Cloning of Leishmania Major P4 Gene

Objective: Leishmania major P4 gene is normally expressed during amastigote form of the parasite and can be good candidate for producing an effective vaccine. In this study we cloned this gene in suitable vector (pQE-30) for further vaccine preparation studies. Materials and Methods: Leishmania promastigotes were grown in N.N.N.medium and culture in RPMI 1640 cell culture medium. Total genomic DNA was extracted by centrifugation of promastigotes. The pellet was suspended in lysis buffer and followed by boiling method. PCR was carried out using P4 gene specific primers. PCR product was detected by agaros gel electrophoresis and cloned into Bluescript plasmid via T/A cloning method. Reaction was transformed into XL1- Blue competent cell and recombinant plasmid screened using agar plate contained X-gal and IPTG. The product was extracted, digested by restriction enzyme and electrophoresed on agarose gel. Results: Plasmid was extracted and cloned gene was released by restriction enzyme and subcloned into pQE-30 expression vector. Conclusion: This construct is ready for protein expression in in-vitro.