Journal of Sericultural and Entomological Science (한국잠사곤충학회지)

Korean Society of Sericultural Science (한국잠사학회)
권호 표지
DOI QR코드

DOI QR Code

Production of the yellow fluorescent silk using the fibroin heavy chain protein expression system in transgenic silkworm

피브로인 H-chain 재조합 단백질 발현시스템을 이용한 황색형광실크의 제작

  • Received : 2014.09.29
  • Accepted : 2014.10.20
  • Published : 2014.10.31
Download PDF
⟨ Previous Next ⟩

Abstract

We constructed the fibroin H-chain expression system to produce enhanced yellow fluorescent proteins (EYFP) in the silk of transgenic silkworm. Fluorescent silk could be made by fusing EYFP cDNA to the heavy chain gene and injecting it into a silkworm. The EYFP fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the EYFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The yellow fluorescence proving that the fusion protein was present in the silk. Accordingly, we suggest that the EYFP fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.

본 연구의 목적은 누에형질전환 기술과 피브로인 재조합 단백질 발현시스템을 이용하여 황색형광실크를 개발하는 것으로서, 본 실험에서는 피브로인 H-chain의 N-말단과 C-말단을 이용하여 피브로인 재조합 단백질 발현 시스템을 제작하였고, 종결코돈이 없는 EYFP 유전자를 위의 발현 시스템에 클로닝하여 황색형광실크를 제작하였다. 누에형질전환체 선발을 위해서는 3xP3 promoter와 EGFP 유전자를 이용하여 선발하였고, 3,060개의 누에알에 microinjection 하여 F1 세대에서 8 bloods의 누에형질전환체를 선발하였다. 선발된 누에형질전환체는 초기배 단계의 눈과 신경조직, 유충과 번데기 그리고 성충의 눈에서 EGFP 형광단백질이 발현되는 것을 확인할 수 있었다. 또한 실크의 피브로인에서 EYFP 단백질이 발현되는 것을 확인하기 위해, F2세대의 누에형질전환체중에서 5령 3일 유충의 견사선을 형광현미경으로 관찰하였고, 중부 견사선에서 황색형광단백질이 발현되는 것을 확인할 수 있었다. 또한 F2 세대의 고치와 저온에서 정련한 실크에서도 황색형광단백질의 발현을 확인할 수 있었고, Western blot 분석에서도 EYFP 재조합 단백질이 피브로인 H-chain과 융합된 형태로 존재하는 것이 확인되었다. 이상의 결과에서 황색형광실크를 생산하는 누에형질전환체가 성공적으로 제작되었음을 확인할 수 있었다.

Keywords

References

  1. Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen JS, Lu H, Richmond J, Kaplan DL (2003) Silk-based biomaterials. Biomaterials 24, 401-416. https://doi.org/10.1016/S0142-9612(02)00353-8
  2. Hino R, Tomita M, Yoshizato K (2006) The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor. Biomaterials 27, 5715-5724. https://doi.org/10.1016/j.biomaterials.2006.07.028
  3. Inoue S, Kanda T, Imamura M, Quan GX, Kojima K, Tanaka H, Tomita M, Hino R, Yoshizato K, Mizuno S, Tamura T (2005) A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins. Insect biochemistry and molecular biology 35, 51-59. https://doi.org/10.1016/j.ibmb.2004.10.002
  4. Inoue S, Tanaka K, Arisaka F, Kimura S, Ohtomo K, Mizuno S (2000) Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6 : 6 : 1 molar ratio. J Biol Chem 275, 40517-40528. https://doi.org/10.1074/jbc.M006897200
  5. Kurihara H, Sezutsu H, Tamura T, Yamada K (2007) Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system. Biochemical and biophysical research communications 355, 976-980. https://doi.org/10.1016/j.bbrc.2007.02.055
  6. Ogawa S, Tomita M, Shimizu K, Yoshizato K (2007) Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin. Journal of Biotechnology 128, 531-544. https://doi.org/10.1016/j.jbiotec.2006.10.019
  7. Tamura T, Iizuka T, Sezutsu H, Tatematsu K, Kobayashi I, Yonemura N, Uchino K, Kojima K, Machii H, Takabayashi C, Yamada K, Kurihara H, Asakura T, Nakazawa Y, Miyawaki A, Karasawa T, Kobayashi H, Yamaguchi J, Kuwabara N, Nakamura T, Yoshii K (2009) Production of high quality silks having different fluorescent colors using transgenic silkworms. AFF Research Journal 32(3), 7-10.
  8. 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, Couble P (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nature biotechnology 18, 81-84. https://doi.org/10.1038/71978
  9. Tanaka K, Kajiyama N, Ishikura K, Waga S, Kikuchi A, Ohtomo K, Takagi T, Mizuno S (1999) Determination of the site of disulfide linkage between heavy and light chains of silk fibroin produced by Bombix mori. Bba-Protein Struct M 1432, 92-103. https://doi.org/10.1016/S0167-4838(99)00088-6
  10. Tatemastu K, Sezutsu H, Tamura T (2012) Utilization of transgenic silkworms for recombinant protein production. Journal of Biotechnology & Biomaterials S9, 1-8.
  11. Thomas JL, Da Rocha M, Besse A, Mauchamp B, Chavancy G (2002) 3xP3-EGFP marker facilitates screening for transgenic silkworm Bombyx mori L. from the embryonic stage onwards. Insect biochemistry and molecular biology 32, 247-253. https://doi.org/10.1016/S0965-1748(01)00150-3
  12. Tomita M, Munetsuna H, Sato T, Adachi T, Hino R, Hayashi M, Shimizu K, Nakamura N, Tamura T, Yoshizato K (2003) Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nature biotechnology 21, 52-56. https://doi.org/10.1038/nbt771
  13. Yamada H, Nakao H, Takasu Y, Tsubouchi K (2001) Preparation of undegraded native molecular fibroin solution from silkworm cocoons. Mat Sci Eng C-Bio S 14, 41-46. https://doi.org/10.1016/S0928-4931(01)00207-7
  14. Zhao Y, Li X, Cao G, Xue R, Gong C (2009) Expression of hIGF-I in the silk glands of transgenic silkworms and in transformed silkworm cells. Sci China C Life Sci 52, 1131-1139. https://doi.org/10.1007/s11427-009-0148-7
  15. Zhou CZ, Confalonieri F, Esnault C, Zivanovic Y, Jacquet M, Janin J, Perasso R, Li ZG, Duguet M (2003) The 62-kb upstream region of Bombyx mori fibroin heavy chain gene is clustered of repetitive elements and candidate matrix association regions. Gene 312, 189-195. https://doi.org/10.1016/S0378-1119(03)00616-4
  16. Zhou CZ, Confalonieri F, Medina N, Zivanovic Y, Esnault C, Yang T, Jacquet M, Janin J, Duguet M, Perasso R, Li ZG (2000) Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acids Res 28, 2413-2419. https://doi.org/10.1093/nar/28.12.2413