Razi Journal of Medical Sciences

    Background and Aim: Leishmania major p4 gene is normally expressed during amastigote form of the parasite and can be a good candidate for producing an effective vaccine. The aim of this study is to clone p4 gene in a suitable vector for further vaccine production studies .

Materials and Methods: This is an experimental study. Leishmania promastigote was grown in N.N.N. medium and later in the form of mass culture in RPMI 1640 cell culture medium. Total leishmania genomic DNA was extracted by centrifugation of promastigote and lysed by lysis buffer 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. After transformation, the recombinant plasmid was screened  and digested by restriction enzyme

Results: PCR reaction and cloning p4 gene in T-vector was done successfully. Recombinant plasmid was extracted and cloned gene was released by restriction enzyme and subcloned into pPQE-30 expression vector and confirmed by restriction analysis and PCR.

Conclusion: This newly expressed combination is p4 gene invitro. Production of p4 recombinant antigen is very much useful especially in the field of vaccine production, drug target and immunological studies.