In Silico Recombinant Plasmid Construction from Ancestral and Consensus Envelope Protein Domain III Sequences of Dengue Virus Serotype 3

Abstract

Dengue, a mosquito-borne disease caused by the dengue virus, is a significant threat to global health, especially in Asia. Observed ADE in FDA licensed dengue vaccine (Dengvaxia) post clinical study leads to the use of ancestral sequence reconstruction and consensus sequence strategy to design new vaccine candidates with an improved vaccine potency. In this study, designated ancestral and consensus protein sequences of Asian DENV3 EDIII were analyzed and optimized for in silico recombinant plasmid construction. An in silico recombinant plasmid for both sequences was successfully constructed. mRNA secondary structure analysis predicted the ancestral EDIII sequence of DENV3 (with an additional two guanine nucleotides to add N-terminal glycine residue) could be optimally inserted into pET-28a (+) using NcoI and XhoI restriction sites to generate accessible RBS and start codon (MFE -25.3 kcal/mol). Meanwhile, mRNA secondary structure analysis predicted the consensus EDIII sequence of DENV3 could be optimally inserted into pET-24a (+) using NdeI and XhoI restriction sites to generate accessible RBS and start codon (MFE -23.6 kcal/mol). The final ancestral (RMSD 1.13 Å, TM-score 0.903) and consensus (RMSD 1.10 Å, TM-score 0.905) proteins were predicted to have close structural homology with the reference DENV3 E protein (PDB:1UZG), based on the RMSD and TM-score generated by I-TASSER. Codon optimization for expression in E. coli BL21 (DE3) shows the CAI of 0.816 and %GC of 48.3% for ancestral sequence and CAI of 0.824 and %GC of 48.1% for consensus sequence.