Cloning and Expression of the Dengue virus Non-structural Protein 1 (NS1) in E. coli
Main Article Content
Abstract
Dengue is an infectious disease that poses a threat to approximately 3.9 billion people in 128 countries in the world. A report in 2013 indicates average of 390 million dengue infections per year. Non-structural protein 1 (NS1) is a highly conversed dengue virus glycoprotein and considered as a specific biomarker for serodiagnosis of dengue infection. In the present study, the ns1 gene encoding the non-structural protein 1 (NS1) from the Vietnam Dengue virus isolate was cloned and sequenced. The isolated ns1 gene was high identity with Dengue virus 1 polyprotein-like gene (gene ID: JQ045627.1). The ns1 gene was successfully inserted into the expression vector pET22b(+) in an open reading frame. The successful expression of recombinant NS1 protein (rNS1) in E. coli BL21 (DE3) was checked by SDS-PAGE and confirmed by Western blot. Some suitable conditions for high expression of rNS1 protein were 0.3 mM IPTG, 4 hours induction and initial inoculum density of 0.2.
Keywords
Dengue virus, E. coli, non-structural protein (NS1), recombinant
Article Details
References
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[2] J. Fan, Y. Liu, Z. Yuan, Critical role of Dengue Virus NS1 protein in viral replication, Virol Sin. 29(3) (2014) 162-169.
[3] I. Gutsche, F. Coulibaly, J.E. Voss, J. Salmon, J. d'Alayer, M. Ermonval, E. Larquet, P. Charneau, T. Krey, F. Mégret, E. Guittet, F.A. Rey and M. Flamand, Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein, Proc. Natl. Acad. Sci. 108(19) (2011) 8003–8008.
[4] F.M. Kassim, M.N. Izati, T.A. TgRogayah, Y.M. Apandi, Z. Saat, Use of dengue NS1 antigen for early diagnosis of dengue virus infection, Southeast Asian J. Trop. Med. Public Health 42(3) (2011) 562-9.
[5] A.K. Falconar, P.R. Young, Immunoaffinity purification of native dimer forms of the flavivirus nonstructural glycoprotein NS1, J. Virol. Methods. 30(3) (1990) 323-332.
[6] J. Sambrook, D.W. Russell, Molecular cloning. A laboratory manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 2001.
[7] C. Robichon, J. Luo, T.B. Causey, J.S. Benner, and J.C. Samuelson, Engineering Escherichia coli BL21(DE3) Derivative Strains To Minimize E. coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography, Appl. Environ. Microbiol. 77(13) (2011) 4634–4646.
[8] N.H. Tolia, L. Joshua-Tor, Strategies for protein coexpression in Escherichia coli, Nat. Methods 3 (2006) 55-64.
[9] Z.Q. Liu, P.C. Yang, Construction of pET32 alpha (+) vector for protein expression and purification, N. Am. J. Med. Sci. 4 (2012) 651-655.
[10] G. Lemos, I. Guillén, J.R. Fernandez, T. Diaz, A.B. Colarte, M.E.F. de Cossio, Expression and purification of a full-length recombinant NS1 protein from a dengue 2 serotype viral isolate, Biotech. Appl. 30 (2013) 187-193.