Aquaculture International

, Volume 26, Issue 6, pp 1401–1413 | Cite as

Evaluation of interferon gamma (IFN-γ) of Labeo rohita as an immunomodulator: in vitro expression model

  • Megha Kadam BedekarEmail author
  • Praveena Soman
  • Sajal Kole
  • Deepika Anand
  • Gayatri Tripathi
  • M. Makesh
  • K. V. Rajendran


Interferon gamma (IFN-γ) or type II interferon is a cytokine that is critical for innate and adaptive immunity against viral and some bacterial and protozoal infections. The importance of IFN-γ in the immune system lies in its ability to inhibit viral replication directly and most importantly from its immunomodulatory effects. Previously, we successfully co-administered IFN-γ along with GAPDH gene of Edwardsiella tarda as bicistronic DNA vaccine in Labeo rohita. In order to ascertain the individual role of IFN-γ, the present study involves cloning and expression of 552-bp IFN-γ open-reading frame (ORF) of L. rohita in striped snakehead (SSN-1) cell line using eukaryotic expression vector system (pQE-TriSystem) followed by transfection in peripheral blood lymphocytes (PBMCs) to evaluate its immunomodulatory ability in comparison to polyinosinic-polycytidylic acid (Poly I:C)-treated PBMCs. The 18.7-kDa protein, expressed in the pQE-IFNγ-transfected SSN-1 cells, reacted with anti-His antibody in Western blot confirming it to be recombinant IFN-γ, whereas the relative expression of IFN-γ, iNOS, Mx, and IL-1β genes in PBMCs was quantified at 24 h and 48 h post treatment by qPCR. The comparative kinetics of all four genes showed significantly (p < 0.05) high upregulation pattern in both pQE-IFNγ-transfected cell group and Poly I:C-treated cell group demonstrating recombinant IFN-γ as an equally efficient inducer like Poly I:C. Thus, our in vitro experiment results highlight the immunomodulatory potential of recombinant IFN-γ as an analogue to synthetic Poly I:C which warranted future studies to further explore the potential of recombinant IFN-γ as an effective vaccine adjuvant against different microbial invasion.


IFN-γ Labeo rohita Peripheral blood mononuclear cells Poly I:C 



The authors thank the present Director Dr. Gopal Krishna and Ex-Director Dr. W S Lakra, ICAR-Central Institute of Fisheries Education, Mumbai, India, for providing necessary facilities and support for this study.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Arts JA, Tijhaar EJ, Chadzinska M, Savelkoul HF, Verburg-van Kemenade BM (2010) Functional analysis of carp interferon-gamma: evolutionary conservation of classical phagocyte activation. Fish Shellfish Immunol 29:793–802CrossRefGoogle Scholar
  2. Caipang CM, Hirono I, Aoki T (2005) Induction of antiviral state in fish cells by Japanese flounder, Paralichthys olivaceus, interferon regulatory factor-1. Fish Shellfish Immunol 19(1):79–91CrossRefGoogle Scholar
  3. Chang CJ, Sun B, Robertsen B (2015) Adjuvant activity of fish type I interferon shown in a virus DNA vaccination model. Vaccine 33:2442–2448CrossRefGoogle Scholar
  4. Chen WQ, Xu QQ, Chang MX, Zou J, Secombes CJ, Peng KM, Nie P (2010) Molecular characterization and expression analysis of the IFN-gamma related gene (IFN gamma rel) in grass carp Ctenopharyngodon idella. Vet Immunol Immunopathol 134:199–207CrossRefGoogle Scholar
  5. Digby MR, Lowenthal JW (1995) Cloning and expression of the chicken interferon gene. J Interf Cytokine Res 15:939–945CrossRefGoogle Scholar
  6. Du Y, Qi J, Lu Y, Wu J, Yoo D, Liu X, Zhang X, Li J, Sun W, Cong X, Shi J, Wang J (2012) Evaluation of a DNA vaccine candidate co-expressing GP3 and GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) with interferon α/γ in immediate and long-lasting protection against HP-PRRSV challenge. Virus Genes 45(3):474–487CrossRefGoogle Scholar
  7. Flaishon L, Hershkoviz R, Lantner F, Lider O, Alon R, Levo Y, Flavell RA, Shachar I (2000) Autocrine secretion of interferon gamma negatively regulates homing of immature B cells. J Exp Med 192(9):1381–1388CrossRefGoogle Scholar
  8. Frucht DM, Fukao T, Bogdan C, Schindler H, O’Shea JJ, Koyasu S (2001) IFN-gamma production by antigen-presenting cells: mechanisms emerge. Trends Immunol 22(10):556–560CrossRefGoogle Scholar
  9. Furnes C, Seppola M, Robertsen B (2009) Molecular characterisation and expression analysis of interferon gamma in Atlantic cod (Gadus morhua). Fish Shellfish Immunol 26:285–292CrossRefGoogle Scholar
  10. Grayfer L, Belosevic M (2009) Molecular characterization, expression and functional analysis of goldfish (Carassius aurutus L.) interferon gamma. Dev Comp Immunol 33(2):235–246CrossRefGoogle Scholar
  11. Harris DP, Haynes L, Sayles PC, Duso DK, Eaton SM, Lepak NM, Johnson LL, Swain SL, Lund FE (2000) Reciprocal regulation of polarized cytokine production by effector B and T cells. Nat Immunol 1(6):475–482CrossRefGoogle Scholar
  12. Igawa D, Sakai M, Savan R (2006) An unexpected discovery of two interferon gamma like genes along with interleukin (IL)-22 and -26 from teleost: IL-22 and -26 genes have been described for the first time outside mammals. Mol Immunol 43:999–1009CrossRefGoogle Scholar
  13. Jimenez N, Coll J, Salguero FJ, Tafalla C (2006) Co-injection of interleukin 8 with the glycoprotein gene from viral haemorrhagic septicemia virus (VHSV) modulates the cytokine response in rainbow trout (Oncorhynchus mykiss). Vaccine 24(27–28):5615–5626CrossRefGoogle Scholar
  14. Kadam M, Shardul S, Bhagath JL, Tiwari V, Prasad N, Goswami PP (2009) Coexpression of 16.8 kDa antigen of Mycobacterium avium paratuberculosis and murine gamma interferon in a bicistronic vector and studies on its potential as DNA vaccine. Vet Res Commun 33(7):597–610CrossRefGoogle Scholar
  15. Kole S, Kumari R, Anand D, Kumar S, Sharma R, Tripathi G, Makesh M, Rajendran KV, Bedekar MK (2018) Nanoconjugation of bicistronic DNA vaccine against Edwardsiella tarda using chitosan nanoparticles: evaluation of its protective efficacy and immune modulatory effects in Labeo rohita vaccinated by different delivery routes. Vaccine 36:2155–2165CrossRefGoogle Scholar
  16. Kumari R, Kole S, Soman P, Rathore G, Tripathi G, Makesh M, Rajendran KV, Bedekar MK (2018) Bicistronic DNA vaccine against Edwardsiella tarda infection in Labeo rohita: construction and comparative evaluation of its protective efficacy against monocistronic DNA vaccine. Aquaculture 485(C):201–209CrossRefGoogle Scholar
  17. Lazarte JMS, Kim YR, Lee JS, Im SP, Kim SW, Jung JW, Kim J, Lee WJ, Jung TS (2017) Enhancement of glycoprotein-based DNA vaccine for viral hemorrhagic septicemia virus (VHSV) via addition of the molecular adjuvant, DDX41. Fish Shellfish Immunol 62:356–365CrossRefGoogle Scholar
  18. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) method. Methods 25:402–408CrossRefGoogle Scholar
  19. Milev-Milovanovic I, Long S, Wilson M, Bengten E, Miller NW, Chinchar VG (2006) Identification and expression analysis of interferon gamma genes in channel catfish. Immunogenetics 58:70–80CrossRefGoogle Scholar
  20. Mosmann TR, Coffman RL (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Ann Rev Immunol 7:145–173CrossRefGoogle Scholar
  21. Parhi J, Sahoo L, Choudhury J, Choudhury TG, Baruah A, PaniPrasad K, Makesh M (2015) Molecular characterization and expression analysis of interferon (IFN-γ) gene in Labeo rohita (Ham.). Aquaculture Rep 2:97–105CrossRefGoogle Scholar
  22. Perussia B (1991) Lymphokine-activated killer cells, natural killer cells and cytokines. Curr Opin Immunol 3:49–55CrossRefGoogle Scholar
  23. Robertsen B (2006) The interferon system of teleost fish. Fish Shellfish Immunol 20:172–191CrossRefGoogle Scholar
  24. Robertsen B, Zou J, Secombes C, Leong JA (2006) Molecular and expression analysis of an interferon-gamma-inducible guanylate-binding protein from rainbow trout (Oncorhynchus mykiss). Dev Comp Immunol 30:1023–1033CrossRefGoogle Scholar
  25. Robertsen B, Chang CJ, Bratland L (2016) IFN-adjuvanted DNA vaccine against infectious salmon anemia virus: antibody kinetics and longevity of IFN expression. Fish Shellfish Immunol 54:328–332CrossRefGoogle Scholar
  26. Sad S, Marcotte R, Mosmann TR (1995) Cytokine-induced differentiation of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or T2 cytokines. Immunity 2:271–279CrossRefGoogle Scholar
  27. Secombes CJ (1996) The nonspecific immune system: cellular defences. In: The fish immune system: organism, pathogen, and environment. Vol 15. Academic PressGoogle Scholar
  28. Skjesol A, Hansen T, Shi CY, Thim HL, Jorgensen JB (2010) Structural and functional studies of STAT1 from Atlantic salmon (Salmo salar). BMC Immunol 11:17CrossRefGoogle Scholar
  29. Stolte EH, Savelkoul HF, Wiegertjes G, Flik G, Lidy Verburg-van Kemenade BM (2008) Differential expression of two interferon-gamma genes in common carp (Cyprinus carpio L.). Dev Comp Immunol 32(12):1467–1481CrossRefGoogle Scholar
  30. Sun B, Skjaeveland I, Svingerud T, Zou J, Jørgensen J, Robertsen B (2011) Antiviral activity of salmonid interferon gamma against infectious pancreatic necrosis virus and salmonid alphavirus and its dependency on type i interferon. J Virol 85(17):9188–9198CrossRefGoogle Scholar
  31. Swain B, Basu M, Lenka SS, Das S, Jayasankar P, Samanta M (2015) Characterization and inductive expression analysis of interferon gamma-related gene in the Indian major carp, Rohu ( Labeo rohita ). DNA Cell Biol 34:1–12CrossRefGoogle Scholar
  32. Wang J, Gujar SA, Cova L, Michalak TI (2007) Bicistronic Woodchuck Hepatitis Virus core and gamma interferon DNA vaccine can protect from hepatitis but does not elicit sterilizing antiviral immunity. J Virol 81(2):903–916CrossRefGoogle Scholar
  33. Zhou Z, Hamming OJ, Ank N, Paludan SR, Nielsen AL, Hartman R (2007) Type III interferon (IFN) induces type I IFN-like response in the restricted subset of cell through signaling pathways involving both the jak- STAT pathway and nitrogen activated protein kinases. J Virol 81:7749–7758CrossRefGoogle Scholar
  34. Zou J, Yoshiura Y, Dijkstra JM, Sakai M, Ototake M, Secombes CJ (2004) Identification of an interferon gamma homologue in Fugu, Takifugu rubripes. Fish Shellfish Immunol 17:403–409CrossRefGoogle Scholar
  35. Zou J, Carrington A, Collet B, Dijkstra JM, Yoshiura Y, Bols N, Secombes C (2005) Identification and bioactivities of IFN-gamma in rainbow trout Oncorhynchus mykiss: the first Th1-type cytokine characterized functionally in fish. J Immunol 175:2484–2494CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Megha Kadam Bedekar
    • 1
    Email author
  • Praveena Soman
    • 1
  • Sajal Kole
    • 1
    • 2
  • Deepika Anand
    • 1
  • Gayatri Tripathi
    • 1
  • M. Makesh
    • 3
  • K. V. Rajendran
    • 1
  1. 1.ICAR-Central Institute of Fisheries EducationMumbaiIndia
  2. 2.Department of Aqualife MedicineChonnam National UniversityGwangjuRepublic of Korea
  3. 3.ICAR-Central Institute of Brackishwater AquacultureChennaiIndia

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