Cloning of Dense Granular (GRA) 7 Gene of Toxoplasma gondii into pTZ57RT Vectors for Sub-Cloning in Prokaryotic and Eukaryotic Plasmids
Background: 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.
Materials and Methods: 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.
Results: 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.
Conclusion: 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.
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