Coronaviruses are vertebrate pathogens mainly associated with respiratory and enteric diseases. In humans, Coronavirus infections manifest usually as mild respiratory tract disease (common cold) that may cause more severe symptoms in elderly or immune-compromised individuals[4, 18]. An international outbreak of severe acute respiratory syndrome (SARS), an atypical pneumonia, spread through more than 30 countries and caused about 8 422 cases and 916 deaths worldwide in 2002-2003. A novel Coronavirus SARS-Coronavirus (SARS-CoV) that can be isolated from the SARS patients and from Vero E6 cells inoculated with clinical specimens was identified to be the causative agent of SARS [3, 10, 11]. The appearance of SARS, exemplified the potential of Coronaviruses to seriously affect human health [5, 13, 14, 15].The frequent detection of SARS-like Corona-viruses in horseshoe bats and the broad range of mammalian hosts that are susceptible to SARS-CoV infection may facilitate a potential reintroduction into the human population. Therefore, the development of system for studying the SARS-CoV biology and pathogenesis is of high medical importance.
Reverse genetic system is a very important tool to the analysis of viral replication and pathogenesis. Recently, reverse genetic systems for a number of Coronaviruses have been established using non-traditional approaches which are based on the use of bacterial artificial chromosomes, the in vitro ligation of Coronavirus cDNA fragments and the use of vaccinia virus as a vector for the propagation of Coronavirus genomic cDNA. With the systems now available, it is possible to genetically modify Coronavirus genomes at will. Recombinant viruses with gene inactivations, deletions or attenuating modifications can be generated and used to study the role of specific gene products in viral replication or pathogenesis. It was reported that the expression of Coronavirus nucleocapsid protein definitely facilitated the efficient rescue of Coronaviruses in all the reverse genetic systems[2, 15, 16].
The Tet-On gene expression systems is a regulated, high-level gene expression system. Maximal expression levels in Tet-On systems are very high and compare favourably with the maximal levels obtainable from strong, constitutive mammalian promoters such as CMV. In this study, we established a series of double stable SARS-CoV nucleocapsid protein-expressing cell lines derived from BHK-21, and the expression was tightly regulated in response to inducer doxycycline in a precise and dose-dependent manner. The constructed double-stable cell strains will play an important role in the reverse genetics research of SARS-CoV, it will facilitate the rescue of SARS-CoV in vitro and the analysis of SARS-CoV RNA synthesis.
Establishment of the Eukaryotic Cell Lines for Inducible Control of SARS-CoV Nucleocapsid Gene Expression*
- Received Date: 11 January 2010
- Accepted Date: 30 April 2010
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.