An anti-amoebic vaccine: generation of the recombinant antigen LC3 from Entamoeba histolytica linked to mutated exotoxin A (PEΔIII) via the Pichia pastoris system.

ABSTRACT

OBJECTIVE: To generate an immunogenic chimeric protein containing the Entamoeba histolytica LC3 fragment fused to the retrograde delivery domains of exotoxin A of Pseudomonas aeruginosa and KDEL3 for use as an effective vaccine. RESULTS: A codon-optimized synthetic gene encoding the PEΔIII-LC3-KDEL3 fusion construct was designed for expression in Pichia pastoris. This transgene was subcloned into the plasmid pPIC9 for methanol-inducible expression. After transformation and selection of positive-transformed clones by PCR, the expression of the recombinant protein PEΔIII-LC3-KDEL3 was elicited. SDS-PAGE, protein glycosylation staining and western blot assays demonstrated a 67 kDa protein in the medium culture supernatant. The recombinant protein was detected with a polyclonal anti-6X His tag antibody and a polyclonal E. histolytica-specific antibody. A specific antibody response was induced in hamsters after immunization with this protein. CONCLUSIONS: We report for the first time the design and expression of the recombinant E. histolytica LC3 protein fused to PEΔIII and KDEL3, with potential application as an immunogen.