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. 2000 Mar 10;45(3):297–310. doi: 10.1016/0165-2427(94)05345-S

Cloning, expression and characterization of biologically active feline tumour necrosis factor-α

Espen Rimstad 1,∗, Gerhard H Reubel 1, Gregg A Dean 1, Joanne Higgins 1, Niels C Pedersen 1
PMCID: PMC7119607  PMID: 7676612

Abstract

We report the cloning, expression and characterization of biologically active feline tumour necrosis factor-α (fTNF-α). Messenger RNA was extracted from feline peritoneal macrophage cultures and used to synthesize cDNA for polymerase chain reaction (PCR) amplification. The PCR products were cloned into the plasmid vector pCRII and sequenced, showing 99.3% homology with a published fTNF-α gene sequence. Subcloning into the vector pGEX-2T and subsequent expression resulted in a 43 kDa fusion protein of fTNF-α and glutathione S-transferase (GST). Thrombin cleavage of the fusion protein yielded a 17 kDa protein. This protein cross-reacted with a monoclonal anti-human TNF-α antibody in Western blotting, but not with a polyclonal anti-murine TNF-α serum. Recombinant fTNF-α (rfTNF-α) and rfTNF-α-GST had a CD50 of 15 ng ml−1 and 230 ng ml−1, respectively, in the L929 cytotoxicity assay. Cats given rfTNF-α-GST intravenously manifested the typical biological effects of TNF-α, including fever, depression, and piloerection. The rfTNF-α-GST upregulated IL-2 receptor and MHC-II antigen expression on peripheral blood mononuclear cells stimulated in vitro, but had no effect on TNF-α receptor and MHC-I antigen expression.

References

  1. Bradford M. A rapid and sensitive method for the quantification of microgram quantities of proteins utilising the principle of protein dye binding. Anal. Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Brunner D., Pedersen N.C. Infection of peritoneal macrophages in vitro and in vivo with feline immunodeficiency virus. J. Virol. 1989;63:5483–5488. doi: 10.1128/jvi.63.12.5483-5488.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carswell E.A., Old L.J., Kassel R.L., Green S., Fiore N., Wiliamson B. Vol. 72. 1975. An endotoxin-induced serum factor that causes necrosis of tumours; pp. 3666–3670. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang J.-Y. Thrombin specificity. Requirement for a polar amino acid adjacent to the thrombin cleavage site of a polypeptide substrate. Eur. J. Biochem. 1985;151:217–224. doi: 10.1111/j.1432-1033.1985.tb09091.x. [DOI] [PubMed] [Google Scholar]
  5. Creagan E.T., Kovach J.S., Moertel C.G., Frytak S., Kvols L.K. A phase I clinical trial of recombinant human tumour necrosis factor. Cancer. 1988;62:2467–2471. doi: 10.1002/1097-0142(19881215)62:12<2467::aid-cncr2820621202>3.0.co;2-5. [DOI] [PubMed] [Google Scholar]
  6. Dewhurst S., Embretson J.E., Anderson D.C., Mullins J.I., Fultz P.N. sequence analysis and acute pathogenicity of molecularly cloned SIVSMM-PBj14. Nature. 1990;345:636–640. doi: 10.1038/345636a0. [DOI] [PubMed] [Google Scholar]
  7. Drews R.T., Coffee B.W., Prestwood A.K., McGraw R.A. Gene sequence of porcine tumour necrosis factor alpha. Nucleic Acids Res. 1990;18:5564. doi: 10.1093/nar/18.18.5564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Flick D.A., Gifford G.E. Comparison of in vitro cell cytotoxic assays for tumour necrosis factor. J. Immunol. Meth. 1984;68:167–175. doi: 10.1016/0022-1759(84)90147-9. [DOI] [PubMed] [Google Scholar]
  9. Folks T.M., Clouse K.A., Justement J., Rabson A., Duh E., Kehrl J.H., Fauci A.D. Vol. 86. 1989. Tumor necrosis factor a induces expression of human immunodeficiency virus in a chronically infected T-cell clone; p. 2365. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Green I.R., Sargan D.R. Sequence of the cDNA encoding ovine tumour necrosis factor-alpha: problems with cloning by inverse PCR. Gene. 1991;109:203–210. doi: 10.1016/0378-1119(91)90610-n. [DOI] [PubMed] [Google Scholar]
  11. Ito H., Shirai T., Yamamoto S., Akira M., Kawahara S., Todd S., Wallace R.B. Molecular cloning of the gene encoding rabbit tumour necrosis factor. DNA. 1986;5:157–165. doi: 10.1089/dna.1986.5.157. [DOI] [PubMed] [Google Scholar]
  12. Jäättelä M. Biologic activities and mechanisms of action of tumour necrosis factor-a/cachectin. Lab. Invest. 1991;64:724–742. [PubMed] [Google Scholar]
  13. Lawrence C.E., Callanen J.J., Jarrett O. Decreased mitogen responsiveness and elevated tumour necrosis factor production in cats shortly after feline immunodeficiency virus infection. In: Dandekar S., Pedersen N., editors. Feline Immunodeficiency Virus. Vol. 35. 1992. pp. 51–59. (Vet. Immunol. Immunopathol.). [DOI] [PubMed] [Google Scholar]
  14. Lehmann R., Joller H., Haagmans B.L., Lutz H. Tumor necrosis factor a levels in cats experimentally infected with feline immunodeficiency virus: effects of immunization and feline leukemia virus infection. In: Dandekar S., Pedersen N., editors. Feline Immunodeficiency Virus. Vol. 35. 1992. pp. 61–69. (Vet. Immunol. Immunopathol.). [DOI] [PubMed] [Google Scholar]
  15. Lowenthal J.W., Ballard D.W., Bogerd H., Bohnlein E., Greene W.C. Tumor necrosis factor-alpha activation of the IL-2 receptor-alpha gene involves the induction of kappa B-specific DNA binding proteins. J. Immunol. 1989;142:3121–3128. [PubMed] [Google Scholar]
  16. Lowenthal J.W., Ballard D.W., Böhlein E., Greene W.C. Vol. 86. 1989. Tumor necrosis factor a induces proteins that bind specifically to kB-like enhancer elements and regulate interleukin 2 receptor a-chain gene expression in primary human T-lymphocytes; p. 2331. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lutz H., Pedersen N.C., Higgiins J., Hübscher U., Troy F.A., Theilen G.H. Humoral immune reactivity to feline leukemia virus and associated antigens in cats naturally infected with feline leukemia virus. Cancer Res. 1980;40:3642–3651. [PubMed] [Google Scholar]
  18. Maniatis T., Goodbourn S., Fisher J.A. Regulation of inducible and tissue-specific gene expression. Science. 1987;236:1237–1245. doi: 10.1126/science.3296191. [DOI] [PubMed] [Google Scholar]
  19. Marmenout A., Fransen L., Tavernier J., van der Hayden J., Tijard R., Kawashima E., Shaw A., Johnson M.-J., Semon D., Muller R., Ruysschaert M.-R., van Vliet A., Fiers W. Molecular cloning and expression of human tumour necrosis factor and comparison with mouse tumour necrosis factor. Eur. J. Biochem. 1985;152:512–522. doi: 10.1111/j.1432-1033.1985.tb09226.x. [DOI] [PubMed] [Google Scholar]
  20. Matsuyama T., Kobayashi N., Yamamoto N. Cytokines and HIV infection: is AIDS a tumour necrosis factor disease? AIDS. 1991;5:1405–1417. doi: 10.1097/00002030-199112000-00001. [DOI] [PubMed] [Google Scholar]
  21. McGraw R.A., Coffee B.W., Otto C.M., Drews R.T., Rawlings C.A. Gene sequence of feline tumour necrosis factor alpha. Nucleic Acids Res. 1990;18:5563. doi: 10.1093/nar/18.18.5563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nicolau D.P., West T.E. Thalidomide: treatment of severe recurrent aphthous stomatitis in patients with AIDS. DICP. 1990;24:1054–1056. doi: 10.1177/106002809002401105. [DOI] [PubMed] [Google Scholar]
  23. Ohno K., Goitsuka R., Kitamura K., Hasegawa A., Tokunaga T., Honda M. Production of a monoclonal antibody that defines the alpha subunit of the feline IL-2 receptor. Hybridoma. 1992;11:595–605. doi: 10.1089/hyb.1992.11.595. [DOI] [PubMed] [Google Scholar]
  24. Osborn L., Kunkel S., Nabel G.J. Vol. 86. 1989. Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancers by activation of the nuclear factor kB; pp. 2336–2340. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pedersen N.C. The feline immunodeficiency virus. In: Levy J., editor. Vol. 2. Plenum Press; New York: 1993. pp. 181–228. (The Retroviridae). [Google Scholar]
  26. Pennica D., Hayflick J.S., Bringham T.S., Palladino M.A., Goeddel D.V. Vol. 82. 1985. Cloning and expression in E. coli of the cDNA for murine tumour necrosis factor; pp. 6060–6064. (Proc. Natl. Acad. Sci.). [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pessara U., Koch N. Tumor necrosis factor a regulates expression of the major histocompatibility complex class II-associated invariant chain by binding of an NF-kB-like factor to apromotor element. Mol. Cell. Biol. 1990;10:4146. doi: 10.1128/mcb.10.8.4146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Poli G., Kinter A., Justement J.S., Kehrl J.H., Bressler P., Stanley S., Fauci A.D. Vol. 87. 1990. Tumor necrosis factor a functions in an autocrine manner in the induction of human immunodeficient virus expression; p. 782. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pollack M.S., Hayes A., Mooney S., Pedersen N.C., Cook R.G. The detection of conventional class I and class II I-E homologue major histocompatibility complex molecules on feline cells. Vet. Immunol. Immunopathol. 1988;19:79–91. doi: 10.1016/0165-2427(88)90048-7. [DOI] [PubMed] [Google Scholar]
  30. Reid G., Rigby M.A., McDonald M., Hosie M.J., Neil J.C., Jarrett O. Immunodiagnosis of feline immunodeficiency virus infection using recombinant viral p17 and p24. AIDS. 1991;5:1477–1483. doi: 10.1097/00002030-199112000-00010. [DOI] [PubMed] [Google Scholar]
  31. Rideout B.A., Moore P.F., Pedresen N.C. Persistent upregulation of MHC class II antigen expression on T lymphocytes from cats experimentally infected with feline immunodeficiency virus. In: Dandekar S., Pedersen N., editors. Feline Immunodeficiency Virus. Vol. 35. 1992. pp. 71–82. (Vet. Immunol. Immunopathol.). [DOI] [PubMed] [Google Scholar]
  32. Shirai T., Yamaguchi H., Ito H., Todd C.W., Wallace R.B. Cloning and expression in Escherichia coli of the gene for human tumour necrosis factor. Nature. 1985;313:803–806. doi: 10.1038/313803a0. [DOI] [PubMed] [Google Scholar]
  33. Smith D.B., Davern K.M., Board P.G., Tiu W.U., Garcia E.G., Mitchell G.F. Vol. 83. 1986. Mr 26000 antigen of Schistosoma japonicum recognized by resistant WEHI 129J mice is a parasite glutathione S-transferase; pp. 8703–8707. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sparger E.E., Shacklett B.L., Renshaw G.L., Barry P.A., Pedersen N.C., Elder J.H., Luciw P.A. Regulation of gene expression directed by the long terminal repeat of the feline immunodeficiency virus. Virology. 1992;187:165–177. doi: 10.1016/0042-6822(92)90305-9. [DOI] [PubMed] [Google Scholar]
  35. Stoddart C.A., Scott F.W. Isolation and identification of feline peritoneal macrophages for in vitro studies of coronavirus-macrophage interactions. J. Leuc. Biol. 1988;44:319–328. doi: 10.1002/jlb.44.5.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Suzuki M., Yamamoto N., Shinozaki F. Augmentation of in vitro HIV replication in peripheral blood mononuclear cells of AIDS and ARC patients by tumour necrosis factor. Lancet. 1989;i:1206. doi: 10.1016/s0140-6736(89)92788-8. [DOI] [PubMed] [Google Scholar]

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