Expression of Mycobacterium tuberculosis Rv0577 gene in Pichia pastoris and antigenicity of the recombinant protein

Rv0577 gene of Mycobacterium tuberculosis strain H37Rv was amplified and cloned into pGEM-T Easy and subcloned into pPICZαA. The correct recombinant plasmid pPICZαA-Rv0577 was identified by double digestion of endonucleases and proved by sequencing, and then linearized and transformed into Pichia pastoris strain X-33 by electroporation. The highly expressed, secreted recombinant protein was sequentially purified by saturated ammonium sulfate precipitation and HPLC. Purified protein was identified by Western blot with anti-His monoclonal antibody and by Dot blot with human sera. The recombinant protein showed its antigenicity by positive Dot blot reaction with sera from TB patients (3/7) and negative reaction with PPD negative sera.