Establishment of the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression |
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Authors: | Guo-hui Chang Andrew Dividson Lei Lin Matt Wilson Stuart G Siddell Qing-yu Zhu |
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Institution: | 1. State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China 2. Department of Cellular and Molecular Medicine, School of Medical and Veterinary Sciences, University of Bristol, Bristol BS8 1TD,United Kingdom |
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Abstract: | In order to establish the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression.The recombinant
plasmid of pTRE-Tight-SARS-N was constructed by using the plasmid p8S as the PCR template which contains a cDNA clone covering
the nucleocapsid gene of SARS-CoV HKU-39449. Restriction enzymes digestion and sequence analysis indicated the recombinant
plasmid of pTRE-Tight-SARS-N contained the nucleocapsid gene with the optimized nucleotide sequence which will improve the
translation efficiency. Positive cell clones were selected by cotransfecting pTRE-Tight-SARS-N with the linear marker pPUR
to BHK-21 Tet-on cells in the presence of puromycin. A set of double-stable eukaryotic cell lines (BHK-Tet-SARS-N) with inducible
control of the SARS-CoV neucleocapsid gene expression was identified by using SDS-PAGE and Western-blot analysis. The expression
of SARS-CoV nucleocapsid protein was tightly regulated by the varying concentration of doxcycline in the constructed double-stable
cell line. The constructed BHK-Tet-SARS-N cell strains will facilitate the rescue of SARS-CoV in vitro and the further reverse
genetic research of SARS-CoV. |
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Keywords: | SARS-CoV Nucleocapsid protein Inducible expression Double stable cell lines |
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