Hosseini, seyed-Younes, Sabahi, farzaneh, Modarresi, Mohamad Hossein, Moazzeni, Seyed Mohammad, Saberi firoozi, mehdi, Ravanshad, Mehrdad, Mehrabani, Davood, Mojarrad, Majid (2011). Cloning and evaluation of expression of a novel overlapping region of NS3 gene of Hepatitis C virus by expressing vector. , 13(4), -.
seyed-Younes Hosseini; farzaneh Sabahi; Mohamad Hossein Modarresi; Seyed Mohammad Moazzeni; mehdi Saberi firoozi; Mehrdad Ravanshad; Davood Mehrabani; Majid Mojarrad. "Cloning and evaluation of expression of a novel overlapping region of NS3 gene of Hepatitis C virus by expressing vector". , 13, 4, 2011, -.
Hosseini, seyed-Younes, Sabahi, farzaneh, Modarresi, Mohamad Hossein, Moazzeni, Seyed Mohammad, Saberi firoozi, mehdi, Ravanshad, Mehrdad, Mehrabani, Davood, Mojarrad, Majid (2011). 'Cloning and evaluation of expression of a novel overlapping region of NS3 gene of Hepatitis C virus by expressing vector', , 13(4), pp. -.
Hosseini, seyed-Younes, Sabahi, farzaneh, Modarresi, Mohamad Hossein, Moazzeni, Seyed Mohammad, Saberi firoozi, mehdi, Ravanshad, Mehrdad, Mehrabani, Davood, Mojarrad, Majid Cloning and evaluation of expression of a novel overlapping region of NS3 gene of Hepatitis C virus by expressing vector. , 2011; 13(4): -.

Cloning and evaluation of expression of a novel overlapping region of NS3 gene of Hepatitis C virus by expressing vector

Pathobiology Reserach
Article 1, Volume 13, Issue 4, 2011    PDF (996.25 K)
Authors
seyed-Younes Hosseini; farzaneh Sabahi* 1; Mohamad Hossein Modarresi; Seyed Mohammad Moazzeni2; mehdi Saberi firoozi; Mehrdad Ravanshad3; Davood Mehrabani; Majid Mojarrad4
1Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran
2Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
3Department of Virology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
4Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran
Abstract
Objective: In this project, our aim was to construct a novel expressing vector harboring a new sequence from overlapping region of NS3 gene of HCV from infected Iranian patient.
Materials and Methods: The partial NS3 (pNS3) gene was amplified by Nested-RT-PCR method using sera of HCV infected patients harboring genotype 1a. After purification and cloning the pNS3 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 pNS3 gene subcloned into the expression vector, IRES vector, followed by selection the suitable clones by 2 different colony-PCRs. The gene expression was evaluated using GFP detection, RT-PCR and western blotting techniques after transfection of the IRES-pNS3 vector into the 293 cell line.
Results: After pNS3 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 1 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 pNS3 gene and production of its protein in 293 cells respectively.
Conclusion: This novel expressing vector harboring partial region of NS3 gene in compare to full NS3 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.
Keywords
HCV Virus; NS3 Gene; DNA Construct
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