Cloning and expression of NS3 helicase fragment of hepatitis C virus and the study of its immunoreactivity in HCV infected patients

Document Type : Original Article

Authors

1 Blood 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, Iran

2 Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran

3 Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

Abstract

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.

Keywords

References

1. Gu M, Rice CM. Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism. Proc Natl Acad Sci USA 2010; 107:521-528.
2. Parfieniuk A, Jaroszewicz J, Flisiak R. Specifically targeted antiviral therapy for hepatitis C virus. World J Gastroenterol 2007; 13:5673-5681.
3. Tang H, Grise H. Cellular and molecular biology of HCV infection and hepatitis. Clin Sci (Lond) 2009; 117:49-65.
4. Zhang SZ, Liang JJ, Qi ZT, Hu YP. Cloning of the non-structural gene 3 of hepatitis C virus and its inducible expression in cultured cells. World J Gastroenterol 1999; 5:125-127.
5. Zhu FL, Lu HY, Li Z, Qi ZT. Cloning and expression of NS3 cDNA fragment of HCV genome of Hebei isolate in E.coli. World J Gastroenterol 1998; 4:165-168.
6. Hanson AM, Hernandez JJ, Shadrick WR, Frick DN. Identification and analysis of inhibitors targeting the hepatitis C virus NS3 helicase. Methods Enzymol 2012; 511:463-483.
7. Yamashita A, Salam KA, Furuta A, Matsuda Y, Fujita O, Tani H, et al. Inhibition of hepatitis C virus replication and viral helicase by ethyl acetate extract of the marine feather star Alloeocomatella polycladia. Mar Drugs 2012; 10:744-761.
8. Yang J, Lei YF, Yin W, Wei SH, An QX, Lv X, et al. Production and characterization of monoclonal antibody specific for NS3 helicase of hepatitis C virus. Hybridoma 2008; 27:181-186.
9. Belon CA, Frick DN. Helicase inhibitors as specifically targeted antiviral therapy for hepatitis C. Future Virol 2009; 4:277-293.
10. Huang ZS, Wang CC, Wu HN. HCV NS3 protein helicase domain assists RNA structure conversion. FEBS Lett 2010; 584:2356-2362.
11. Krekulova L, Rehak V, Riley LW. Structure and functions of hepatitis C virus proteins: 15 years after. Folia Microbiol (Praha( 2006; 51:665-680.
12. Moradpour D, Penin F. Hepatitis C virus proteins: from structure to function. Curr Top Microbiol Immunol 2013; 369:113-142.
13. Beran RK, Pyle AM. Hepatitis C viral NS3-4A protease activity is enhanced by the NS3 helicase. J Biol Chem 2008; 283:29929-29937.
 
14. Mukherjee S, Hanson AM, Shadrick WR, Ndjomou J, Sweeney NL, Hernandez JJ, et al. Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays. Nucleic Acids Res 2012; 40:8607-8621.
15. Prabhu R, Khalap N, Burioni R, Clementi M, Garry RF, Dash S. Inhibition of hepatitis C virus nonstructural protein, helicase activity, and viral replication by a recombinant human antibody clone. Am J Pathol 2004; 165:1163-1173.
16. Alter MJ, Kruszon-Moran D, Nainan OV, McQuillan GM, Gao F, Moyer LA, et al. The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. N Engl J Med 1999; 341:556-562.
17. Furuta A, Salam KA, Akimitsu N, Tanaka J, Tani H, Yamashita A, et al. Cholesterol sulfate as a potential inhibitor of hepatitis C virus NS3 helicase. J Enzyme Inhib Med Chem 2014; 29:223-229.
18. Alaee M, Rajabi P, Sharifi Z, Farajollahi MM. Immunoreactivity assessment of hepatitis C virus NS3 protease and NS5A proteins expressed in TOPO cloning system. J Microbiol Immunol Infect 2014 ;47:282-291.
19. Frick DN. The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target. Curr Issues Mol Biol 2007; 9:1-20.
20. Raney KD, Sharma SD, Moustafa IM, Cameron CE. Hepatitis C virus non-structural protein 3 (HCV NS3): a multifunctional antiviral target. J Biol Chem 2010; 285:22725-22731.
21. Sillanpaa M, Melen K, Porkka P, Fagerlund R, Nevalainen K, Lappalainen M, et al. Hepatitis C virus core, NS3, NS4B and NS5A are the major immunogenic proteins in humoral immunity in chronic HCV infection. Virol J 2009; 6:84.
22. Alaee M, Rajabi P, Sharifi Z, Farajollahi MM. Immunoreactivity assessment of hepatitis C virus NS3 protease and NS5A proteins expressed in TOPO cloning system. J Microbiol Immunol Infect 2014;47:282-291.
Iranian Journal of Basic Medical Sciences
Volume 18, Issue 2 - Serial Number 2
February 2015
Pages 159-163

Files

Share

How to cite

Statistics