Abstract: This study was to examine TLR-4 gene expression in HEK 293 cells. Total RNA was isolated from human umbilical vein endothelial cells(HUVECs) by using trizol re agent. The sequences coding for TLR-4 were amplified and inserted into eukaryot ic expression vector pcDNA3 at HindⅢ and BamHΙsites. The pcDNA3-TLR-4 was tran sfected into HEK 293 cells by using Dosper liposomal transfectional reagent. The expression of TLR-4 was determined on the HEK 293 cells transfected with pcDNA 3 -TLR-4 by fluorescence immunocytochemisty and flow cytometry. 2 726 bp TLR-4 cD NA fragment was amplified from HUVECs by RT-PCR. The recombinant plasmid pcDNA3 -TLR-4 was confirmed by PCR, enzyme digestion and sequencing. Both immunocytoflu orescent staining and flow cytometric analysis showed TLR-4 expression in the H EK 293 cells that was transfected with pcDNA3-TLR-4. This study shows that when tran s fected with pcDNA3-TLR-4, the HEK 293 cells express TLR-4, in order to furthr d efine its ligands and signal pathway.

Key words: TLR-4, clone, eukaryotic expression