Cloning and expression of Leishmania infantum K26 antigen in E. coli and evaluation of its potential for serodiagnosis of visceral leishmaniasis

The objective of this study was preparation and evaluation of recombinant 26 antigen of Leishmania infantum (L. infantum) for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. Genomic DNA was extracted from L. infantum promastigotes by phenol-chloroform method and used for PCR amplification of k26 gene. The PCR product was purified, cloned into the Bluescript vector and subjected to DNA sequencing. For production of recombinant 26 protein, the insert was removed by restriction digestion, subcloned into the pQE-80L vector and expressed in E. coli. The recombinant protein was purified by Ni-NTA column and used for evaluation of response of VL patients by immunoblotting. PCR amplification of K26 gene using L. infantum genomic DNA as a template was resulted in amplification of a 756 bp fragment. Cloning and sequencing of amplified fragment showed that there is a 98 % homology to L. chagasi and 95 % to L. donovani 26 gene sequence. Recombinant expression and purification of L. infantum K26 gene produced a highly pure protein appeared as a 45 kDa band in SDS-PAGE analysis. Western blot analysis showed that the sera from visceral leishmaniasis patients contain a high titer of antibody against K26 antigen. Western blot analysis using purified recombinant antigen showed that K26 antigens are recognized by sera from VL patient due to L. infantum and that this antigen can be exploited for serodiagnosis of VL. EMRO/tdr Grant SGS 05/95.