Gene Cloning of Leishmania Major Thiol-Specific Antioxidant Antigen (TSA) Gene from Pakistan: A Step towards Vaccine Development

The objective of this study was cloning of Thiol-Specific Antioxidant Antigen (TSA) gene, in suitable vector for further vaccine studies. The study has been conducted at Centre for Molecular Genetics University of Karachi, Karachi. The samples for this study were collected from a local hospital in Karachi, Pakistan. These samples were identified as Leishmania major spp at our institute, using PCR based investigations. These strains were maintained invitro. Among the vaccine candidates, TSA (thiol-specific antioxidant protein) has been reported as one of the major vaccine candidates, which elicits a Th1 response. TSA antigen gene was successfully amplified from identified species, a band of 600bp showed TSA antigen gene. TSA antigen gene sequencing results has been submitted to GenBank and obtained accession numbers KC758697 to KC758702. Amplified and purified TSA antigen gene product was used for gene cloning. The TSA cloning was done in Vector pTZ57R/T. The ligation mixture was transformed in XL1-Blue Competent Cells. For blue/white screening LB agar plates containing the antibiotic ampicillin solution, IPTG and X-gal were used. Transformants were obtained as white colonies. Recombinant plasmids were purified and restriction analysis was done. Restriction analysis of the transformed purified plasmid produces a band of 600bp corresponds to TSA antigen gene. Presence of TSA Antigen gene was also confirmed through PCR with specific primers from recombinant plasmid and a band of 600bp was obtained corresponding to TSA antigen gene. In conclusion we have successfully cloned TSA gene, which can be used for gene expression and for further vaccine development studies.