Construction and Sequencing of Dense Granular14 (GRA14) Gene of Toxoplasma gondii (RH) in Expression Prokaryotic Plasmid PET32a: A Preliminary Study in Vaccine Production

Document Type : Original Article


1 Department of Parasitology and Mycology, Faculty of Medicine, Baqiyatallah University of Medical Sciences, Tehran, Iran

2 Health Research Center, Life Style Institute, Baqiyatallah Universityof Medical Sciences, Tehran, Iran

3 Department of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran



Introduction: Toxoplasma gondii is an obligatory intracellular protozoan parasite, which infects human beings. Since the current antigens used for diagnosis or vaccination are contaminated with non -parasitic material in which the parasite is grown, it is tried to produce recombinant antigens to design vaccines against toxoplasmosis, or make diagnostic kits. Choosing the type of antigen to produce recombinant vaccine or diagnostic kits is considerably important. The dense granule protein 14 (GRA14) gene is one of the excretory-secretary antigens of Toxoplasma which seems to be an appropriate candidate in production of recombinant vaccines and diagnostic kits. The current study aimed to clone GRA14 gene of T. gondii (RH) in a cloning vector for further production of dense granular proteins.
Materials and Methods: Genomic DNA was isolated from tachyzoite of parasite by phenol chloroform method and gene fragment was amplified by polymerase chain reaction (PCR). The PCR products were ligated into restriction enzymes sites of pTG19-T cloning vector. Then transformed into Escherichia coli Top10 strain and screened by IPTG and X-Gal. Then recombinant plasmid confirmed by the colony-PCR and restriction enzyme digestion using SacI and NotI was done followed by sequencing .After isolation of this gene from pTG19-T, it was subcloned into a prokaryotic expression plasmid (pET32a). The pET32a - GRA14 constructs were analyzed by PCR, restriction analysis and sequencing.
Results: Evaluation of PCR products by agarose gel electrophoresis and analysis of nucleotide sequencing of 1227 bp gene encoding the protein GRA14, revealed the complete homology with the recorded sequences in the gene bank. After enzyme restriction and electrophoresis a fragment about 1227 bp was separated from pET32a.
Conclusions: The result of this study showed that recombinant GRA14 Toxoplasma was constructed successfully and ready for future study which seems like the antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit.


  1. Weiss LM, Kim K. Toxoplasma gondii: The model apicomplexan. Perspectives and methods. 1st ed. USA: Academic Press; 2007.
  2. Remington JS, McLeod R, Thulliez P, Desmonts G. Toxoplasmosis. In: Remington JS, Klein JO, Wilson CB, Baker CJ, eds. Infectious Diseases of the Fetus and Newborn Infant. Philadelphia: Elsevier Saunders; 2006;947-1091. doi:10.1016/B0-72-160537-0/50033- 5.
  3. Bhopale GM. Development of a vaccine for toxoplasmosis: current status. Microbes Infect. 2003;5(5):457-462.
  4. Dalton JP, Mulcahy G. Parasite vaccines--a reality? Vet Parasitol. 2001;98(1-3):149-167.
  5. Garcia JL, Gennari SM, Navarro IT, et al. Partial protection against tissue cysts formation in pigs vaccinated with crude rhoptry proteins of Toxoplasma gondii. Vet Parasitol. 2005;129(3-4):209- 217. doi:10.1016/j.vetpar.2005.01.006.
  6. Crampton A, Vanniasinkam T. Parasite vaccines: the new generation. Infect Genet Evol. 2007;7(5):664-673. doi:10.1016/j. meegid.2007.06.004.
  7. Jacobs D, Vercammen M, Saman E. Evaluation of recombinant dense granule antigen 7 (GRA7) of Toxoplasma gondii for detection of immunoglobulin G antibodies and analysis of a major antigenic domain. Clin Diagn Lab Immunol. 1999;6(1):24-29.
  8. Li S, Maine G, Suzuki Y, et al. Serodiagnosis of recently acquired Toxoplasma gondii infection with a recombinant antigen. J Clin Microbiol. 2000;38(1):179-184.
  9. Jongert E, Roberts CW, Gargano N, Forster-Waldl E, Petersen E. Vaccines against Toxoplasma gondii: challenges and opportunities. Mem Inst Oswaldo Cruz. 2009;104(2):252-266.
  10. Li S, Galvan G, Araujo FG, Suzuki Y, Remington JS, Parmley S. Serodiagnosis of recently acquired Toxoplasma gondii infection using an enzyme-linked immunosorbent assay with a combination of recombinant antigens. Clin Diagn Lab Immunol. 2000;7(5):781- 787.
  11. Cesbron-Delauw MF. Dense-granule organelles of Toxoplasma gondii: their role in the host-parasite relationship. Parasitol Today. 1994;10(8):293-296.
  12. Ching XT, Lau YL, Fong MY, Nissapatorn V, Andiappan H. Recombinant dense granular protein (GRA5) for detection of human toxoplasmosis by Western blot. Biomed Res Int. 2014;2014:690529. doi:10.1155/2014/690529.
  13. Mercier C, Cesbron-Delauw MF, Ferguson DJP. Dense granules of the infectious stages of Toxoplasma gondii: their central role in the host/parasite relationship. In: Soldati D, Ajioka JW, eds. Toxoplasma: Molecular and Cellular Biology. Horizon Scientific Press; 2007.
  14. Hughes HP, van Knapen F. Characterisation of a secretory antigen from Toxoplasma gondii and its role in circulating antigen production. Int J Parasitol. 1982;12(5):433-437.
  15. Holec-Gasior L, Kur J. Toxoplasma gondii: Recombinant GRA5 antigen for detection of immunoglobulin G antibodies using enzyme-linked immunosorbent assay. Exp Parasitol. 2010;124(3):272-278. doi:10.1016/j.exppara.2009.10.010.
  16. Peyron F, Lobry JR, Musset K, et al. Serotyping of Toxoplasma gondii in chronically infected pregnant women: predominance of type II in Europe and types I and III in Colombia (South America). Microbes Infect. 2006;8(9-10):2333-2340. doi:10.1016/j. micinf.2006.03.023.
  17. Switaj K, Master A, Skrzypczak M, Zaborowski P. Recent trends in molecular diagnostics for Toxoplasma gondii infections. Clin Microbiol Infect. 2005;11(3):170-176. doi:10.1111/j.1469- 0691.2004.01073.x.
  18. Wang Y, Wang G, Ou J, Yin H, Zhang D. Analyzing and identifying novel B cell epitopes within Toxoplasma gondii GRA4. Parasit Vectors. 2014;7:474. doi:10.1186/s13071-014-0474-x.
  19. Rome ME, Beck JR, Turetzky JM, Webster P, Bradley PJ. Intervacuolar transport and unique topology of GRA14, a novel dense granule protein in Toxoplasma gondii. Infect Immun. 2008;76(11):4865- 4875. doi:10.1128/iai.00782-08.
  20. Nam HW. GRA proteins of Toxoplasma gondii: maintenance of host-parasite interactions across the parasitophorous vacuolar membrane. Korean J Parasitol. 2009;47 Suppl:S29-37. doi:10.3347/ kjp.2009.47.S.S29.
  21. Ahmadpour E, Sarvi S, Daryani A, et al. Cloning and sequencing the plasmid encoding dense granule antigen 14 (GRA14) of Toxoplasma gondii RH strain. J Mazandaran Univ Med Sci. 2014;24(112):42-49.
  22. Rome ME, Beck JR, Turetzky JM, Webster P, Bradley PJ. Intervacuolar transport and unique topology of GRA14, a novel dense granule protein in Toxoplasma gondii. Infect Immun. 2008;76(11):4865- 4875. doi:10.1128/iai.00782-08.
  23. Kalani H, Daryani A, Sharif M, et al. Comparison of eight cell-free media for maintenance of Toxoplasma gondii Tachyzoites. Iran J Parasitol. 2016;11(1):104-109.
  24. Sudan V, Tewari AK, Singh H. An insight into the behavior, course and kinetics of acute infection of Toxoplasma gondii human RH strain in experimentally infected murine model. Iran J Parasitol. 2014;9(1):114-119.
  25. Sambrook J, Russell DW. Preparation and transformation of competent E. coli using calcium chloride. CSH Protoc. 2006;2006(1). doi:10.1101/pdb.prot3932.
  26. Arab-Mazar Z, Fallahi S, Koochaki A, Mirahmadi H, Tabaei SJ. Cloning, expression and immunoreactivity of recombinant Toxoplasma gondii GRA5 protein. Iran J Microbiol. 2016;8(5):331- 337.
  27. Rasti S, Haghighi A, Kazemi B, Rezaian M. Cloning and characterization of Serine-rich Entamoeba histolytica protein gene from an Iranian E. histolytica Isolate. Pak J Biol Sci. 2006;9(4):654- 658. doi:10.3923/pjbs.2006.654.658.
  28. Hoseinian Khosroshahi K, Ghaffarifar F, D’Souza S, Sharifi Z, Dalimi A. Evaluation of the immune response induced by DNA vaccine cocktail expressing complete SAG1 and ROP2 genes against toxoplasmosis. Vaccine. 2011;29(4):778-783. doi:10.1016/j.vaccine.2010.11.012.
  29. Wang Y, Yin H. Research progress on surface antigen 1 (SAG1) of Toxoplasma gondii. Parasit Vectors. 2014;7:180. doi:10.1186/1756- 3305-7-180.
  30. Johnson AM, Roberts H, Tenter AM. Evaluation of a recombinant antigen ELISA for the diagnosis of acute toxoplasmosis and comparison with traditional antigen ELISAs. J Med Microbiol. 1992;37(6):404-409. doi:10.1099/00222615-37-6-404.
  31. Eslamirad Z, Ghaffarifar F, Shojapour M, Khansarinejad B, Sadraei J. A preliminary Study: Expression of Rhoptry Protein 1 (ROP1) Toxoplasma gondii in Prokaryote System. Jundishapur J Microbiol. 2013;6(6):e10089. doi:10.5812/jjm.10089.
  32. Nigro M, Gutierrez A, Hoffer AM, et al. Evaluation of Toxoplasma gondii recombinant proteins for the diagnosis of recently acquired toxoplasmosis by an immunoglobulin G analysis. Diagn Microbiol Infect Dis. 2003;47(4):609-613.
  33. Yawen Li & Huaiyu Zhou. Moving towards improved vaccines for Toxoplasma gondii. Expert Opin Biol Ther. 2018;18(3):273-280. doi:10.1080/14712598.2018.1413086.
  34. Arab-Mazar Z, Seyyed-Tabaei SJ, Mirahmadi H. Cloning of dense granular (GRA) 7 gene of Toxoplasma gondii into pTZ57RT vectors. Nov Biomed. 2014;2(4):114-119.
  35. Ahmadpour E, Sarvi S, Hashemi Soteh MB, et al. Evaluation of the immune response in BALB/c mice induced by a novel DNA vaccine expressing GRA14 against Toxoplasma gondii. Parasite Immunol. 2017;39(4). doi:10.1111/pim.12419.
  36. Bhopale GM. Development of a vaccine for toxoplasmosis: current status. Microbes Infect. 2003;5(5):457-462.
  37. Guo H, Chen G, Zheng H, Zhou Y, Lu F. [Immune responses in mice vaccinated with recombinant plasmid pcDNA3 containing ROP1 gene from Toxoplasma gondii]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 1999;17(6):334-337.
  38. Meek B, Diepersloot RJ, van Gool T, Speijer D, Peek R. Igm recognition of recombinant Toxoplasma gondii antigens by sera of acutely or latently infected humans. Diagn Microbiol Infect Dis. 2003;45(1):45-52.
  39. Holec-Gasior L, Kur J, Hiszczynska-Sawicka E. GRA2 and ROP1 recombinant antigens as potential markers for detection of Toxoplasma gondii-specific immunoglobulin G in humans with acute toxoplasmosis. Clin Vaccine Immunol. 2009;16(4):510-514. doi:10.1128/cvi.00341-08.
  40. Selseleh MM, Keshavarz H, Mohebali M, et al. Production and evaluation of Toxoplasma gondii recombinant surface antigen 1 (SAG1) for serodiagnosis of acute and chronic toxoplasma infection in human sera. Iran J Parasitol. 2012;7(3):1-9.
  41. Chahed Bel-Ochi N, Bouratbine A, Mousli M. Enzyme-linked immunosorbent assay using recombinant SAG1 antigen to detect Toxoplasma gondii-specific immunoglobulin G antibodies in human sera and saliva. Clin Vaccine Immunol. 2013;20(4):468- 473. doi:10.1128/cvi.00512-12.
  42. Igarashi M, Kano F, Tamekuni K, et al. Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7. Genet Mol Res. 2008;7(2):305-313.
  43. Mohabati R, Babaie J, Amiri S, et al. Expression and Purification of Recombinant ROP1 of Toxoplasma gondii in Bacteria. Avicenna J Med Biotechnol. 2013;5(4):227-233.
  44. Eslamirad Z, Ghorbanzadeh B, Hajihossein R, et al. Cloning of 1183 bp Fragment from Rhoptry Protein I (ROPI) Gene of Toxoplasma gondii (RH) in Expression Prokaryote Plasmid PET32a. Zahedan J Res Med Sci. 2013;15(10):32-36.
  45. Wang Z, Ge W, Huang SY, Li J, Zhu XQ, Liu Q. Evaluation of recombinant granule antigens GRA1 and GRA7 for serodiagnosis of Toxoplasma gondii infection in dogs. BMC Vet Res. 2014;10:158. doi:10.1186/1746-6148-10-158.
  46. Sadeghiani G, Zare M, Babaie J, et al. Heterologous production of dense granule GRA7 antigen of Toxoplasma gondii in Escherichia coli. Southeast Asian J Trop Med Public Health. 2009;40(4):692- 700.
  47. Hruzik A, Asif AR, Gross U. Identification of Toxoplasma gondii SUB1 antigen as a marker for acute infection by use of an innovative evaluation method. J Clin Microbiol. 2011;49(7):2419- 2425. doi:10.1128/jcm.00464-11.