Establishment of a HEK-293 cell line stably expressing human TLR5 and NF-κB responsive reporter
10.13220/j.cnki.jipr.2017.05.007
- Author:
Tong SHI
1
Author Information
1. Beijing Research Institute of Pharmaceutical Chemistry
- Publication Type:Journal Article
- Keywords:
hTLR5;
Lentivirus;
NF-κB;
Stably expressing
- From:
Journal of International Pharmaceutical Research
2017;44(5):420-426
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the lentivirus plasmids of human Toll like receptor-5(TLR5)and nuclear factor(NF)-κB signaling pathway responsive luciferase and green fluorescent protein(GFP)reporters, and establish a HEK-293 cell line stably expressing human TLR5 and NF-κB responsive reporters. Methods The TLR5 and 5×NF-κB-binding sites genes were cloned to lentiviral expressing plasmid pWSLV-puro and pWSLV-nanluc-GFP, respectively. The recombinant plasmids, pWSLV-hTLR5-puro and pWSLV-5×NF-κB-nanluc-GFP were respectively transfected with packaging plasmid into HEK293T cells in order to package lentivirus. The lentivirus was used to coinfect HEK-293 cells, and the positive clones stably expressing TLR5 and NF-κB responsive reporter (HEK293-N-T)were generated using colony-purification in present of puromycin and fluorescence activated cell sorting. The cells expressing TLR5 were confirmed by Western blot and immunofluorescence. Additionally, the cells expressing NF-κB responsive reporter were confirmed by fluorescence microscopy and luciferase quantitative assay. Results The results of recombinant plasmids digestion identification and the sequencing of cDNA showed that the sequences inserted into the plasmid and the expected sequences of hTLR5 and 5×NF-κB-binding sites genes were completely consistent. It was confirmed that the hTLR5 and 5×NF-κB-binding sites genes were correctly inserted into the vectors, and that the recombinant plasmids, pWSLV-hTLR5-puro and pWSLV-5×NF-κB-nanluc-GFP, were successfully constructed. Additionally, Western blot and immunofluorescence analysis showed that TLR5 were stably expressed in a high level in the HEK293-N-T cells and located in the cell membranes. Moreover, the results of fluorescence microscopy and luciferase quantitative assay indicated that the NF-κB responsive reporter could reflect the activation of NF-κB signaling in a dose-dependent manner. Conclusion The HEK-293 cell line stably expressing human TLR5 and NF-κB responsive reporter has been successfully established and the HEK293-N-T cells can be used for the screening and evaluation of drugs target to TLR5.
