Cloning and sequencing the plasmid encoding dense granule antigen 14 (GRA14) of Toxoplasma gondii RH strain

Background and Toxoplasmosis is a common parasitic disease throughout the world and ‎one-third of the population has antibodies to Toxoplasma gondii. This disease causes serious medical ‎problems in fetuses and immunocompromised individuals. As gene encoding protein GRA14 can be ‎considered as a suitable target for DNA vaccine and designing diagnostic kits; the aim of this study was to do ‎cloning and sequencing the gene encoding GRA14 protein of Toxoplasma gondii RH strain.‎ DNA extraction was performed on harvested tachyzoites from mouse ‎peritoneal fluid, then PCR carried out and amplification products were analyzed by gel electrophoresis. ‎GRA14 gene was cloned in pTG19-T as a cloning vector, then recombinant plasmid confirmed by the colony-‎PCR and restriction enzyme digestion using HindIII and EcoRI, followed by sequencing.‎ Evaluation of PCR products by agarose gel electrophoresis and analysis of nucleotide ‎sequencing of 1227 bp gene encoding the protein GRA14, revealed the complete homology with the recorded ‎sequences in the gene bank. Furthermore, cloning of GRA14 gene in pTG19-T vector was confirmed with ‎colony PCR and restriction enzyme digestion.‎ The results showed that the GRA14 gene was successfully cloned into the pTG19-T ‎vector and this plasmid can be used to design DNA vaccines and diagnostic kits in further studies.‎