7. Wurm FM (2003) Human therapeutic proteins from silkworms. Nature biotechnology 21(1), 34-35.
  8. Christensen B, Fink J, Merrifield RB and Mauzerall D (1988) Channelforming properties of cecropins and related model compounds incorporated into planar lipid membranes. Proceedings of the National Academy of Sciences of the United States of America 85(14), 5072-5076.
  9. Hara S and Yamakawa M (1995) Moricin, a novel type of antibacterial peptide isolated from the silkworm, Bombyx mori. J Biol Chem 270(50), 29923-29927.
  10. Hoffmann JA (2003) The immune response of Drosophila. Nature 426(6962), 33-38.
  11. Kaneko Y, Furukawa S, Tanaka H and Yamakawa M (2007) Expression of antimicrobial peptide genes encoding Enbocin and Gloverin isoforms in the silkworm, Bombyx mori. Biosci Biotechnol Biochem 71(9), 2233-2241.
  12. Kaneko Y, Tanaka H, Ishibashi J, Iwasaki T and Yamakawa M (2008) Gene expression of a novel defensin antimicrobial peptide in the silkworm, Bombyx mori. Biosci Biotechnol Biochem 72(9), 2353-2361.
  13. Lemaitre B and Hoffmann J (2007) The host defense of Drosophila melanogaster. Annu Rev Immunol 25, 697-743.
  14. Looft T, Johnson TA, Allen HK, Bayles DO, Alt DP, Stedtfeld RD, Sul WJ, Stedtfeld T M, Chai B, Cole JR, Hashsham SA, Tiedje JM and Stanton TB (2012) In-feed antibiotic effects on the swine intestinal microbiome. Proceedings of the National Academy of Sciences of the United States of America 109(5), 1691-1696.
  15. Morishima I, Suginaka S, Ueno T and Hirano H (1990) Isolation and structure of cecropins, inducible antibacterial peptides, from the silkworm, Bombyx mori. Comp Biochem Physiol B 95(3), 551-554.
  16. Mourgues F, Brisset MN and Chevreau E (1998) Strategies to improve plant resistance to bacterial diseases through genetic engineering. Trends Biotechnol 16(5), 203-210.
  17. Andreu D and Rivas L (1998) Animal antimicrobial peptides: an overview. Biopolymers 47(6), 415-433.<415::AID-BIP2>3.0.CO;2-D
  18. Baltzer SA and Brown MH (2011) Antimicrobial peptides: promising alternatives to conventional antibiotics. Journal of molecular microbiology and biotechnology 20(4), 228-235 .
  19. Barton MD (2000) Antibiotic use in animal feed and its impact on human healt. Nutrition research reviews 13(2), 279-299.
  20. Berghammer AJ, Klingler M and Wimmer EA (1999) A universal marker for transgenic insects. Nature 402(6760), 370-371.