cloning, expression and immunoreactivity of recombinant toxoplasma gondii gra5 protein

Authors

zahra arab-mazar infectious diseases and tropical medicine research center, shahid beheshti unversity of medical sciences, tehran, iran

shirzad fallahi department of parasitology and mycology, lorestan university of medical sciences, khorramabad, iran

ameneh koochaki department of biotechnology, faculty of medicine, shahid beheshti university of medical sciences, tehran, iran

hadi mirahmadi infectious diseases and tropical medicine research center, zahedan university of medical sciences, zahedan, iran

abstract

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 xho i and bam hi 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

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Journal title:
iranian journal of microbiology

جلد ۸، شماره ۵، صفحات ۳۳۱-۳۳۷

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