Molecular identification of phoshphoglyceate kinase gene from the secretory-excretory antigens of Toxocara canis

Toxocara canis has a worldwide distribution and is regarded as the main cause of human toxocariasis. Infection in humans, particularly children, is frequently caused by accidental ingestion of embryonated Toxocara eggs present in soil, water, food, dirty hands. In this study, C-terminus part of an expressed sequence tag (EST) encoded phoshphoglycerate kinase gene was selected from the NCBI Genbank (accession number HO348231) and amplified using semi-nested RT-PCR. CDNA was synthesized with the extracted total RNA as template and the modified oligo (dT) as primer. The product of RT-PCR and pMalc2x expression vector both were digested by BamHI and HindIII restriction enzymes and ligated. This construct was eventually transferred to bacterium E.coli, strain TG1. After plasmid purification, the insert was sequenced and characterized. Sequence comparisons with GenBank database were done using the BLAST software. Based on the sequence data, the complete sequence contained 304 nucleotides encoding an open reading frame of 101 amino acid residues with a predicted molecular mass of 10.497 kDa and theoretical pI of 5. The results of this study indicated that the C-terminus part of phosphoglycerate kinase was successfully cloned and expressed in E. coli. Analysis of recombinant protein showed that part of this protein belongs to "phosphoglycerate kinase superfamily". Multiple aligenment shows that TcPGK had 95% and 85% similarity with phosphoglycerate kinase from Ascaris suum and also Loa loa and Burigia malayi, respectively.