ABSTRACT
Serological assay based on dense granular [GRA] proteins of Toxoplasma gondii [T. gondii] is actually the most popular laboratory diagnostic tool to detection of toxoplasmosis. We aimed to construct a recombinant GRA7-pTZ57RT plasmid vectors that it is suitable for sub-cloning and GRA7 protein production. Souris mice were used for maintaining of T. gondii tachyzoites by serial intraperitoneal passage. The tachyzoites' DNA was extracted, and the GRA7 gene was amplified by PCR. The purified DNA was inserted into pTZ57RT cloning vectors, and then transformed into TOP10 competent cells. Finally, cloning and transformation were confirmed by restriction enzymatic digestion and gene sequencing. Agarose gel electrophoresis analysis on PCR products of genomic DNA, revealed 726 bp bands that were equal to the GRA7 gene. Both white [recombinant] and blue [non-recombinant] colonies appeared on ampicillin-LB agar. Results of enzymatic digestion and gene sequencing confirmed successful cloning and transformation procedures. The GRA7 gene of T. gondii was cloned into pTZ57RT plasmid, which is suggested to be further used as DNA vaccine or sub-cloned for production of recombinant GRA7 protein
ABSTRACT
The aim of this study was to detect the genotype of Fasciola spp. in Meshkin-Shahr, Ardabil Province, northwestern Iran in different hosts using PCR-RFLP. The parasite hosts included cattle, and sheep. Overall, 70 adult flukes from livers of slaughtered animals were collected from the abattoirs of aforementioned area. The included 35 samples from infected sheep and 35 samples from 35 infected cattle. PCR-RFLP and sequence analysis of the first nuclear ribosomal internal transcribed spacer [ITS 1] region from Fasciola species were used to conduct the study. The fragment of approximately 700bp in all of the Fasciola samples was amplified. PCR products of ITS 1 were subjected for digestion by restriction enzyme. RsaI restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species. Amplicons with the sequences of F. hepatica had a pattern of about 360, 100, and 60 bp band size, whereas F. gigantica worms had a profile of 360, 170, and 60 bp in size, respectively. Results based on PCR-RFLP analysis were confirmed by sequence analysis of representative ITS 1 amplicons. No hybrid forms were detected in the present study. All sheep were infected with F. hepatica but cattle were infected with both species. Both species of Fasciola are present in Ardabil. The method described here can be valuable for identification of Fasciola species in endemic parts for fasciolosis, regions with intermediate species and in that overlapping distribution area