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

作者：Luz Martinez Hernandez Sandra; Cervantes Garcia Daniel; Munoz Ortega Martin; Aldaba Muruato Liseth R; Loera Muro Victor M; Ascacio Martinez Jorge A; de Jesus Loera Arias Maria; Montes de Oca Luna Roberto; Ventura Juarez Javier^*

来源：Biotechnology Letters, 2017, 39(8): 1149-1157.

DOI：10.1007/s10529-017-2341-2

• Entamoeba histolytica
• Anti-amoebic vaccine
• Exotoxin A
• KDEL
• LC3
• Pichia pastoris
• Recombinant protein
• Vaccine

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 PEDIII-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 PE Delta IIILC3-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 PEDIII and KDEL3, with potential application as an immunogen.

• 出版日期2017-8