Abstract
Paratuberculosis or Johne’s disease is a chronic gastric disease of ruminants. For this disease there is no effective treatment or preventive measure available. 16.8 kDa protein is an immunogenic protein of Mycobacterium avium paratuberculosis and can be an ideal candidate for developing a DNA vaccine construct. In present study a bicistronic DNA vaccine construct pIR16.8/IFN was developed using eukaryotic vector pIRES 6.1. Two genes MPT (expressing 16.8 kDa protein) and murine IFNγ were cloned, expressed and immunoreactivity was studied in murine model. Immunoreactivity was also compared with monocistronic construct pIR16.8 expressing 16.8 kDa protein. Both pIR16.8 and pIR16.8/IFN showed eukaryotic expression of respective proteins in BHK21 cells. The expressed proteins also showed immunoreactivity when reacted with hyperimmune sera raised against recombinant 16.8 kDa protein in western blot assay and immunofluorence assay. Both constructs were used as DNA vaccine in murine model and immunogenecity was studied by DTH, lymphocyte proliferation assay and NO determination. DTH reaction was significantly high in pIR16.8/IFN than pIR16.8 group, similarly lymphocyte proliferation and NO release was higher in pIR16.8/IFN group than pIR16.8 group. This indicated T cell epitopic nature of 16.8 kDa protein. The study also showed that co-expression of IFNγ with mycobacterial gene can enhance immunogenecity of DNA vaccine and can be used as immunoadjuvant.
Similar content being viewed by others
Abbreviations
- MPT:
-
Mycobacterium avium paratuberculosis
- DTH:
-
Delayed Type Hypersensitivity
- h-hours, rpm:
-
revolutions per minute, min-minute
- FCS:
-
foetal calf serum
- DMEM:
-
Dulbacco’s Modified Eagle Medium
- MTT:
-
4,5-dimethyl thiazol-2-4 diphenyl tetrazolium bromide
- NO:
-
Nitric oxide
References
Adams JL, Czuprynski CJ. Mycobacterial cell wall components induce the production of TNF-, IL-1, and IL-6 by bovine monocytes and the murine macrophage cell line RAW 264.7. Microb. Pathog 1994; 16: 401–411. doi:10.1006/mpat.1994.1040
Barouch DH, Santra S, Tenner-Racz P, Kuroda MJ, Schimitz JE, Jackson SS, Lifton MA, Freed DC, Perry HC, Davis ME, Shiver JW, Letvin NL. Potent CD4+ T cell response elicited by a bicistronic HIV-1 DNA vaccine expressing gp120 and GM-CSF. J immunol 2002; 1682: 562–568
Basagoudanawar SH, Goswami PP, Tiwari V, Cellular immuneresponse to 35 kDa recombinant antigen of Mycobacterium avium paratuberculosis, Vet. Res. Comm 2005;28:209–224. doi:10.1023/B:VERC.0000017371.68083.04
Burrells C, Inglis NF, Davies RC, Sharp JM. Detection of specific T cell reactivity in sheep infected with Mycobacterium avium subspecies silvaticum and paratuberculosis using two defined mycobacterial antigens. Vet. Immunol. and Immunopathol 1995; 45: 311–320. doi:10.1016/0165-2427(94)05347-U
Chow YH, Huang W, Chi WK, Chu Y, Tao MH, Improvement of hepatitis B virus DNA vaccine by plasmids co-expressing hepatiis B surface antigen and interleukin-2. J Virol 1997; 71 1:169–178.
Cooper AM, Dalton DK, Stwert TA, Griffin JP, Russell DG Orme IM. Disseminated tuberculosisin interferon gamma gene disrupted mice. J.Exp.Med 1993; 178:2243–2247. doi:10.1084/jem.178.6.2243
Dalton DK, Meek SP, Keshav S, Figari SI, Bradley A, Stweart TA, Multiple defects of immune cell function in mice with disrupted interferon gamma genes. Science 1993; 259:1739–1742. doi:10.1126/science.8456300
Flynn JL Chan J. Immunology of tuberculosis. Annu Rev Immunol 2001; 19:93–129. doi:10.1146/annurev.immunol.19.1.93
Flynn JL, Chan J, Tribold KJ, Dalton DK, Stewart TA Bloom BR. An essential role of interferon gamma in resistance of mycobacterium tuberculosis infection. J. Exp Med 1993;178:2249–2254. doi:10.1084/jem.178.6.2249
Gilmour NJL, Angus KW, Mitchell B. Intestinal infection and host response to oral administration of Mycobacterium johnei in sheep. Vet Microbiol 1978; 2:223–235. doi:10.1016/0378-1135(77)90015-3
Goswami PP, Rupa P, Prihar NS Garg LC. Molecular cloning of Clostridium perfringens epsilon-toxin gene and its high level expression in E. coli. Biochem Biophys Res Commun 1996; 2263:735–40. doi:10.1006/bbrc.1996.1422
Grey PW, Goeddel DV. Cloning and expression of murine interferon cDNA. Proc Natl Acad Sci USA 1983; 80 19: 5842–5846. doi:10.1073/pnas.80.19.5842
Herman-Taylor J, Bull T, Sheridan J, Cheng J, Stellakis M, Sumar N. The causation of Crohn’s disease by Mycobacterium avium subsp. paratuberculosis. Can. J. Gastroenterol 2000; 146: 521–539.
Horwitz MA, Lee BW, Dillon BJ, Harth G. Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis. Proc Natl Acad Sci USA 1995; 92:1530–1534. doi:10.1073/pnas.92.5.1530
Huygen K, Content J, Olivier D, Montogomery DL, Yawman AM, Deck RR, De Witt CM, Orme M, Baldwin S, D’Souza C, Drowart E, Lozes P, Vandenbussche P, Van Vooren JP, Liu MA, Ulmer JB. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat Med 1996; 2: 893–898. doi:10.1038/nm0896-893
Kamath AT, Feng CG, Mac Donald M, Briscoe H, Britton WJ. Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis. Infect. Immun 1999; 67: 1702–1707.
Lai WC, Bennel M, Johnston SA, Barry MA, Pakes SP. Protection against Mycobacterium pulmonis infection by genetic vaccination. Cell Biol 1995; 14: 643–648.
Moss MT, Green EP, Tizard ML, Malik ZP, Hermon-Taylor J. Specific detection of Mycobacterium paratuberculosis by DNA hybridisation with a fragment of the insertion element IS900. Gut 1991; 32:395–398. doi:10.1136/gut.32.4.395
Mukhija R, Rupa P, Pillai D, Garg LC. High-level production and one-step purification of biologically active human growth hormone in Escherichia coli. Gene 1995; 1652:303–6. doi:10.1016/0378-1119(95)00525-B
Mullerad JH, Hovav R, Nahary Y, Bercovier H. immunogenicity of 16.7 kDa Mycobacterium paratuberculosis antigen. Microb. Pathogenesis 2003; 34:81–90. doi:10.1016/S0882-4010(02)00209-7
Pillai D, Dixit A, Garg LC. Translational fusion of heat labile enterotoxin chain B and β-subunit of human chorionic gonadotropin: periplasmic expression in Escherichia coli and its immunogenicity. FEBS Lett 1996; 387: 23–26. doi:10.1016/0014-5793(96)00428-0
Portillo PD, Murillo LA, Patarroyo ME. Amplification of species-specific DNA fragment of Mycobacterium tuberculosis and its possible use in diagnosis. J Clin Microbiol 1991; 29:2163–2168.
Rainczuk A, Scorza T, Spithill TW, Smooker PM. A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoit surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria. Infect and Immun 2004; 72 4:5565–5573. doi:10.1128/IAI.72.10.5565-5573.2004
Renu. DNA vaccine a ray of hope, Crit Rev Biochem Mol Bio 1999;341:1–24. doi:10.1080/10409239991209165
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual 2nd edn. Cold Spring Harbor Laboratory Press, Cold spring Harbor, NY. 1989
Schluger NW, Rom WN. The host immune response to tuberculosis. Am J Respir Crit Care Med 1998; 157:679–691.
Stable JR. Production of γ interferon by peripheral blood mononuclear cells: an important diagnostic tool for detection of subclinical paratuberculosis. J Vet Diagn Investig 1996; 8: 345–350
Tascon RE, Colston MJ, Ragno S, Stavropoulos E, Gregory DB, Lowrie DB. Vaccination against tuberculosis by DNA injection. Nat Med 1996; 2: 888–92. doi:10.1038/nm0896-888
Thompson DE. The role of mycobacteria in Crohn’s disease. J Med Microbiol 1994; 41: 74–94
Weldingh K, Rosenkrands I, Jacobsen S, Rasmussen PB, Elhay MJ, Andersen P. Two dimensional electrophoresis for analysis of mycobacterium tuberculosis culture filtrate and purification and characterization of six novel proteins. Infect immun 1998; 66:3492–3500.
Zhao B, Collins MT, Czuprynski CJ. Effects of gamma interferon and nitric oxide on the interaction of Mycobacterium avium subsp. paratuberculosis with bovine monocytes. Infect Immun 1997; 655: 1761–1766.
Zhu X, Venkataprasad N, Thangaraj HS, Hill M, Singh M, Ivanyi J, Vordermeier HM. Functions and specificity of T cells following nuclei and vaccination of mice against Mycobacterium tuberculosis infection. J Immunol 1997; 58:5921–5926.
Acknowledgements
This research work was supported by Research grant No. 9/563(70)/2K4-EMR.I from
Center for Scientific and Industrial Research, New Delhi, India 110012.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kadam, M., Shardul, S., Bhagath, J.L. et al. 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, 597–610 (2009). https://doi.org/10.1007/s11259-009-9207-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11259-009-9207-6