4.1. Gene Synthesis, Cloning and Plasmid Construction

The nucleotide sequences of the whole HEV genotype 3 nucleoprotein HEV ORF2 1–660 and chimeric M2 HEV 1–610 (lacking the 50 amino acids (aa) from the C-terminus) (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"DQ079627.1","term_id":"71379723","term_text":"DQ079627.1"}}DQ079627.1) were synthesized by Life technologies, (Carlsbad, CA, USA). These genes were used as master genes to design six HEV genes with different lengths of ORF2 capsid proteins and three chimeric genes bearing the influenza M2e peptide (see Figure 1). The M2 HEV 1–610 master gene has an AgeI restriction site at position 333–336 bp, and was used to generate the M2 HEV 110–610 construct. Additionally, more restriction sites were added from both sides of the M2e sequence (1675–1680 bp and 1753–1758 bp MfeI), allowing the production of the HEV 1–610 and the HEV 110–610 versions of the gene without the M2e epitope to occur. Amplification of DNA fragments for the production of the HEV 33–660, HEV 110–660, and HEV 33–610 constructs was carried out by PCR. All primers used for cloning are listed in Table 1.

List of primers used in this work.

The PCR fragments were flanked by AgeI and XhoI restriction sites (New England Biolabs, Ipswich, MA, USA), which were used for the cloning of PCR fragments into the pEAQ-HT vector digested with the same enzymes. The constructs HEV 1–660, HEV 1–610, and HEV 110–610 were cloned into pEAQ-HT from the master genes using the restriction sites AgeI/XhoI, NruI/ XhoI, and AgeI/XhoI respectively; E. coli XL1 blue strain was used for all cloning experiments [42]. Finally, the plasmid DNA was purified and the sequence was confirmed by PCR and DNA sequencing.

For the cloning of the HEV 110–610 and M2 HEV 110–610 constructs in the pEff vector, the corresponding sequences were amplified by PCR using primers HEV110_Asc-F/ HEV_Sma-R, and cloned into pEff vector at AscI and SmaI restriction sites. The corresponding recombinant pEAQ vectors were used as templates.

All pEAQ and pEff recombinant vectors were transformed into the electrocompetent A. tumefaciens strain LBA4404 or GV3101 [43].