title: Design, Construction and Cloning of Truncated ORF2 and tPAsp-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector.
abstract: BACKGROUND
  Hepatitis E Virus (HEV) is the causative agent of enterically transmitted acute hepatitis and has high mortality rate of up to 30% among pregnant women. Therefore, development of a novel vaccine is a desirable goal.
  
  
  OBJECTIVE
  The aim of this study was to construct tPAsp-PADRE-truncated open reading frame 2 (ORF2) and truncated ORF2 DNA plasmid, which can assist future studies with the preparation of an effective vaccine against Hepatitis E Virus.
  
  
  METHODS
  A synthetic codon-optimized gene cassette encoding tPAsp-PADRE-truncated ORF2 protein was designed, constructed and analyzed by some bioinformatics software. Furthermore, a codon-optimized truncated ORF2 gene was amplified by the polymerase chain reaction (PCR), with a specific primer from the previous construct. The constructs were sub-cloned in the pVAX1 expression vector and finally expressed in eukaryotic cells.
  
  
  RESULTS
  Sequence analysis and bioinformatics studies of the codon-optimized gene cassette revealed that codon adaptation index (CAI), GC content, and frequency of optimal codon usage (Fop) value were improved, and performance of the secretory signal was confirmed. Cloning and sub-cloning of the tPAsp-PADRE-truncated ORF2 gene cassette and truncated ORF2 gene were confirmed by colony PCR, restriction enzymes digestion and DNA sequencing of the recombinant plasmids pVAX-tPAsp-PADRE-truncated ORF2 (aa 112-660) and pVAX-truncated ORF2 (aa 112-660). The expression of truncated ORF2 protein in eukaryotic cells was approved by an Immunoﬂuorescence assay (IFA) and the reverse transcriptase polymerase chain reaction (RT-PCR) method.
  
  
  CONCLUSIONS
  The results of this study demonstrated that the tPAsp-PADRE-truncated ORF2 gene cassette and the truncated ORF2 gene in recombinant plasmids are successfully expressed in eukaryotic cells. The immunogenicity of the two recombinant plasmids with different formulations will be evaluated as a novel DNA vaccine in future investigations.
authors: Department of Microbiology and Parasitology, School of Medicine, Bushehr University of Medical Sciences, Bushehr, IR Iran; Health Research Institute, Infectious and Tropical Disease Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran.
authors: Fatemeh
authors: F
authors: Farshadpour
authors: Health Research Institute, Infectious and Tropical Disease Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran.
authors: Manoochehr
authors: M
authors: Makvandi
authors: Department of Microbiology and Parasitology, School of Medicine, Bushehr University of Medical Sciences, Bushehr, IR Iran; Persian Gulf Tropical Medicine Research Center, Bushehr University of Medical Sciences, Bushehr, IR Iran.
authors: Reza
authors: R
authors: Taherkhani
keywords: DNA Vaccine; Hepatitis E Virus; ORF2 Protein; PADRE Peptide; Signal Sequence