[Cloning and sequencing of granular antigen 7 [GRA7] in TOPO vector and transformation in TOP10]

Toxoplasmosis, caused by an intracellular protozoan parasite, and the Toxoplasma gondii, is widespread throughout the world. In recent years, significant progress has been made in the identification of vaccine candidates which could induce a protective response. GRA7, an excretory 29 kDa Toxoplasma gondii a dense granular antigen released by infected host cells. In tachyzoite-infected cells, p29 accumulates within the parasitophorous vacuole and co-localizes with its delimiting membrane. In the present work, first genomic DNA of Toxoplasma gondii was extracted and used for amplifying of GRA7 gene as a template. Then PCR product was extracted from agarose gel and cloned into TOPO vector. The plasmid containing GRA7 gene was extracted from the transformed bacteria [TOP10 strain] and sequenced. Sequence analysis of GRA7 gene cloned into TOPO vector showed only one base difference when composed with the gene bank sequence for RH strain was only one base. The results indicated that this clone is suitable for subcloning in Prokaryotic and Eukaryotic plasmid

effect of betamethasone and IFN-gamma on replication of toxoplasma gondii [RH strain] and nitric oxide production in HeLa cell culture

Toxoplasmosis is a protozoal infection caused by Toxoplasma gondii. Toxoplasmosis produce severe damage in patients who are immunosuppresed. In those who are immunosupressed, latent infection can be reactivated resulting in acute disseminating disease. Betamethasone is a synthetic glycocorticoid, used as an anti-inflamatory and immunosuppressant in a wide variety of disorders. The aim of this study was evaluation of betamethasone as an immunosuppressor drug on infected cells by Toxoplasma gondii. In this study, at first HeLa cells were grown in 24 well culture plates in culture medium .When confluent monolayer was obtained, we compared 6 groups to evaluate the effect of betamethasone as a corticosteroid drug [two concentrations 4 and 40microg/ml] and the effect of IFN-gamma [100 IU/ml] on growth, replication and Nitric Oxide [NO] production. The results showed, that high number of plaques were seen in group with 40 microg/ml of betamethasone and the lowest number of plaques were seen in group with 100 IU of IFN-gamma. The difference between plaque number in control and groups treated with IFN-gamma and betamethasone was significant [P<0.05]. The groups with betamethasone or IFN-gamma without tachyzoites did not show any effect on cell structures. Replication rates in the wells treated with IFN-gamma were decreased significantly 72h post inoculation in comparison with control group [P< 0.05]. There was no significant difference among different groups in NO production. The results indicated that betamethasone increase the invasion of tachyzoites to host cells in vitro