State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, 100071, China.
Virol Sin. 2010 Oct;25(5):361-8. doi: 10.1007/s12250-010-3124-2. Epub 2010 Oct 8.
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.
为了建立诱导控制 SARS-CoV 核衣壳基因表达的真核细胞系。使用包含 SARS-CoV HKU-39449 核衣壳基因 cDNA 克隆的质粒 p8S 作为 PCR 模板，构建了 pTRE-Tight-SARS-N 重组质粒。酶切和序列分析表明，pTRE-Tight-SARS-N 重组质粒包含优化的核苷酸序列的核衣壳基因，这将提高翻译效率。在嘌呤霉素存在下，将 pTRE-Tight-SARS-N 与线性标记物 pPUR 共转染到 BHK-21 Tet-on 细胞中，选择阳性细胞克隆。通过 SDS-PAGE 和 Western-blot 分析，鉴定了一组具有诱导控制 SARS-CoV neucleocapsid 基因表达的双稳定真核细胞系（BHK-Tet-SARS-N）。在构建的双稳定细胞系中，SARS-CoV 核衣壳蛋白的表达受 doxcycline 浓度的严格调控。构建的 BHK-Tet-SARS-N 细胞株将有助于 SARS-CoV 的体外拯救和 SARS-CoV 的进一步反向遗传学研究。
