Establishment of the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression.
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.
Chang
Guo-Hui
GH
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, 100071, China. changguohui999@yahoo.com.cn
Dividson
Andrew
A
Lin
Lei
L
Wilson
Matt
M
Siddell
Stuart G
SG
Zhu
Qing-Yu
QY
D000818:Animals; D002460:Cell Line; D019295:Computational Biology; D000086462:Coronavirus Nucleocapsid Proteins; D006224:Cricetinae; D004318:Doxycycline / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D019590:Nucleocapsid Proteins / Q000096:biosynthesis* / Q000235:genetics*; D010957:Plasmids; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D017421:Sequence Analysis
D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D004318:Doxycycline