Cloning, expression and immunoreactivity of recombinantToxoplasma gondiiGRA5 protein.
OBJECTIVE
Toxoplasma gondiiis 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 fromT. gondii(TgGRAs) are potential antigens for the development of diagnostic tools.


METHODS
DNA was extracted fromT. 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 intoXhoI andBamHI digested pGEX6p-1 expression vector. Recombinant plasmid was transformed intoE. 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 ofT. gondiiGRA5 gene from different isolates. The recombinant plasmid pGEX-6p-1/GRA5 induced by IPTG was expressed inE. coli.It was a GST fusion protein and could react with human positive sera analyzed by western blot.


CONCLUSIONS
The GRA5 gene ofT. gondiiisolates is highly conservative. This antigen as a recombinant protein was successfully expressed inE. coli,which showed high immunoreactivity.
Infectious Diseases and Tropical Medicine Research Center, Shahid Beheshti Unversity of Medical Sciences, Tehran, Iran; Department of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Zahra
Z
Arab-Mazar
Razi Herbal Medicine Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran; Department of Parasitology and Mycology, Lorestan University of Medical Sciences, Khorramabad, Iran.
Shirzad
S
Fallahi
Department of Biotechnology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Ameneh
A
Koochaki
Infectious Diseases and Tropical Medicine Research Center, Zahedan University of Medical Sciences, Zahedan, Iran.
Hadi
H
Mirahmadi
Department of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Seyyed Javad Seyyed
SJ
Tabaei
Dense granule antigen; GRA5; Immunoreactivity; Toxoplasma gondii