Cytotechnology

, Volume 11, Issue 2, pp 121–131 | Cite as

Purification and characterization of interferon-like antiviral protein derived from flatfish (Paralichthys olivaceus) lymphocytes immortalized by oncogenes

  • Tadakazu Tamai
  • Sanetaka Shirahata
  • Nobuyuki Sato
  • Shoji Kimura
  • Michio Nonaka
  • Hiroki Murakami
Original Research Papers

Abstract

Flatfish leukocytes were transfected with the expression plasmids of the v-myc, c-myc, c-fos, v-myb and c-Ha-ras oncogenes. Only cotransfection of c-Ha-ras with c-myc or c-fos resulted in complete immortalization of the cells. Interferon-like anti-viral protein was found in the cultured medium of the immortalized lymphocytes. The protein was purified by DEAE-Toyopearl 650 M ion exchange chromatography and WGA agarose affinity chromatography. The protein was a glycoprotein of about 16 kDa. The antiviral activity of the protein was trypsin-sensitive and was fairly stable at pH values from 4 to 8. The protein retained about 60% of the activity even at 60°C and showed a broad antiviral activity for various fish cells and viruses.

Key words

cytokines fish leukocytes immortalization fish interferon oncogenes 

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References

  1. Allen G and Fantes KH (1980) A family of structural genes for human lympho blastoid leukocyte type interferon. Nature 287: 408–411.Google Scholar
  2. Bergmann DG, Souza LM and Baluda MA (1981) Vertebrate DNA species contain nucleotide sequences related to the transforming gene of avian myeloblastosis virus. J. Virol. 40: 450–455.Google Scholar
  3. Caspi RR, Sharabani R and Artalion RR (1980) The cells involved in the immune response of fish. I. The separation and study of lymphocyte subpopulation in carp — a new approach. In: Manning MJ (ed.) Phylogeny of Immunological Memory. (pp. 131) Elsevier/North Holland Biomed. Press, Amsterdam.Google Scholar
  4. Curran T, Macconnell WP, Van Straten F and Verma IM (1983) Structure of the FBJ murine osteo sarcoma virus genome molecular cloning of its associated helper virus and the cellular homologue of the v-fos gene from mouse and human cells. Mol. Cell. Biol. 3: 914–921.Google Scholar
  5. De Kinkelin P, Dorson M and Hattenberger-Baudouy AM (1982) Interferon synthesis in trout and carp after viral infection. Dev. Comp. Immun. Suppl. 2: 167–174.Google Scholar
  6. De Maeyer E and De Maeyer-Guignard J (1988) Interferon and Other Regulatory Cytokines, John Wiley and Sons, New York.Google Scholar
  7. De Sena J and Rio GJ (1975) Partial purification and characterization of RTG-2 fish cell interferon. Infect. Immunol. 11: 815–822.Google Scholar
  8. Dorson M, Barde A and de Kinkelin P (1975) Egtved virus induced rainbow trout serum interferon: some physicochemical properties. Ann. Microbial. (Inst. Pasteur) 126: 485–489.Google Scholar
  9. Dubois M, Gilles, KA, Hamilton, JK, Rebers, PA and Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350–356.Google Scholar
  10. Epstein I (1984) The special significance of interferon-gamma. In: Vilcek J and DeMaeyer E (eds) Interferon, Vol. 2: Interferons and the Immune System (pp. 195–220) Elsevier Science Publishers, Amsterdam.Google Scholar
  11. Graham D and Secombes CJ (1990) Do fish lymphocytes secrete interferon-γ? J. Fish Biol. 36: 563–573.Google Scholar
  12. Gravell M and Malsberger RG (1965) A permanent cell line from the fathead minnow (Pimephales promelas). Ann. NY Acad. Sci. 126: 555–565.Google Scholar
  13. Grob PM and Chadha KC (1979) Separation of human leukocyte interferon components by concanavalin A-agarose affinity chromatography and their characterization. Biochemistry 18: 5782–5786.Google Scholar
  14. Hashizume S, Rashid A, Shoji M and Kuroda K (1984) Electrophoretic extraction — concentration of proteins from polyacrylamide gels under alkaline, neutral, and acidic conditions. Electrophoresis 5: 30–34.Google Scholar
  15. Hosoi K, Utsumi J, Kitagawa T, Shimizu H and Kobayashi S (1988) Structural characterization of fibroblast human interferon-beta 1. J. Interferon Res. 8: 375–384.Google Scholar
  16. Jenuwein T and Muller R (1987) Structure-function analysis offos protein: a single amino acid change activates the immortalizing potential of v-fos. Cell 48: 647–657.Google Scholar
  17. Kelekar A and Cole MD (1987) Immortalization by c-myc, H-ras and Ela oncogenes induces differential cellular gene expression and growth factor responses. Mol. Cell. Biol. 7: 3899–3907.Google Scholar
  18. Kiener PA and Spitalny GL (1987) Induction, production and purification of murine gamma interferon. In: Clemens MJ, Morris AG and Gearing AJH (eds.) Lymphokines and Interferons. A Practical Approach (pp. 15–28) IRL Press, Oxford.Google Scholar
  19. Knight E, Jr. (1976) Interferon: Purification and initial characterization from human diploid cells. Proc. Nat. Acad. Sci. UsA 73: 520–523.Google Scholar
  20. Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature 227: 680–685.Google Scholar
  21. Land H, Parada LF and Weinberg RA (1983) Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature 304: 596–602.Google Scholar
  22. Langer JA and Pestka S (1988) Interferon receptors. Immunology Today 9: 393–400.Google Scholar
  23. Ledwith BJ, Manam S, Kraynak AR, Nichols WW and Bradley MO (1990) Antisense-fos RNA causes partial reversion of the transformed phenotypes induced by the c-Ha-ras oncogene. Mol. Cell. Biol. 10: 1545–1555.Google Scholar
  24. Merril CR (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211: 1437–1438.Google Scholar
  25. Mougneau E, Lemieux L, Rassoulzadegan M and Cuzin F (1984) Biological activities of v-myc and rearranged c-myc oncogenes in rat fibroblast cells in culture. Proc. Natl. Acad. Sci. 81: 5758–5762.Google Scholar
  26. Murakami H, Masui H, Sato GH, Sueoka N, Chow TP and Kano-Sueoka T (1982) Growth of hybridoma cells in serumfree medium: Ethanolamine is an essential component. Proc. Natl. Acad. Sci. USA 79: 1158–1162.Google Scholar
  27. Neumann E, Schaefer-Ridder M, Wang Y and Hofschneider PH (1982) Gene transfer into mouse myeloma cells by electroporation in high electric fields. EMBO J. 1: 841–845.Google Scholar
  28. Okamoto N, Shirakura T, Nagakura Y and Sano T (1983) The mechanism of interference with fish viral infection in the RTG-2 cell line. Fish Pathol. 18: 7–12.Google Scholar
  29. Rawson C, Shirahata S, Collodi P, Natsuno T and Barnes D (1991) Oncogene transformation frequency of nonsenescent SFME cells is increased by c-myc. Oncogene 6: 487–489.Google Scholar
  30. Sano E, Okano K, Sawaca R, Naruto M, Sudo T, Kamata K, Iizuka M and Kobayashi S (1988) Constitutive long-term production and characterization of recombinant human interferon-gamma from two different mammalian cells. Cell Struct. Funct. 13: 143–160.Google Scholar
  31. Sekiya T, Fushimi M, Hori H, Hirohashi S and Nishimura S (1983) Molecular cloning and the total nucleotide sequence of the human c-Ha-ras-1 gene activated in a melanoma from a Japanese patient. Proc. Natl. Acad. Sci. USA 81: 4771–4775.Google Scholar
  32. Shih C and Weinberg RA (1982) Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell 29: 161–169.Google Scholar
  33. Shirahata S, Rawson C, Loo D, Chang Y-J and Barnes D (1990)Ras andneu oncogenes reverse serum inhibition and epidermal growth factor dependence of serum-free mouse embryo cells. J. Cell. Physiol. 144: 69–76.Google Scholar
  34. Stewart WE (1980) Interferon nomenclature. Nature 286: 110.Google Scholar
  35. Tamai T, Sato N, Kimura S, Nonaka M, Shirahata S and Murakami H (1992) Cloning and expression of flatfish interleukin 2 gene. In: Murakami H, Shirahata S and Tachibana H (Eds.) Animal Cell Technology: Basic and Applied Aspects (pp. 509–514). Kluwer Academic Publishers. Dordrecht.Google Scholar
  36. Taniguchi T, Mantei N, Schwarzstein M, Nagata S, Muramatsu M and Weissmann C (1980) Human leukocyte and fibroblast interferons are structurally related. Nature 285: 547–549.Google Scholar
  37. Taya Y, Hosogai K, Hirohashi S, Shimosato Y, Tsuchiya R, Tsuchida N, Fushimi M, Sekiya T and Nishimura S (1984) A novel combination of K-ras andmyc amplification accompanied by point mutational activation of K-ras in a human lung cancer. EMBO J. 3: 2943–2946.Google Scholar
  38. Trinchieri G and Perussia B (1985) Immune interferon: a pleiotropic lymphokine with multiple effects. Immunology Today 6: 131–136.Google Scholar
  39. Weber K and Kuter DJ (1971) Reversible denaturation of enzymes by sodium dodecyl sulfate. J. Biol. Chem. 246: 4504–4509.Google Scholar
  40. Yip YK, Barrowclough BS, Urban C and Vilcek J (1982) Molecular weight of human gamma interferon is similar to that of other human interferons. Science 215: 411–413.Google Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Tadakazu Tamai
    • 1
  • Sanetaka Shirahata
    • 2
  • Nobuyuki Sato
    • 1
  • Shoji Kimura
    • 1
  • Michio Nonaka
    • 1
  • Hiroki Murakami
    • 2
  1. 1.Taiyo Central R&D InstituteTaiyo Fishery Co. LtdTsukuba-shi, IbarakiJapan
  2. 2.Graduate School of Genetic Resources TechnologyKyushu UniversityFukuokaJapan

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