Parasitology Research

, Volume 112, Issue 7, pp 2487–2494 | Cite as

Molecular cloning, sequencing, and biological characterization of GRA4 gene of Toxoplasma gondii

  • Hira Ram
  • J. R. Rao
  • A. K. Tewari
  • P. S. Banerjee
  • A. K. Sharma
Original Paper


In the present study, GRA4 (dense granule antigen) gene of Toxoplasma gondii was cloned, sequenced, and biologically characterized. The nucleotide sequence data obtained were analyzed and submitted in GenBank database (accession no. EU660037). Analysis of nucleotide sequence of GRA4 gene revealed 99.2 % homology with the published sequence (accession no. M76432). The gene segment (open reading frame) of 1,054 bp was further amplified and re-cloned in expression vector pET-32a. The recombinant protein obtained following the expression in prokaryotic system had a molecular mass of approx. 50 kDa and showed good immunoreactivity with T. gondii sera collected from infected goats. The immunization study of the recombinant protein performed in laboratory mice and live challenge with T. gondii revealed a high level of IgG response against the tachyzoite lysate antigen (TLA) by an indirect ELISA. Protection against T. gondii challenge infection was not evident in immunized mice except for the prolongation of survival period by 2 days. Humoral immune response profile revealed initially a high level of IgG antibody, but at 1 week post-challenge, a sudden drop in the level of the antibody was appreciable. Cytokine profiling by enzyme-linked immunosorbent spot method revealed relatively high level of IFN-γ production by the rodent spleen cells followed by IL-10 and IL-4. Increase in IFN-γ production by spleen cells of immunized mice following TLA stimulation suggested direct correlation to the up-regulated Th1 cells. However, the present immunization trial failed to show any positive relationship with the protection of mice following T. gondii challenge infection.



The authors are thankful to the director of the Indian Veterinary Research Institute, Izatnagar, and Station In charge, IVRI, Mukteswar Campus, for providing all the necessary research facilities to carry out this research work.


  1. Benita MB, Laloup M, Versteyhe S, Dewit J, Braekeleer JD, Jongert E, Borchard G (2003) Generation of Toxoplasma gondii GRA1 protein and DNA vaccine loaded chitosan particles: preparation, characterization and preliminary in vivo studies. Int J Pharm 266:17–27CrossRefGoogle Scholar
  2. Bhopale GM (2003) Development of a vaccine for toxoplasmosis: current status. Microbes Infect 5:457–462PubMedCrossRefGoogle Scholar
  3. Bourguin I, Chardes T, Bout D (1993) Oral immunization with Toxoplasma gondii antigens in association with cholera toxin induces enhance protective and cell mediated immunity in C57BL/6 mice. Infect Immun 61:2082–2088PubMedGoogle Scholar
  4. Brown CR (1995) Definitive identification of a gene that confers resistance against Toxoplasma cyst burden and encephalitis. Immunology 85:419–428PubMedGoogle Scholar
  5. Buxton D, Uggala A, Lovgren K, Thomson K, Kunden A, Morein B, Blewett DA (1989) Trial of a novel ISCOM vaccine in pregnant sheep. Br Vet J 145:451–457PubMedCrossRefGoogle Scholar
  6. Debard N, Bujoni-Gatel D, Bout D (1996) Intranasal immunization with SAG1 protein of Toxoplasma gondii in association with cholera toxin dramatically reduces development of cerebral cysts after oral infection. Infect Immun 64:2158–2166PubMedGoogle Scholar
  7. Desolme B, Mevelec MN, Buzoni-Gatel D, Bout D (2000) Induction of protective immunity against toxoplasmosis in mice by DNA immunization with a plasmid encoding Toxoplasma gondii GRA4 gene. Vaccine 18:2512–2521PubMedCrossRefGoogle Scholar
  8. Dlugonska H (2008) Toxoplasma Rhoptries: unique secretary organelles and source of promising vaccine proteins for immunoprevention of toxoplasmosis. J Biomed Biotech. doi:10.1155/2008/632424 Google Scholar
  9. Gazzinelli RT, Hakim FT, Hieny S, Shearer GM, Sher A (1991) Synergistic role of CD4+ and CD8+ T lymphocytes in IFN-gamma production and protective immunity induced by an attenuated Toxoplasma gondii vaccine. J Immunol 146:286–292PubMedGoogle Scholar
  10. Gazzinelli RT, Wysocka M, Denkers EY, Hieny S, Caspar P, Trichieri G, Sher A (1994) Parasite-induced IL-12 stimulates early IFN-γ synthesis and resistance during acute infection with Toxoplasma gondii. J Immunol 153:2533–2543PubMedGoogle Scholar
  11. Haumont M, Delhaye L, Garcia L, Juardo M, Mazzu P, Daminet V, Verlant V, Bollen A, Biemans R, Jacqent A (2000) Protective immunity against congenital toxoplasmosis with recombinant SAG1 protein in guinea pig model. Infect Immun 68:4948–4953PubMedCrossRefGoogle Scholar
  12. Huaiyu Z, Qinmin G, Qunli Z, Jiaqin Z, Hua C, Yi L, Shenyi H (2007) Toxoplasma gondii: expression and characterization of a recombinant protein containing SAG1 and GRA2 in Pichia pastoris. Parasitol Res 100:829–835CrossRefGoogle Scholar
  13. Igarashi M, Kano F, Tamekuni K, Machado RZ, Navarro IT, Vidotto O, Vidotto MC, Garcia JL (2008) Toxoplasma gondii: evaluation of an intranasal vaccine using recombinant proteins against brain cyst formation in BALB/c mice. Exp Parasitol 118:386–392PubMedCrossRefGoogle Scholar
  14. Johnson M, Broady K, Angelici MC, Johnson A (2003) The relationship between nucleoside triphosphate hydrolase (NTPase) isoform and Toxoplasma strain virulence in rat and human toxoplasmosis. Microbes Infect 5:797–806PubMedCrossRefGoogle Scholar
  15. Jongert E, Roberts CW, Gargano N, Forster-Wald E, Petersen E (2009) Vaccines against Toxoplasma gondii: challenges and opportunities. Mem Inst Oswaldo Cruz Rio de Janeiro 104:252–266CrossRefGoogle Scholar
  16. Li WS, Chen QX, Ye JX, Xie ZX, Chen J, Zhang LF (2011) Comparative evaluation of immunization with recombinant protein and plasmid DNA vaccines of fusion antigen ROP2 and SAG1 from Toxoplasma gondii in mice: cellular and humoral immune response. Parasitol Res 109:637–644PubMedCrossRefGoogle Scholar
  17. Liu MM, Yuan ZG, Peng GH, Zhou DH, He XH, Yan C, Yin CC, He Y, Lin RQ, Song HQ, Zhu XQ (2010) Toxoplasma gondii microneme protein 8 (MIC8) is a potential vaccine candidate against toxoplasmosis. Parasitol Res 106:1079–1084PubMedCrossRefGoogle Scholar
  18. Lunden A (1995) Immune response in sheep after immunization with Toxoplasma gondii antigen incorporated into ISCOMS. Vet Parasitol 56:23–35PubMedCrossRefGoogle Scholar
  19. Martin V, Supanitsky A, Echevrria PC, Litwin S, Tanos T, De Roodt AR, Guarnera EA, Angel SO (2004) Recombinant GRA4 and ROP2 protein combined with alum or the GRA4 gene provide partial protection in chronic murine models of toxoplasmosis. Clin Diag Lab Immunol 11:704–710Google Scholar
  20. Mevelec MN, Bout D, Desolme B, Marchand H, Magne R, Bruneel O, Buzoni-Gatel D (2005) Evaluation of protective effect of DNA vaccination with gene encoding GRA4 and SAG1 associated with GM-CSF plasmid, against acute, chronic and congenital toxoplasmosis in mice. Vaccine 23:4489–4499PubMedCrossRefGoogle Scholar
  21. Morris MT, Coppin A, Tomavo S, Carruthers VB (2002) Functional analysis of Toxoplasma gondii protease inhibitor 1. J Biol Chem 227:45259–45266CrossRefGoogle Scholar
  22. Nam HW (2009) GRA proteins of Toxoplasma gondii: maintenance of host-parasite interactions across the parasitophorous vacuolar membrane. Korean J Parasitol 47:S29–S37PubMedCrossRefGoogle Scholar
  23. Parker SJ, Roberts CW, Alexander J (1991) CD8+ T cells are the major lymphocyte subpopulation involved in the protective immune response to Toxoplasma gondii in mice. Clin Exp Immunol 84:207–212PubMedCrossRefGoogle Scholar
  24. Petersen E, Nielsen HV, Christiansen L, Spenter J (1998) Immunization with E. coli produced recombinant Toxoplasma gondii SAG1 with alum as adjuvant protects mice against lethal infection with Toxoplasma gondii. Vaccine 16:1283–1289PubMedCrossRefGoogle Scholar
  25. Pszenny V, Ledesma BE, Matrajt M, Duschak VG, Bontempi EJ, Dubremetz JF, Angel SO (2002) Subcellular localization and post-secretory targeting of TgPI, a serine proteinase inhibitor from Toxoplasma gondii. Mol Biochem Parasitol 121:283–286PubMedCrossRefGoogle Scholar
  26. Suzuki Y, Orellana MA, Schreiber RD, Remington JS (1988) Interferon gamma: the major mediator of resistance against Toxoplasma gondii. Science 240:516–518PubMedCrossRefGoogle Scholar
  27. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from acrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354PubMedCrossRefGoogle Scholar
  28. Velge-Roussel F, Marcelo P, Lepage AC, Buzoni-Gatel D, Bout DT (2000) Intranasal immunization with Toxoplasma gondii SAG1 induces protective cells into both NALT and GALT compartments. Infect Immun 68:969–972PubMedCrossRefGoogle Scholar
  29. Wu XN, Lin J, Lin X, Chen J, Chen ZL, Lin JY (2012) Multicomponent DNA vaccine-encoding Toxoplasma gondii GRA1 and SAG1 primes: anti-Toxoplasma immune response in mice. Parasitol Res 111:2001–2009PubMedCrossRefGoogle Scholar
  30. Xi-Meng Sun Jun Z, Elashram Saeed AA, Wen-Chao Y, Xian-Yong L, Suo X, Heng W, Qi-Jun C (2011) DNA vaccination with a gene encoding Toxoplasma gondii GRA6 induces partial protection against toxoplasmosis in BALB/c mice. Parasit Vectors 4:213CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Hira Ram
    • 1
  • J. R. Rao
    • 1
  • A. K. Tewari
    • 1
  • P. S. Banerjee
    • 1
  • A. K. Sharma
    • 2
  1. 1.Division of ParasitologyIndian Veterinary Research InstituteIzatnagarIndia
  2. 2.Division of Temperate Animal HusbandryIVRINainitalIndia

Personalised recommendations