RESUMO
The present work aimed at establishing a platform to enable frequent characterization of the HCV RNA-dependent-RNA-polymerase from Egyptian clinical isolates. Subjecting amplified HCV-NS5B coding gene from Egyptian patient's serum to sequencing, multiple alignment, and phylogenetic analysis confirmed its subtype 4a origin. Nucleotide sequence analysis revealed presence of an additional start codon at the beginning of the NS5B gene. Peptide sequence alignment demonstrated presence of unique amino acid residues in our 4a-NS5B sequence distinct from the JFH-1-NS5B sequence as well as unique amino acids compared to other genotypes. The distinct molecular structure of the herein characterized 4a-NS5B from the 2a-JFH-1-NS5B was further demonstrated both in the built 3D models and the Ramachandran plots corresponding to each structure. Both the unique amino acid residues and 3D structure of the 4a-NS5B may influence both genotype 4a replication rate and response to therapy in comparison to other genotypes. Many resistance mutations to polymerase inhibitors were found both in ours and other genotypes' sequences. The presence of the required amino acid motifs for the RNA dependent RNA polymerase activity encouraged to clone the NS5B570-encoding sequence downstream CMV promotor in a mammalian expression vector. Such construct was used for both prokaryotic expression in bacteria and for DNA immunization. Successful mammalian expression and induction of specific immune response were demonstrated by ELISA and Western blotting. The potential of both the raised antibodies and the expressed NS5B to differentiate between HCV-infected and control human sera were demonstrated which reflect their diagnostic value.