Cloning of Leishmania Major P4 Gene

Objective: leishmania major p4 gene is normally expressed during amastigote form ofthe parasite and can be good candidate for producing an effective vaccine. in this study wecloned 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 culturein rpmi 1640 cell culture medium. total genomic dna was extracted by centrifugationof 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 byagarosgel electrophoresis and cloned into bluescript plasmid via tia cloning method. reactionwas transformed into xl1- blue competent cell and recombinant plasmid screened usingagar plate contained x-gal and iptg. the product was extracted, digested by restrictionenzyme and electrophoresed on agarose gel.results: plasmid was extracted and cloned gene was released by restriction enzyme andsubcloned into pqe-30 expression vector.conclusion: this construct is ready for protein expression in in-vitro.