TY  - JOUR
T1  - Eukaryotic Expression of Functionally Active Recombinant Porcine Toll-Like 
  Receptor 5 from Flp-In-293 Cells
AU - Pan, Zhiming AU - Kang, Xilong AU - Sun, Xiaolin AU - Li, Qiuchun AU - Wang, Jing AU - Jiao, Xinan 
JO  - Journal of Animal and Veterinary Advances
VL  - 13
IS  - 4
SP  - 267
EP  - 273
PY  - 2014
DA  - 2001/08/19
SN  - 1680-5593
DO  - javaa.2014.267.273
UR  - https://makhillpublications.co/view-article.php?doi=javaa.2014.267.273
KW  - Porcine TLR5
KW  -Flp-In-293 cell line
KW  -expression
KW  -identification
KW  -bacterial pathogens
AB  - 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 <I>TLR5</I> 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. &#946;-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 <I>S. typhimurium</I> 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.
ER  -