Eukaryotic Expression Of The Extracellular Domain Of Human TLR4 And Its Effect On Inflammation Induced By LPS

Excessive gram-negative infection is universal among serious trauma patients, major operation patients and other critically ill patients, which can result in endotoxemia, even endotoxin shock. How to treat these patients is very difficult in the field of intensive therapy at present. The causative and pathogenic factor of gram-negative bacteria is lipopolysaccharide(LPS), named endotoxin. Recent studies have shown that most of LPS-related inflammation signal is transduced into cells by Toll-like receptor 4(TLR4), a transmembrane protein, which leads to signal transduction within cells. If one step of the signal transduction pathway is blocked at the level TLR4, excessive inflammation responses could be inhibited effectively. In this study, the extracellular domain of human TLR4 was gained by eukaryotic expression system to observe its effects on LPS-related inflammation response.Methods: The cDNA products of the extracellular domain of human TLR4 was amplified from the plasmid pCMV-TLR4 with human TLR4 gene by polymerase chain reaction(PCR). The cDNA fragment was cloned into eukaryotic expression plasmid pcDNA3.1(+) to construct recombinant expression plasmid pcDNA3.1(+)-eTLR4. The later was identified by restriction enzyme digestion analysis and sequencing. The plasmid pcDNA3.1(+)-eTLR4 was introduced into HEK293 cells through lipofection transfection. The mRNA expression of the extracellular domain of human TLR4 was detected by reverse transcriptase-PCR (RT-PCR). The HEK293 cells which stably expressed the extracellular domain of human TLR4 was gained by G418 filtration. U937 cells induced differentiation was affected by the supernatant of cultured HEK293 cells to measure the concentration of tumor necrosis factor-a (TNF-a) by enzyme linked immunosorbent assay(ELISA).Results: 1. The cDNA products of the extracellular domain of human TLR4 was successfully amplified from the plasmid pCMV-TLR4 with human TLR4 gene by PCR. The...