Cloning, expression and purification of recombinant A2 protein from Leishmania infantum for diagnosis of visceral leishmaniasis in Iran

Abstract

Visceral leishmaniasis (VL) is a fatal disease caused by Leishmania infantum in the Mediterranean basin and Iran. Different methods are used for diagnosis of VL. The aims of this study were expression and purification of recombinant A2 (rA2) protein of L.infantum and its application in the diagnosis of VL. The serological diagnosis of VL was applied using rA2 protein. In this study, A2 gene of L.infantum sequence was ordered for the synthesis, cloned in E.coli strain TOP10F' and proliferated in pET22-b vector. The expression and purification of rA2 proteins applied in host via BL21 and Ni-NTA respectively. The A2 gene sequences were synthesized and the construct transformed to pET22-b vector. A 102 Sanaz Naderi et al. 520bp fragment was identified in digested pEASY-A2 plasmid. The gene was successfully cloned in to pET22-b standard expression vector and transformed in E.coli BL21. Expression of rA2 was confirmed by SDS-PAGE and a 27KD protein was detected. The antigenicity of A2 protein was assessed using both pooled dog sera and C9 anti-A2 monoclonal Ab. This study recommends rA2ELISA as alternative assay to detect VL. More evaluation should be made to develop a cheap and reliable serologic test for detection of L.infantum among infected hosts.