Establishment of the Eukaryotic Cell Lines for Inducible Control of SARS-CoV Nucleocapsid Gene Expression<sup>*</sup>

Guo-hui CHANG; Andrew Dividson; Lei LIN; Matt Wilson; Stuart G Siddell; Qing-yu ZHU

doi:10.1007/s12250-010-3124-2

October 2010

Guo-hui CHANG, Andrew Dividson, Lei LIN, Matt Wilson, Stuart G Siddell and Qing-yu ZHU. Establishment of the Eukaryotic Cell Lines for Inducible Control of SARS-CoV Nucleocapsid Gene Expression*[J]. Virologica Sinica, 2010, 25(5): 361-368. doi: 10.1007/s12250-010-3124-2

Citation: Guo-hui CHANG, Andrew Dividson, Lei LIN, Matt Wilson, Stuart G Siddell, Qing-yu ZHU. Establishment of the Eukaryotic Cell Lines for Inducible Control of SARS-CoV Nucleocapsid Gene Expression^* .VIROLOGICA SINICA, 2010, 25(5) : 361-368. http://dx.doi.org/10.1007/s12250-010-3124-2

稳定诱导表达SARS-CoV核衣壳蛋白真核细胞系的建立

cstr: 32224.14.s12250-010-3124-2

1.
病原微生物生物安全国家重点实验室, 北京微生物流行病研究所北京 100071;2Department of Cellular and Molecular Medicine, School of Medical and Veterinary Sciencese, University of Bristol, Bristol BS8 1TD United Kingdom

通讯作者： 常国辉, changguohui999@yahoo.com.cn;
收稿日期： 2010-01-11
录用日期： 2010-04-30

摘要

为了建立可稳定诱导表达SARS-CoV核衣壳蛋白的真核细胞系。以含有SARS-CoV HKU-39449核衣壳蛋白基因的质粒p8S为摸板，利用密码子优化和PCR、克隆等手段构建重组质粒pTRE-Tight-SARS-N，通过共转染和Western Blot等方法建立并鉴定单克隆细胞系。限制性内切酶酶切消化和序列测定等鉴定结果表明，构建的pTRE-Tight-SARS-N 重组质粒含有密码子成功优化的SARS-CoV核衣壳蛋白基因。嘌呤霉素的筛选结果表明，通过重组质粒与筛选线形质粒pPUR共转染BHK-21 TET-ON细胞，获得了大量的阳性细胞克隆。SDS-PAGE蛋白电泳和Western Blot杂交图谱显示，诱导剂强力霉素可严格调控SARS-CoV核衣壳蛋白表达的开启及其蛋白的表达量。通过该研究建立了一系列可通过强力霉素定量诱导而稳定表达SARS-CoV核衣壳蛋白的细胞系，该结果的取得为体外拯救SARS-CoV和进一步开展其反向遗传学研究奠定了基础。
- SARS-CoV
- , Nucleocapsid protein
- , Inducible expression
- , Double stable cell lines

Establishment of the Eukaryotic Cell Lines for Inducible Control of SARS-CoV Nucleocapsid Gene Expression^*

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

Corresponding author: Guo-hui CHANG, changguohui999@yahoo.com.cn
Received Date: 11 January 2010
Accepted Date: 30 April 2010

Fund Project: This work was supported by the European Commission (SARS-DTV) SP22-CT-2004–511064the State Key Laboratory of Pathogen and Biosecunity SKLPBS0918

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.
- SARS-CoV
- , Nucleocapsid protein
- , Inducible expression
- , Double stable cell lines

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