Cloning and Sequencing of Truncated Toxoplasma gondii Subtilisin-Like 1 Antigen
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Date
2016-08-31
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Brieflands
Abstract
Background: Toxoplasma gondii is an obligate intracellular protozoan parasite which has significant medical and veterinary impact on all around the world. Tracking of specific antigens or antibodies for toxoplasmosis is the main choice in its diagnosis. Recombinant proteins will improve sensitivity and specificity and reduce problems of standardization and reproducibility of diagnostic kits. Toxoplasma gondii Subtilisin-like protein (TgSUB1) is a novel example of a glycosylphosphatidylinositol) GPI (-anchored protein which can be considered as a potential marker for serodiagnosis of toxoplasmosis. Objectives: The aims of this study were to find out major antigenic parts of this whole protein and to develop a recombinant prokaryotic plasmid. Methods: In this experimental study, using bioinformatics softwares Parker Hydrophilicity prediction and Bepipred linear Epitope prediction to select best highly antigenic region of this protein, a 744 bp fragment was amplified by polymerase chain reaction (PCR) on cDNA obtained from T. gondii RNA. The PCR product was cloned in PCR2.1 vector and subcloned into expression pET28a vector. The PCR2.1-SUB1 and PET28a-SUB1constructs were analyzed by PCR, restriction analysis and sequencing. Results: A highly antigenic region in the hydrophilic part of the protein including amino acid residues 549 to 795 was successfully cloned and the sequences were confirmed. All nucleotide sequences in the PCR product have 100% homology with the published reference sequence. Conclusions: Pairing bioinformatics tools and cloning of the candidate molecules in vaccine development studies and diagnostic approaches will have powerful impact on promotion of research in infectious diseases. This strategy is considered as available and inexpensive technology even in less developed countries where the infectious diseases like toxoplasmosis is prevalent.