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 Coronaviruses 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 [12] . 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 nontraditional approaches which are based on the use of bacterial artificial chromosomes [1] , the in vitro ligation of Coronavirus cDNA fragments [22] and the use of vaccinia virus as a vector for the propagation of Coronavirus genomic cDNA [17] . 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 [21] . In this study, we established a series of double stable SARS-CoV nucleocapsid proteinexpressing 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.
The template plasmid p8S was kindly provided by 
The PCR product was recovered by gel extraction 
The Because of the small size, pPUR is highly effective at generating stable transfectants.
After 48 h of cotransfection, add 0.5-2.0μg/mL puromycin to the complete DMEM medium, examined the viable cells under microscope every two days. As shown in Fig.3 , after 144 h of cotransfection, 90% cells enteric disorders of diverse severity [11] . HCoV-229E
(Human coronavirus 229E), is a prototypic group 1
Coronaviruses, works on HCoV-229E and MHV (mouse hepatitis virus) provided good models for research on SARS-CoV.
It was reported that the HCoV 229E N proteinexpressing cell line, which was derived from the BHK-21 cells, designated BHK-HCoV-N, has been produced for the HCoV 229E reverse genetic system.
The cell line was based on the Tet-On expression system [18] and allowed the controlled expression of N protein on induction with doxycycline. It was observed that the rescue of the recombinant HCoV TCID 50 /mL [15] . Similar results had been obtained RNA, this will facilitate the rescue of the SARS-CoV RNA [16] .
Our goal in this study is to generate a cell line that gives low background and high expression of SARS-CoV nucleocapsid protein. The Tet-On system has the advantages of high inducibility and fast response times. With this systems, induction can be detected within 30 minutes and induction levels up to 10 000fold have been observed [21] . We have screened as many as 100 clones to obtain one that exhibits suitably high induction and low background.
We have shown here that the BHK-21 derived 