Cloning and evaluation of expression of a novel overlapping region of NS۳ gene of Hepatitis C virus by expressing vector

Objective: In this project, our aim was to construct a novel expressing vector harboring a new sequence from overlapping region of NS۳ gene of HCV from infected Iranian patient. Materials and Methods: The partial NS۳ (pNS۳) gene was amplified by Nested-RT-PCR method using sera of HCV infected patients harboring genotype ۱a. After purification and cloning the pNS۳ into TA-cloning vector, the best colony was selected based on Blue/White screening and colony-PCR following by confirmation with sequencing and restriction digestion with BglII. The sequenced gene was compared with other reference sequences using alignment softwares. The resultant pNS۳ gene subcloned into the expression vector, IRES vector, followed by selection the suitable clones by ۲ different colony-PCRs. The gene expression was evaluated using GFP detection, RT-PCR and western blotting techniques after transfection of the IRES-pNS۳ vector into the ۲۹۳ cell line. Results: After pNS۳ sequence amplification by RT-PCR, sequencing results showed high homology among the sequences with other reference sequences. This result also showed that it belonged to genotype ۱ of HCV. Colony-PCR showed the insertion of gene into expressing vector with the right orientation. GFP expression, RT-PCR and western blotting confirmed transfection of vector, expression of pNS۳ gene and production of its protein in ۲۹۳ cells respectively. Conclusion: This novel expressing vector harboring partial region of NS۳ gene in compare to full NS۳ gene maybe more useful in immune induction by antigen presenting cells due to absence of genes responsible for protease activity of the protein in the setting of HCV vaccine.

کلیدواژه ها:

HCV Virus ، NS۳ Gene ، DNA Construct ، ویروس هپاتیت C ، سازه DNA ، ژن NS۳