Optimization and In-silico Evaluation of Protein Fusion Antigen

The sequences coding for the protein portions selected to be included in the fusion antigen were joined contiguously, respecting the open reading frames of interest. Sequences coding for a Flag-Tag and a His-Tag were added to the 3′ end, followed by the introduction of a stop codon. Correct translation was confirmed using the ExPASy translate tool (https://web.expasy.org/translate/), followed by codon optimization to a mammalian codon usage bias. The originated sequence was synthetized (GeneArt Strings—Thermo Fischer Scientific, MA, USA) and cloned into the VR2001-TOPO vector as previously described^{29 (link)}. The VR-2001-SfSPChimera plasmid was then purified as previously reported^{29 (link)}, and used either as a DNA vaccine or as a vector in the attempt of protein expression using a mammalian system, described elsewhere^{25 (link)}.
Additional in-silico determinations were performed now using the multiepitope antigen protein sequence as template. The prediction of antigenicity probability (ANTIGENpro), solubility upon overexpression (SOLpro), formation of disulphide bonds (DIpro) and presence of transmembrane domains (ABTMpro) were performed using the SCRATCH protein predictor online tool (https://scratch.proteomics.ics.uci.edu/). The prediction of allergenic potential was performed using the AlgPred online tool (https://crdd.osdd.net/raghava/algpred/)^{90 (link)}.