Eukaryotic Expression of Functionally Active Recombinant Porcine Toll-Like
Receptor 5 from Flp-In-293 Cells
Toll-Like Receptor 5 (TLR5), a member of the TLR family, specifically recognizes flagellin which contributes to the motility of bacterial pathogens. Therefore, TLR5 plays a crucial role in host defense against flagellated bacteria. In the current study, researchers cloned the TLR5 gene from Jiangquhai pigs (pTLR5). The pTLR5 open reading frame was fused with a FLAG tag by cloning amplified pTLR5 into the pcDNA3.1-FLAG vector. Flp-In-293 cells expressing pTLR5-FLAG (designated pTLR5-Flp-In-293) were obtained using the Flp recombinase system. β-galactosidase activity and zeocin resistance were lost in the pTLR5-Flp-In-293 cells but hygromycin B resistance was acquired. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and western blot analysis confirmed that pTLR5-FLAG was expressed in pTLR5-Flp-In-293 cells at both the transcriptional and protein levels. Indirect immunofluorescence assay showed that the pTLR5-FLAG protein was located in the cell membrane of pTLR5-Flp-In-293 cells. Stability analysis by fluorescence-activated cell sorting showed that compared with Flp-In-293 cells, the 1st, 10th and 20th generations of the recombinant cell line expressed consistent levels of pTLR5-FLAG protein. Recombinant pTLR5-Flp-In-293 cells stimulated with flagellin from S. typhimurium expressed high levels of IL-8 which indicated that pTLR5 protein expressed in the pTLR5-Flp-In-293 cell line was a functional TLR5 homolog.