Chloroplast-Expressed SAV3 E2-Ecto Vaccine

The sequence of the SAV vaccine was based on the E2 external glycoprotein domain of the structural protein sequence derived from the genome of a Norwegian isolate of the SAV3 subtype (SAV3-4-SF/10; Genbank Accession number KC122923) [25 (link)]. The protein sequence was designed as follows: residues 353 to 730 of the structural polyprotein were fused to the C terminus of the Cholera toxin B subunit sequence [26 (link)] via a (GGGGS)x3 flexible linker, and an HA tag (YPYDVPDYA) was added to the E2 C-terminus via a single GGGGS linker. The chimeric protein was termed E2-ecto, and the sequence was back-translated using the codon preference table for the C. reinhardtii chloroplast to synthesize a level 0 coding sequence (CDS) part for start–stop assembly [27 (link)], a variation of the Modular Cloning (MoClo) assembly system [28 (link)]. Full sequence details are given in supplementary Figure S1. The CDS part was then fused to the C. reinhardtii rrnS promoter, psaA 5′ untranslated region (5′UTR) and rbcL 3′UTR using start–stop assembly to create a level 1 transcriptional unit, which was then assembled with left and right homology arms derived from the chloroplast genome, in order to create a level 2 plasmid termed pE2-Ecto that would target the transcriptional unit into the genome, downstream of psbH (see Figure 1 and Figure S1).