Iranian Journal of Microbiology 2016. 8(5):331-337.

Cloning, expression and immunoreactivity of recombinant Toxoplasma gondii GRA5 protein
Zahra Arab-Mazar, Shirzad Fallahi, Ameneh Koochaki, Hadi Mirahmadi, Seyyed Javad Seyyed Tabaei


Background and Objectives: Toxoplasma gondii is an obligatory intracellular parasite which causes severe diseases in the fetus of pregnant women and immunocopmromised patients. Serological tests based on recombinant protein are one of the main diagnosis methods for the detection of specific antibodies in serum samples. Dense granule antigenic proteins derived from T. gondii (TgGRAs) are potential antigens for the development of diagnostic tools.

Materials and Methods: DNA was extracted from T. gondii (RH-strain) tachyzoites and PCR reaction was done using corresponding primers for GRA5 antigen. The PCR product was purified and ligated into pTG19-t vector and then subcloned into XhoI and BamHI digested pGEX6p-1 expression vector. Recombinant plasmid was transformed into E. coli (BL21 DE3) and induced by 1mM IPTG and analyzed by 15% SDS-PAGE. Expressed protein was confirmed by western blot analysis.

Results: There was no difference among the sequences of T. gondii GRA5 gene from different isolates. The recombinant plasmid pGEX-6p-1/GRA5 induced by IPTG was expressed in E. coli. It was a GST fusion protein and could react with human positive sera analyzed by western blot.

Conclusion: The GRA5 gene of T. gondii isolates is highly conservative. This antigen as a recombinant protein was successfully expressed in E. coli, which showed high immunoreactivity.

Keywords: Toxoplasma gondii, Dense granule antigen; GRA5, Immunoreactivity


Toxoplasma gondii; Dense granule antigen; GRA5; Cloning; Immunoreactivity

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McConkey G A, Martin H L, Bristow G C, Webster J P.Toxoplasma gondii infection and behaviour–location, location, location? J Exp Biol 2013; 216: 113-119.

Hill D, Dubey J. Toxoplasma gondii prevalence in farm animals in the United States. Int J Parasitol 2013; 43:107-113.

Dubey J, Lindsay D, Speer C. Structures of Toxoplas- ma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clin Mi- crobiol Rev 1998 Apr; 11(2):267-299.

Dubey J. Pathogenicity and infectivity of Toxoplasma gondii oocysts for rats. J Parasitol 1996; 951-956.

Indrasanti D, Haryanto A, Artama W. Expression and isolation of recombinant microneme 3 (MIC3) protein of Toxoplasma gondii local isolate on Eschericia coli (BL21). Anim Prod 2011; 13: 131-137.

Selseleh M, Keshavarz H, Mohebali M, Shojaee S, Selseleh M, Eshragian M R, et al. Production and eval- uation of Toxoplasma gondii recombinant GRA7 for serodiagnosis of human infections. Korean J Parasitol 2012; 50: 233-238.

Aubert D, Maine G, Villena I, Hunt J, Howard L, Sheu M, et al. Recombinant antigens to detect Toxoplasma gondii-specific immunoglobulin G and immunoglobu- lin M in human sera by enzyme immunoassay. J Clin Microbiol 2000; 38: 1144-1150.

Golkar M, Azadmanesh K, Khalili G, Khoshkhol- gh-Sima B, Babaie J, Mercier C, et al. Serodiagnosis of recently acquired Toxoplasma gondii infection in preg- nant women using enzyme-linked immunosorbent as- says with a recombinant dense granule GRA6 protein. Diagn Microbiol Infect Dis 2008 May;61(1):31-39.

Holec L, Gąsior A, Brillowska-Dąbrowska A, Kur J.Toxoplasma gondii: enzyme-linked immunosorbent assay using different fragments of recombinant mi- croneme protein 1 (MIC1) for detection of immuno- globulin G antibodies. Exp Parasitol 2008; 119: 1-6.

Arab-Mazar Z, Seyyed-Tabaei S J, Mirahmadi H.Cloning of dense granular (GRA) 7 gene of Toxoplas- ma gondii into pTZ57RT vectors for sub-cloning in prokaryotic and eukaryotic plasmids. Novelty in Bio- medicine 2014; 2: 114-119.

Fallahi Sh, Seyyed Tabaei SJ, Pournia Y, Zebardast N, Kazemi B. Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gon- dii in blood samples of children with leukaemia. Diagn Microbiol Infect Dis 2014 Jul; 79(3):347-354.

Hughes H , Van Knapen F. Characterisation of a se- cretory antigen from Toxoplasma gondii and its role in circulating antigen production. Int J Parasitol 1982; 12:433-437.

Holec-Gąsior L , Kur J. Toxoplasma gondii: Recombi- nant GRA5 antigen for detection of immunoglobulin G antibodies using enzyme-linked immunosorbent assay. Exp Parasitol 2010; 124: 272-278.

Arab-Mazar Z, Fallahi Sh, Koochaki A, Haghighi A, Tabaei S J S. Immunodiagnosis and molecular valida- tion of Toxoplasma gondii-recombinant dense granular (GRA) 7 protein for the detection of toxoplasmosis in patients with cancer. Microbiol Res 2016; 183: 53-59.

Fallahi Sh, Mazar Z A, Ghasemian M, Haghighi A.Challenging loop-mediated isothermal amplification (LAMP) technique for molecular detection of Toxo- plasma gondii. Asian Pac J Trop Med 2015; 8(5):366-372.

McAuley J B. Congenital toxoplasmosis. J Ped Infect Dis 2014; 3(suppl 1): S30-S35.

Coppens I, Andries M, Liu J L, Cesbron-Delauw M-F.Intracellular trafficking of dense granule proteins in Toxoplasma gondii and experimental evidences for a regulated exocytosis. Europ J Cell Biol 1999; 78: 463-472.

Velmurugan GV, Tewari AK, Rao JR, Baidya S, Ku- mar MU, Mishra AK. High-level expression of SAG1 and GRA7 gene of Toxoplasma gondii (Izatnagar iso- late) and their application in serodiagnosis of goat toxo- plasmosis. Vet Parasitol 2008; 154: 185-192.

Lau Y-L, Fong M-Y, Mohd Idris M, Ching X-T. Clon- ing and expression of Toxoplasma gondii dense granule antigen 2 (GRA2) gene by Pichia pastoris. Southeast Asian J Trop Med Pub Health 2012; 43(1): 10.

Golkar M, Rafati S, Abdel-Latif M S, Brenier-Pinchart M-P, Fricker-Hidalgo H, Sima B K, et al. The dense granule protein GRA2, a new marker for the serodiag- nosis of acute Toxoplasma infection: comparison of sera collected in both France and Iran from pregnant wom- en. Diagn Microbiol Infect Dis 2007 Aug;58(4):419-426.

Nam H-W. GRA proteins of Toxoplasma gondii: main- tenance of host-parasite interactions across the para- sitophorous vacuolar membrane. Korean J Parasitol 2009; 47(Suppl): S29-S37.

Lecordier L, Mercier C, Sibley L D, Cesbron-Delauw M-F. Transmembrane insertion of the Toxoplasma gon- dii GRA5 protein occurs after soluble secretion into the host cell. Mol Biol Cell 1999; 10: 1277-1287.

Lecordier L, Mercier C, Torpier G, Tourvieille B, Dar-cy F, Li L J, et al. Molecular structure of a Toxoplasma gondii dense granule antigen (GRA 5) associated with the parasitophorous vacuole membrane. Mol Biochem Parasitol 1993; 59: 143-153.

Ching X T, Lau Y L, Fong M Y. Hterologous expres- sion of Toxoplasma gondii dense granule protein 2 and 5. Southeast Asian J Trop Med Public Health 2015; 46:375-387.

Hiszczynska-Sawicka E, Oledzka G, Holec-Gasior L,Li H, Xu J B, Sedcole R, et al. Evaluation of immune responses in sheep induced by DNA immunization with genes encoding GRA1, GRA4, GRA6 and GRA7 antigens of Toxoplasma gondii. Vet Parasitol 2011;177(3-4): 281-289.

Ching X T, Lau Y L, Fong M Y, Nissapatorn V, Andi- appan H. Recombinant dense granular protein (GRA5) for detection of human toxoplasmosis by Western blot. Biomed Res Int 2014; 2014: 690529


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