Expression of Recombinant Heat-Shock Protein 70 of MCAN/IR/96/LON-49, a Tool for Diagnosis andFuture Vaccine Research

Heat shock protein 70 (HSP70) is present in all organisms studied so far,and is a major immunogen in infections caused by pathogens including Leishmania spp. The aim of this study was to clone and express HSP70 from L. infantumstrain MCAN/IR/96/LON-49 and evaluate antibody response against HSP70 in visceralleishmaniasis (VL). The L. infantum HSP70 gene segment was amplified byspecific primers. It was cloned into pTZ57R vector and subcloned into pET32a (+) expressionvector. The new construct was transformed in the E.coli Rosetta strain, andHSP70 protein was expressed in the presence of 1 mM IPTG and purified using a Hi-Trap chelating column. Antibody responses against HSP70 were determined by ELISAin 37 patients with visceral leishmaniasis and 63 healthy controls. Expressionof HSP70 protein was confirmed using SDS-PAGE electrophoresis and dot blot with ananti-His tag antibody. There was no difference between the sequence of nucleotides ofthe HSP70 gene in the present study and other reported sequences. The ELISA resultsindicated that the sera of 81.1% (30/37) of the patients and 6.3% (5/63) of controls reactedto L. infantum HSP70. The conservative nature of the HSP70 moleculeis an advantage in vaccine studies, because of minor differences (6%) between thenucleotide sequences and consequently the similarity in amino acid sequences in variousstrains of L. infantum. It could therefore be used in vaccine research againstleishmaniasis and also as a tool for serodiagnosis.