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

Biotechnol Lett. 2017 Aug;39(8):1149-1157. doi: 10.1007/s10529-017-2341-2. Epub 2017 May 3.

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.

Keywords: Anti-amoebic vaccine; Entamoeba histolytica; Exotoxin A; KDEL; LC3; Pichia pastoris; Recombinant protein; Vaccine.

MeSH terms

  • ADP Ribose Transferases / genetics*
  • ADP Ribose Transferases / immunology
  • Animals
  • Bacterial Toxins / genetics*
  • Bacterial Toxins / immunology
  • Entamoeba histolytica / genetics*
  • Entamoeba histolytica / immunology
  • Exotoxins / genetics*
  • Exotoxins / immunology
  • Pichia / genetics
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / immunology
  • Vaccines*
  • Virulence Factors / genetics*
  • Virulence Factors / immunology

Substances

  • Bacterial Toxins
  • Exotoxins
  • Recombinant Fusion Proteins
  • Vaccines
  • Virulence Factors
  • ADP Ribose Transferases
  • toxA protein, Pseudomonas aeruginosa