2.3  Construction of Multi-Epitopes Peptide (MEP) and Adjuvanting
Overlapping immunodominant epitopes were used to construct a multi-epitope peptide (MEP) which is considered a promising strategy to stop viral infections [24]. One of the key issues with the design of peptide vaccine is its weak immunogenicity that can be resolved by designing a MEP with appropriate adjuvants [25]. In the current study, MEP was designed using AAY linkers to combine screened multiple epitopes [26]. To the N-terminal of MEP, an adjuvant in the form of B subunit of cholera toxin was linked to the MEP thus creating a multi-epitope peptide vaccine construct (MEPVC) [27]. The tertiary structure of the MEPVC was created through a software called 3Dpro of SCRATCH protein predictor [28], I-tasser [29], and Swiss-Model [30]. The best model was further loop modelled using GalaxyLoop [31] and refined using GalaxyRefine [32] of GalaxyWeb. To improve the construct's stability, disulfide bonds were introduced in the structure [33] using Design 2.0 [34]. The sequence of the MEPVC was translated in reverse and then optimized for codon usage according to the Escherichia coli, which will end up in the increased expression of the MEPVC sequence cloned in the mentioned expression system [35]. The entire activity was accomplished using Java Codon Adaptation Tool (JCat) server [36]. In order to assess the expression of sequence that have been cloned, the GC content and codon adaptation index (CAI) were measured. The value of 1 CAI is contemplated ideal [37,38] whereas the appropriate GC content should be fluctuated between 30-70% due to favorable transcriptional and translational efficiencies [27]. There were other input factors carefully calculated to prevent rho-independent transcription termination, the binding sites of the prokaryotic ribosome, and the cleavage sites of restriction enzyme. As the final step of this phase, the cloning of the engineered construct was carried out into pET-28a (+) expression vector using SnapGene (https://www.snapgene.com/).