Cloning and Expression of a Secretory form of Truncated ORF2 (aa 112-607) from Hepatitis E Virus in the pVAX1 Vector

Abstract

Background: The hepatitis E virus (HEV) accounts for the hepatitis E infection with a high mortality rate in pregnant women. Therefore, the design of the novel and effective vaccines seems essential and the DNA vaccine approach could be useful to achieve this anticipated goal. Objectives: The aim of this study was the cloning of a secretory form of truncated open reading frame 2 (ORF2) of HEV containing amino acids (aa) 112 -607 into the eukaryotic expression pVAX1 Vector and evaluating of the expression of this recombinant protein in eukaryotic cells. Methods: The truncated ORF2 gene (aa 112 -607) was cloned in the pVAX1 plasmid by restriction enzyme digest and confirmed by digestion and sequencing. Then, the recombinant plasmid was transfected into eukaryotic cells to express the recombinant protein.The expressed protein in the cell lysate and supernatant was evaluated by immunofluorescence assay (IFA) and western blot assay. Results: Colony polymerase chain reactions (PCR), restriction enzyme digestion, along with DNA sequencing of the recombinant plasmid were performed for confirmation of cloning tPA-PADRE-truncated ORF2 gene (aa 112 -607) into pVAX1 eukaryotic expression vector. The appearance of the truncated ORF2 protein (56 kDa) in the eukaryotic cells was accepted by the western blot assay, reverse transcription polymerase chain reaction (RT-PCR) method, as well as IFA. Conclusions: All outcomes of the present research showed that pVAX1-tPA-PADRE-truncated ORF2 (aa 112 -607, 56 kDa) recombinant plasmid was able to express truncated ORF2 from HEV as a potential candidate vaccine.