Production of biologically active feline interferon beta in insect larvae using a recombinant baculovirus


Feline interferon beta is a cytokine that belongs to the type I IFN family, with antitumor, antiviral and immunomodulatory functions. In this work, recombinant feline interferon beta (rFeIFNβ) was expressed in insect larvae that constitute important agronomic plagues. rFeIFNβ accumulated in the hemolymph of Spodoptera frugiperda larvae infected with recombinant baculovirus and was purified by Blue-Sepharose chromatography directly from larval homogenates on day 4 post-infection. rFeIFNβ was recovered after purification with a specific activity of 1 × 106 IU mg−1. By this method, we obtained 8.9 × 104 IU of purified rFeIFNβ per larva. The product was biologically active in vitro, with an antiviral activity of 9.5 × 104 IU mL−1, as well as a potent antitumor activity comparable to that of the commercial FeIFNω. The glycosylation of rFeIFNβ was confirmed by peptide-N-glycosidase F digestion. Our findings provide a cost-effective platform for large-scale rFeIFNβ production in laboratory research or veterinary medicine applications.

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Autographa californica multiple nucleopolyhedrovirus


Feline interferon


Recombinant feline interferon


Molecular weight


Fetal bovine serum


Vesicular stomatitis virus


Modified Eagle’s medium


Multiplicity of infection


Day(s) post-infection


  1. Argyle DJ, Harris M, Lawrence C, McBride K, Barron R, McGillivray C, Onions DE (1998) Expression of feline recombinant interferon-gamma in baculovirus and demonstration of biological activity. Vet Immunol Immunopathol 64(2):97–105

    Article  CAS  PubMed  Google Scholar 

  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  3. Burges HD, Croizier G, Huber J (1980) A review of safety tests on baculoviruses. Entomophaga 25(4):329–340

    Article  Google Scholar 

  4. Chamorey AL, Magne N, Pivot X, Milano G (2002) Impact of glycosylation on the effect of cytokines. A special focus on oncology. Eur Cytok Netw 13(2):154–160

    CAS  Google Scholar 

  5. Chawla-Sarkar M, Leaman DW, Borden EC (2001) Preferential induction of apoptosis by interferon (IFN)-beta compared with IFN-alpha2: correlation with TRAIL/Apo2L induction in melanoma cell lines. Clin Cancer Res 7(6):1821–1831

    CAS  PubMed  Google Scholar 

  6. Coelho LF, Magno de Freitas Almeida G, Mennechet FJ, Blangy A, Uze G (2005) Interferon-alpha and -beta differentially regulate osteoclastogenesis: role of differential induction of chemokine CXCL11 expression. Proc Natl Acad Sci USA 102(33):11917–11922.

    Article  CAS  PubMed  Google Scholar 

  7. Damdinsuren B, Nagano H, Wada H, Kondo M, Ota H, Nakamura M, Noda T, Natsag J, Yamamoto H, Doki Y, Umeshita K, Dono K, Nakamori S, Sakon M, Monden M (2007) Stronger growth-inhibitory effect of interferon (IFN)-beta compared to IFN-alpha is mediated by IFN signaling pathway in hepatocellular carcinoma cells. Int J Oncol 30(1):201–208

    CAS  PubMed  Google Scholar 

  8. de Weerd NA, Vivian JP, Nguyen TK, Mangan NE, Gould JA, Braniff SJ, Zaker-Tabrizi L, Fung KY, Forster SC, Beddoe T, Reid HH, Rossjohn J, Hertzog PJ (2013) Structural basis of a unique interferon-beta signaling axis mediated via the receptor IFNAR1. Nat Immunol 14(9):901–907.

    Article  CAS  PubMed  Google Scholar 

  9. Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA (2009) Spheroid-based drug screen: considerations and practical approach. Nat Protoc 4(3):309–324.

    Article  CAS  PubMed  Google Scholar 

  10. Gronowski AM, Hilbert DM, Sheehan KC, Garotta G, Schreiber RD (1999) Baculovirus stimulates antiviral effects in mammalian cells. J Virol 73(12):9944–9951

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Hervas-Stubbs S, Rueda P, Lopez L, Lecrerc C (2007) Insect baculoviruses strongly potentiate adaptative immune responses by inducing Type I IFN. J Immunol 178(4):2361–2369.

    Article  CAS  PubMed  Google Scholar 

  12. Isaacs A, Lindenmann J (1957) Virus interference. I. The interferon. Proc R Soc Lond Ser B Biol Sci 147(927):258–267

    Article  CAS  Google Scholar 

  13. Iwata A, Saito T, Mizukoshi-Iwata N, Fujino M, Katsumata A, Hamada K, Sokawa Y, Ueda S (1996) Cloning and expression of the canine interferon-beta gene. J Interferon Cytok Res 16(10):765–770.

    Article  CAS  Google Scholar 

  14. Kost TA, Condreay JP (2002) Innovations—biotechnology: baculovirus vectors as gene transfer vectors for mammalian cells: biosafety considerations. Appl Biosaf 7(3):167–169

    Article  Google Scholar 

  15. Kruth SA (1998) Biological response modifiers: interferons, interleukins, recombinant products, liposomal products. Vet Clin N Am Small Anim Pract 28(2):269–295

    Article  CAS  Google Scholar 

  16. Kulakosky PC, Hughes PR, Wood HA (1998) N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells. Glycobiology 8(7):741–745

    Article  CAS  PubMed  Google Scholar 

  17. Lindner DJ (2002) Interferons as antiangiogenic agents. Curr Oncol Rep 4(6):510–514

    Article  PubMed  Google Scholar 

  18. Maeda S, Kawai T, Obinata M, Fujiwara H, Horiuchi T, Saeki Y, Sato Y, Furusawa M (1985) Production of human alpha-interferon in silkworm using a baculovirus vector. Nature 315(6020):592–594

    Article  CAS  PubMed  Google Scholar 

  19. Martina JA, Daniotti JL, Maccioni HJ (1998) Influence of N-glycosylation and N-glycan trimming on the activity and intracellular traffic of GD3 synthase. J Biol Chem 273(6):3725–3731

    Article  CAS  PubMed  Google Scholar 

  20. Mochizuki M, Nakatani H, Yoshida M (1994) Inhibitory effects of recombinant feline interferon on the replication of feline enteropathogenic viruses in vitro. Vet Microbiol 39(1–2):145–152

    Article  CAS  PubMed  Google Scholar 

  21. Na Z, Huipeng Y, Lipan L, Cuiping C, Umashankar ML, Xingmeng L, Xiaofeng W, Bing W, Weizheng C, Cenis JL (2008) Efficient production of canine interferon-alpha in silkworm Bombyx mori by use of a BmNPV/Bac-to-Bac expression system. Appl Microbiol Biotechnol 78(2):221–226.

    Article  CAS  PubMed  Google Scholar 

  22. Nagai A, Taira O, Ishikawa M, Hiramatsu K, Hohdatsu T, Koyama H, Arai S, Sato H, Nakano K, Maehara N (2004) Cloning of cDNAs encoding multiple subtypes of feline interferon-alpha from the feline epitherial cell line. J Vet Med Sci 66(6):725–728

    Article  CAS  PubMed  Google Scholar 

  23. O’Reilly DR, Miller LK, Luckow VA (1994) Baculovirus expression vectors: a laboratory manual. Oxford University Press, Oxford

    Google Scholar 

  24. Okano F, Satoh M, Ido T, Okamoto N, Yamada K (2000) Production of canine IFN-gamma in silkworm by recombinant baculovirus and characterization of the product. J Interferon Cytok Res 20(11):1015–1022.

    Article  CAS  Google Scholar 

  25. Pestka S (2007) The interferons: 50 years after their discovery, there is much more to learn. J Biol Chem 282(28):20047–20051.

    Article  CAS  PubMed  Google Scholar 

  26. Rodriguez J, Spearman M, Huzel N, Butler M (2005) Enhanced production of monomeric interferon-beta by CHO cells through the control of culture conditions. Biotechnol Progress 21(1):22–30.

    Article  CAS  Google Scholar 

  27. Romero LV, Targovnik AM, Wolman FJ, Cascone O, Miranda MV (2011) Rachiplusia nu larva as a biofactory to achieve high level expression of horseradish peroxidase. Biotechnol Lett 33(5):947–956.

    Article  CAS  PubMed  Google Scholar 

  28. Rubinstein S, Familletti PC, Pestka S (1981) Convenient assay for interferons. J Virol 37(2):755–758

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Sakurai T, Ueda Y, Sato M, Yanai A (1992) Feline interferon production in silkworm by recombinant baculovirus. J Vet Med Sci 54(3):563–565

    Article  CAS  PubMed  Google Scholar 

  30. Schmeisser H, Kontsek P, Esposito D, Gillette W, Schreiber G, Zoon KC (2006) Binding characteristics of IFN-alpha subvariants to IFNAR2-EC and influence of the 6-histidine tag. J Interferon Cytok Res 26(12):866–876.

    Article  CAS  Google Scholar 

  31. Shi X, Jarvis DL (2007) Protein N-glycosylation in the baculovirus-insect cell system. Curr Drug Targets 8(10):1116–1125

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sparks AN (1979) A review of the biology of the fall armyworm. Fla Entomol 62(2):82–87.

    Article  Google Scholar 

  33. Stifter SA, Gould JA, Mangan NE, Reid HH, Rossjohn J, Hertzog PJ, de Weerd NA (2014) Purification and biological characterization of soluble, recombinant mouse IFNbeta expressed in insect cells. Prot Express Purif 94:7–14.

    Article  CAS  Google Scholar 

  34. Targovnik AM, Villaverde MS, Arregui MB, Fogar M, Taboga O, Glikin GC, Finocchiaro LME, Cascone O, Miranda MV (2014) Expression and purification of recombinant feline interferon in the baculovirus-insect larvae system. Process Biochem 49(6):917–926.

    Article  CAS  Google Scholar 

  35. Targovnik AM, Arregui MB, Bracco LF, Urtasun N, Baieli MF, Segura MM, Simonella MA, Fogar M, Wolman FJ, Cascone O, Miranda MV (2016) Insect larvae: a new platform to produce commercial recombinant proteins. Curr Pharm Biotechnol 17(5):431–438

    Article  CAS  PubMed  Google Scholar 

  36. Usami A, Ishiyama S, Enomoto C, Okazaki H, Higuchi K, Ikeda M, Yamamoto T, Sugai M, Ishikawa Y, Hosaka Y, Koyama T, Tobita Y, Ebihara S, Mochizuki T, Asano Y, Nagaya H (2011) Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm. J Biochem 149(2):219–227.

    Article  CAS  PubMed  Google Scholar 

  37. van Oers MM (2011) Opportunities and challenges for the baculovirus expression system. J Invertebr Pathol 107 Suppl:S3–S15.

    Article  CAS  PubMed  Google Scholar 

  38. Villaverde MS, Targovnik AM, Miranda MV, Finocchiaro LME, Glikin GC (2016) Cytotoxic effects induced by interferon-ω gene lipofection through ROS generation and mitochondrial membrane potential disruption in feline mammary carcinoma cells. Cytokine 84(Supplement C):47–55.

    Article  CAS  PubMed  Google Scholar 

  39. Weiss RC, Toivio-Kinnucan M (1988) Inhibition of feline infectious peritonitis virus replication by recombinant human leukocyte (alpha) interferon and feline fibroblastic (beta) interferon. Am J Vet Res 49(8):1329–1335

    CAS  PubMed  Google Scholar 

  40. Yang LM, Xue QH, Sun L, Zhu YP, Liu WJ (2007) Cloning and characterization of a novel feline IFN-omega. J Interferon Cytok Res 27(2):119–127.

    Article  CAS  Google Scholar 

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This work was supported by Grants from Agencia Nacional de Promoción Científica y Tecnológica de Argentina and from Universidad de Buenos Aires (PICT 2014-3350; UBACyT 2016-20020150100145BA). AMT, MSV, FJW and MVM are career researchers of the Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET). MBA is a research fellow of CONICET. GJM is a research fellow of Agencia de Promoción Científica y Tecnológica (ANPCyT).

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MBA worked out almost all of the technical details and performed the numerical calculations; GJM and IS contributed with analytical methods; MSV performed antitumor activity assays; FJW contributed in downstream processing; AMT contributed in discussion; MVM was involved in planning and supervised the work. All authors discussed the results and commented on the manuscript.

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Correspondence to María Victoria Miranda.

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Arregui, M.B., Mc Callum, G.J., Smith, I. et al. Production of biologically active feline interferon beta in insect larvae using a recombinant baculovirus. 3 Biotech 8, 341 (2018).

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  • Feline interferon beta
  • Baculovirus
  • Insect larvae
  • Veterinary medicine