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

• Published in: Biotechnology letters 2017-08, Vol.39 (8), p.1149-1157
• Main Authors: Martínez-Hernández, Sandra Luz, Cervantes-García, Daniel, Muñoz-Ortega, Martín, Aldaba-Muruato, Liseth R., Loera-Muro, Victor M., Ascacio-Martínez, Jorge A., de Jesús Loera-Arias, María, de Oca-Luna, Roberto Montes, Ventura-Juárez, Javier
• Format: Article
• Language: English
• Subjects: ADP Ribose Transferases - genetics; ADP Ribose Transferases - immunology; Animals; Antibody response; Antigens; Applied Microbiology; Assaying; Bacterial Toxins - genetics; Bacterial Toxins - immunology; Biochemistry; Biomedical and Life Sciences; Biotechnology; Entamoeba histolytica - genetics; Entamoeba histolytica - immunology; Exotoxin A; Exotoxins - genetics; Exotoxins - immunology; Fragmentation; Gel electrophoresis; Gene expression; Genetic transformation; Glycosylation; Hamsters; Immunization; Immunogenicity; Life Sciences; Methanol; Microbiology; Microorganisms; Mutation; Original Research Paper; Pichia - genetics; Pichia pastoris; Polymerase chain reaction; Proteins; Pseudomonas; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Sodium lauryl sulfate; Staining; Toxins; Vaccines; Virulence Factors - genetics; Virulence Factors - immunology; Yeast
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-017-2341-2

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.