Construction of an Expression Vector Containing a Novel Fusion Sequence from Middle Region of NS3 and Truncated Core Genes of Hepatitis C Virus

Abstract

Background and Aims: DNA constructs containing HCV antigens have become one of the vaccine candidates for induction of anti-HCV cellular and humoral immunity. In this study, we constructed a novel expressing vector harboring a fusion sequence derived from an overlapping fragment in the middle of NS3 and a truncated core fragment to avoid troubles reported to be associated with full gene expression. Methods: The partial NS3 (pNS3) and core genes were amplified by RT-PCR method using serum of HCV infected patient harboring genotype 1a of virus. After purification and cloning the genes into TA-cloning vector, they were evaluated by sequencing and restriction digestion analysis. The resultant pNS3 and core gene subcloned into expression vector separately followed by expression evaluation using RT-PCR and western blotting. The core expressing vector exploited for amplification of a new truncated core (50-160aa) sequence using PCR. Truncated core fragment was first cloned into TA vector at a natural restriction site downstream of pNS3 fragment. The resulting fused sequence was cut and subcloned into expression vector. The integrity and ability of expression of this fused sequence was evaluated by sequencing followed by RT-PCR analysis after DNA transfection into 293 cells. Results: The repeated sequencing data showed sequence integrity among the gene fragments as well as homology among them and reference 1a sequences. The colony-PCR, RT-PCR and western blotting confirmed insertion of genes into expressing vector, expression of genes in 293 cell line and production of protein in 293 respectively. Conclusion: This new expressing vector harboring a novel fused fragments of NS3 and core genes may overcome shortcomings in vaccine design in the setting of HCV disease.

Construction and Preparation of Three Recombinant Adenoviruses Expressing Truncated NS3 and Core Genes of Hepatitis C Virus for Vaccine Purposes

“…However, the NS3 gene from the Iranian patient had more differences in gene sequence, albeit a high homology in amino acid sequence (unpublished data) in comparison to other reference sequences. Evaluation of the amplified NS3 sequence by phylogenetic analysis strongly confirmed that it belonged to 1a genotype as was previously described in detail ( 30 ).…”

“…Amplification of new partial core (aa 50-160), new overlapping region of NS3 gene covering protease/helicase domains and a fusion of both truncated fragments from HCV genotype 1a were previously described ( 30 , 31 ). In brief, full core and partial NS3 sequences were propagated from the serum of a HCV patient by RT-PCR.…”

“…The transfer/shuttle vectors contained partial NS3 or partial core and fusion of both genes designated as IR-pNS3, IRpCore, and IR-pNS3-pCore ( Figure 1 ) respectively. Phylogenetic analysis of sequences was also used to confirm true cloning of the desired genotype as described previously ( 30 , 31 ).…”

Truncated Core/NS3 Fusion Protein of HCV Adjuvanted with Outer Membrane Vesicles of Neisseria meningitidis Serogroup B: Potent Inducer of the Murine Immune System

“…Three adenoviral constructs expressing the middle region of the core gene and consisting of some parts of D1 and D2 (aa 50-160) and the middle region of NS3 consisting of protease and helicase (aa 1095-1387) have been developed by Hosseini et al . [15,16]. In order to retain the conformational structure and more immune response of the recombinant core, the full-length of core D1 (aa 1-118) with a flexible linker (AAY) and proteasome cleavable site and also the middle region of NS3 in fused form (rC/N) were produced without C-terminal domains of core, and without N and C-terminal sequences of NS3.…”