Recombinant HCV type 1 Nonstructural Protein 5 (a.a. 2212-2313) [GST]

Peptides were synthesized with very high purity and yield on a highly solvating copolymer of 1,4-butanediol dimethacrylate cross-linked polystyrene support (PS-BDODMA). The polymer was synthesized by radical aqueous suspension polymerization using toluene as the diluent and 1% polyvinyl alcohol as the suspension stabilizer. The supports were highly resistant to various chemical reagents and solvents that are frequently used in polypeptide synthesis. The peptides synthesized on this support were designed from the core (C), envelope (E1 and E2) and nonstructural protein (NS1-NS5) regions of the prototype hepatitis C viral (HCV) polyprotein, and were used to develop a peptide-based immunoassay (PBEIA) for the detection of HCV antibodies. The purity of these peptides was tested by HPLC. Peptide identity was confirmed by amino acid analysis and MALDI-TOF-MS. A single peptide chosen from a conserved area (E2/NS1) at the C-terminus of the hypervariable region (HVRI) was found to be sufficient for effective and reliable diagnosis of HCV infection in infected individuals, as well as also apparently healthy individuals. The CD spectrum of the peptide shows no preference for any ordered secondary structure. When used, peptide mixtures from various protein regions of HCV reduced the sensitivity and reliability of the diagnosis partly because of epitope masking.

The Japanese variant of the hepatitis C virus (HCV-N) genome, consisting of 9440 nucleotides in length, was cloned from a small amount (2 ml) of plasma from a single Japanese carrier by using RT-PCR and modified RT-PCR. The HCV-N genome has a long open reading frame that encodes a 3014 amino acid polyprotein with 340 and 57 bases of 5' and 3' non-coding sequences, respectively. HCV-N has a 4-amino-acid insertion in the NS5 region as compared to other HCV isolates, but this insertion is found to be very rare upon direct sequencing of that region. Comparative sequence analysis of all the complete and partial HCV sequences that were reported indicates that HCV can be subdivided into at least 4 groups. The HCV-N isolate has a high homology with HCV-J and HCV-BK (> 90%) and so belongs to group II, but shows less similarity to HCV-1 (> 78%, group I) and least to HC-J6 (> 67%, group III). Among these HCV isolates, the 5' non-coding region was the most conserved (> 93%) since it plays an important role in replication. The RT-PCR assay to detect HCV-RNA, using the primers deduced from this region, was very sensitive and specific. The putative core protein could become an important target for immunoassay because of a high degree of amino acid sequence similarity in that region. A high degree of diversity and a low similarity between each HCV isolate in the putative envelope protein play an important role in the chronicity of HCV infection and development of immunopreventive agents, such as immunoglobulin and vaccine for that infection.