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. 2010 Oct 8;25(5):361–368. doi: 10.1007/s12250-010-3124-2

Establishment of the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression

Guo-hui Chang 1,✉, Andrew Dividson 2, Lei Lin 1, Matt Wilson 2, Stuart G Siddell 2, Qing-yu Zhu 1
PMCID: PMC7091148  PMID: 20960182

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.

Key words: SARS-CoV, Nucleocapsid protein, Inducible expression, Double stable cell lines

Footnotes

Fundation Items: This work was supported by the European Commission (SARS-DTV) SP22-CT-2004-511064), and the State Key Laboratory of Pathogen and Biosecunity SKLPBS0918.

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Articles from Virologica Sinica are provided here courtesy of Wuhan Institute of Virology, Chinese Academy of Sciences

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