Cloning and Expression of Leishmania infantum P4 gene for production of recombinant P4 antigen

Visceral leishmaniasis (VL) or Kala-azar caused by Leishmania infantum is a severe endemic disease in the Mediterranean basin countries including Iran. The Drugs available for treatment of VL are toxic and drug resistance is increasing in many parts of the world, thus, it is believed that vaccine development is an ideal method for prevention and control of VL. The aim of this study was to prepare recombinant P4 protein from Leishmania infantum and evaluate its immunogenicity in VL patients. DNA was extracted from Leishmania infantum and used for amplification of P4 gene by PCR. The PCR product was cloned, sequenced and expressed in E. coli using pET 28a expression vector. Recombinant P4 protein were purified and used for analysis of sera of VL patients. Analysis of the sequence of Leishmania infantum P4 gene (Li-P4) revealed that the gene consists of an ORF of 951bp with a 89% homology with P4 gene of cutaneous leishmaniasis agents. Expression of Li-P4 gene in E. coli resulted in high levels of recombinant proteins with molecular weight of 33 KD in SDS-PAGE. Immunoblotting analysis of the purified Li-P4 with sera of VL patients indicated that Li-P4 protein is a highly immunogenic protein expressed in the amastigote-stage of Leishmania infantum. This is the first study on isolation and characterization of P4 antigen from Leishmania infantum and indicates that Li-P4 protein is highly immunogenic and could be considered as a potent vaccine candidate against VL caused by Leishmania infantum.