Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients
Objective(s): Hepatitis C is a major cause of liver failure worldwide. Current therapies applied for this disease are not fully effective and produce side effects in most cases. Non-structural protein 3 helicase (NS3) of HCV is one of the key enzymes in viral replication and infection. Therefore, th...
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| Format: | Article |
| Language: | English |
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Mashhad University of Medical Sciences
2015-02-01
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| Series: | Iranian Journal of Basic Medical Sciences |
| Subjects: | |
| Online Access: | http://ijbms.mums.ac.ir/pdf_4019_bbd20947db6955a68c5c4bb35297d268.html |
| _version_ | 1818486861228670976 |
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| author | Mahrou Sadri Mohammad M Farajollahi Zohreh Sharifi |
| author_facet | Mahrou Sadri Mohammad M Farajollahi Zohreh Sharifi |
| author_sort | Mahrou Sadri |
| collection | DOAJ |
| description | Objective(s): Hepatitis C is a major cause of liver failure worldwide. Current therapies applied for this disease are not fully effective and produce side effects in most cases. Non-structural protein 3 helicase (NS3) of HCV is one of the key enzymes in viral replication and infection. Therefore, this region is a promising target to design new drugs and therapies against HCV infection. The aim of this study was cloning and expression of HCV NS3 helicase fragment in Escherichia coli BL21 (DE3) using pET102/D-TOPO expression vector and studying immunoreactivity of the expressed antigen in Iranian infected with hepatitis C. Materials and Methods: The viral RNA was extracted from the serum of HCV infected patient. The NS3 helicase region was amplified by RT-PCR. The PCR product was directionally cloned into the expression vector pET102/D-TOPO and transformed into the BL21 strain of E. coli (DE3). The transformed bacteria were then induced by adding 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) into the culture medium to enhance the protein expression. SDS-PAGE and western blotting were carried out to identify the protein under investigation, and finally purified recombinant fusion protein was used as the antigen for ELISA method. Results: Theinsertion of theDNA fragment of the NS3 regioninto the expression vectorwas further confirmed by PCR and sequencing. SDS-PAGE analysis showed the successful expression of the recombinant protein of interest. Furthermore, immunoreactivity of fusion NS3 helicase was confirmed by ELISA and western blotting. Conclusion: It seems that this recombinant protein could be a useful source of antigen for future studies on HCV diagnosis and therapy. |
| first_indexed | 2024-12-10T16:28:28Z |
| format | Article |
| id | doaj.art-02aff88b379443ba88cd345462457f93 |
| institution | Directory Open Access Journal |
| issn | 2008-3866 2008-3874 |
| language | English |
| last_indexed | 2024-12-10T16:28:28Z |
| publishDate | 2015-02-01 |
| publisher | Mashhad University of Medical Sciences |
| record_format | Article |
| series | Iranian Journal of Basic Medical Sciences |
| spelling | doaj.art-02aff88b379443ba88cd345462457f932022-12-22T01:41:35ZengMashhad University of Medical SciencesIranian Journal of Basic Medical Sciences2008-38662008-38742015-02-011821591634019Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patientsMahrou Sadri0Mohammad M Farajollahi1Zohreh Sharifi2Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, IranDepartment of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, IranBlood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, IranObjective(s): Hepatitis C is a major cause of liver failure worldwide. Current therapies applied for this disease are not fully effective and produce side effects in most cases. Non-structural protein 3 helicase (NS3) of HCV is one of the key enzymes in viral replication and infection. Therefore, this region is a promising target to design new drugs and therapies against HCV infection. The aim of this study was cloning and expression of HCV NS3 helicase fragment in Escherichia coli BL21 (DE3) using pET102/D-TOPO expression vector and studying immunoreactivity of the expressed antigen in Iranian infected with hepatitis C. Materials and Methods: The viral RNA was extracted from the serum of HCV infected patient. The NS3 helicase region was amplified by RT-PCR. The PCR product was directionally cloned into the expression vector pET102/D-TOPO and transformed into the BL21 strain of E. coli (DE3). The transformed bacteria were then induced by adding 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) into the culture medium to enhance the protein expression. SDS-PAGE and western blotting were carried out to identify the protein under investigation, and finally purified recombinant fusion protein was used as the antigen for ELISA method. Results: Theinsertion of theDNA fragment of the NS3 regioninto the expression vectorwas further confirmed by PCR and sequencing. SDS-PAGE analysis showed the successful expression of the recombinant protein of interest. Furthermore, immunoreactivity of fusion NS3 helicase was confirmed by ELISA and western blotting. Conclusion: It seems that this recombinant protein could be a useful source of antigen for future studies on HCV diagnosis and therapy.http://ijbms.mums.ac.ir/pdf_4019_bbd20947db6955a68c5c4bb35297d268.htmlHCV Molecular cloning NS3 helicase Recombinant protein |
| spellingShingle | Mahrou Sadri Mohammad M Farajollahi Zohreh Sharifi Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients Iranian Journal of Basic Medical Sciences HCV Molecular cloning NS3 helicase Recombinant protein |
| title | Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients |
| title_full | Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients |
| title_fullStr | Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients |
| title_full_unstemmed | Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients |
| title_short | Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients |
| title_sort | cloning and expression of ns3 helicase fragment of hepatitis c virus and the study of its immunoreactivity in hcv infected patients |
| topic | HCV Molecular cloning NS3 helicase Recombinant protein |
| url | http://ijbms.mums.ac.ir/pdf_4019_bbd20947db6955a68c5c4bb35297d268.html |
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