title: [Development and characterization of a stable cell line expressing respiratory syncytial virus non-structural protein NS1].
abstract: To develop a stable cell line that could express the RSV NS1, the full-length RSV NS1 gene was generated by RT-PCR amplification from respiratory syncytial virus. NS1 gene was ligated with pBABE-puro to construct the recombinant retroviral expression plasmid pBABE-NS1, which was cotransfected into 293FT packaging cells with PIK packaging plasmid by calcium phosphate co-precipitation. The supernatant of 293FT was collected to infect HEp-2 cells, the resulting cell clones stably expressing NS1 were screened by puromycin. Using QPCR, CPE staining method and indirect immunofluorescence assay, the expression of NS1 at both gene and protein levels was identified. The recombinant plasmid pBABE-NS1 was identified by EcoRI and BamHI endonuclease digestion and the sequence analysis. QPCR results showed that the NS1 gene amplification in HEp-2-NS1 cells was 8483 fold higher than that in HEp-2 cells. Although the exogenous interferon was added, all cells were destroyed after 48 hours post infection using CPE staining method, showing that HEp-2-NS1 cells remained sensitive to the VSV virus. The results of RT-PCR and indirect immunofluorescence assay showed that the NS1 gene in HEp-2 cells could not only transcribe mRNA, but also express NS1 protein steadily. We had successfully established HEp-2-NS1 cell lines with stable expression of respiratory syncytial virus non-structural protein NS1.
authors.affiliation: Guangzhou Institute of Respiratory Disease, State Key Laboratory of Respiratory Diseases, Guangzhou Medical University, Guangzhou 510230, China. b96106@163.com
authors.forename: Sheng
authors.initials: S
authors.lastname: Qin
authors.forename: Yu-Tao
authors.initials: YT
authors.lastname: Wang
authors.forename: Zi-Feng
authors.initials: ZF
authors.lastname: Yang
authors.forename: Qiao-Lian
authors.initials: QL
authors.lastname: Chen
authors.forename: Wen-Da
authors.initials: WD
authors.lastname: Guan
authors.forename: Shi-Guan
authors.initials: SG
authors.lastname: Wu
authors.forename: Wen-Kuan
authors.initials: WK
authors.lastname: Liu
authors.forename: Zhao-Guang
authors.initials: ZG
authors.lastname: Zhen
authors.forename: Hai-Tao
authors.initials: HT
authors.lastname: Li
authors.forename: Rong
authors.initials: R
authors.lastname: Zhou
mesh_terms: D002461:Cell Line, Transformed; D057809:HEK293 Cells; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012136:Respiratory Syncytial Viruses / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins