فهرست مطالب سید جواد سیدطبایی

One of the most important complications of toxoplasmosis is its early diagnosis. It seems that GRA7 protein can be a good candidate for detection of the acute phase in Toxoplasmosis. Accordingly, the present study aimed to diagnose toxoplasmosis via a newly immunochromatographic test using recombinant antigen gra7.

The parasite was cultured in mice and then were used for DNA extraction. The gra7 gene was amplified by PCR and cloned into the pET-32a (+) plasmid. Thereafter, the recombinant vector was transferred into the Escherichia coli Rosetta strain and gra7 was detected via SDS-PAGE and western blotting. The bacterial lysate was used to purify the protein by Ni-NTA affinity chromatography .Anti-human gold conjugated antibody, test line and control line were injected to conjugate pad and nitrocellulose membrane, respectively, and all the layer were assembled. By using serum of patients and healthy individuals, manufactured kits were evaluated.

Our results indicated that the selected gene was correctly cloned and the protein of interest was produced and purified. The test revealed sensitivity and specificity of 100 and 96.7 percent, respectively. The kit was also shown to be stable over 16 weeks in 37°C.

The choice of antigen based on cellular and clinical features of the parasite, as well as the use of previous outcomes yielded to develop a rapid diagnostic test for toxoplasmosis.

Backgraound: Entamoeba histolytica antigenic markers such as Serine-Rich E. histolytica protein (SREHP) have recently been used for vaccine preparation, genetic diversity studies of Entamoeba histolytica isolates and for differentiation between E. histolytica and E. dispar species. This study was carried out with the aim of expression of a recombinant Serine Rich E. histolytica protein in the laboratory to use it in the ELISA kit.

In this study which is an exploration method, an Iranian isolate of Serine-Rich E. histolytica gene which had previously been cloned in bluescript plasmid (pBSc), was cut using BamHI restriction enzyme. After extracting and purification from gel, the SREHP gene was sub cloned into pET32a expression vector. The inserted gene was confirmed with Rosconis solution, PCR and sequencing methods. PCR was performed with the SREHP specific primers as well as pET T7 promoter primer. The cloned gene was also digested with HindIII and BamHI restriction enzymes. Recombinant plasmid was conveyed to competent cell BL21 (DE3). A colony of the plasmid including SREHP gene was cultivated and induced with IPTG. The result of expressed protein was observed on the SDS-PAGE gel. The SREHP gene was sub cloned into pET32a expression vector. A recombinant plasmid including an inserted SREHP gene was screened and confirmed with quick check method using Ruscoins solution, as well as PCR by special primers (SREHP and universal pET primer), digested with BamHI and HindIII restriction enzymes. Finally an open reading frame of 666 nucleotides from inserted SREHP gene was obtained with the sequencing method.

The recombinant protein of Serine-Rich E. histolytica in presence of IPTG was expressed in five hours and the result of expressed protein in the length of 44 KDa was observed on SDS-PAGE gel.

SREHP protein was successfully cloned and expressed in this study. However additional studies are recommended for preparation and purification of the SREHP in a large quantity and the using it for the ELISA test.