Cloning Of Extracellular Domain Of Toll-like Receptor 4(TLR4)and The Mechanisms Involved In Lipopolysaccharide(LPS)Mediated Cellular Reactions

Background Lipopolysaccharide(LPS), or endotoxin, is the major component of the outer membrane of the cell wall of Gram-negative bacteria. LPS is a potent activator of cells of the immune and inflammatory systems, including macrophages, monocytes, and endothelial cells, and contributes to the systemic pathophysiological changes seen in septic shock. Studies conducted over the past two years have demonstrated that the Toll-like receptors (TLRs) family as signaling receptors plays a crucial role in mammalian host defense. Analysis of the signaling activated by human Tolls revealed the pathway involvement of the adapter protein MyD88, and have been shown to transduce signals through intracellular components shared by the IL-1 signaling machinery, ultimately leading to activation of NE- K B and subsequent cellular events. Two members of the mammalian TLR family, TLR2 and TLR4, have been implicated as receptors mediating cellular activation in response to bacterial LPS. TLR4 from the C3H/HeJ mice has a single point mutation at amino acid 712 (Pro to His)which results in nonresponsiveness to LPS. Furthermore, Chinese hamster ovary (CHO) flbroblast cells genomically encode a nonfunctional gene for TLR2 even though these cells are fully LPS responsive; and TLR2 knockout mice have no significant defects in LPS responsiveness. These observations strongly support the concept that TLR4, and not TLR2, is the dominant LPS receptor in mammals and the hypothesis that TLR4 is a cell-surface component of the LPS signaling pathway. Objective To clone, express extracellular domain of TLR4; and transfect TLR4 into HL6O and human dermal flbroblast cells, observe the LPS responsiveness of these two cell lines. Methods 1. RT-PCR was applied to amplify the extracellular domain of TLR4, with total RNA of primary human umbilical vein endothelial cells as the template. RT-PCR products were directly cloned into pMD18-T plasmid. The identified recombinants were sent for automated sequencing. 2. The extracellular domain of TLR4 was cloned into an expressing vector pET-11a, and the recombinant protein of TLR4 was expressed in E. coli.BL21. 3. Digested the full length TLR4 from pCMVSPORT3-TLR4 plasmid by Kpn I and Not I, and subcloned into mammalian cell expression vector pcDNA3. 1 (＋). The recombinant expression plasmids were used to tranfect HL6O and human dermal fibroblast cells and stable expression cell lines were established by using G418 selection. 4. Expose the transfected cells in LPS, and observe roles of TLR4 in LPS action. Results 1. The DNA encoded for extracellular domain of TLR4 were Obtained, and its sequence was correct in comparasion with the published data. 2. The extraceilular domain of TLR4 was cloned into pET-11a and the expressed protein was determined to be 7lkDa (SDS-PAGE) as that of prospective. 3. The full length TLR4 was subcloned into mammalian cell expression vector pcDNA3.i( + ),and was tranfected into HL6O, the stable expression cell line of HL6O was obtained 4.. TLR4 mediates the restrained effect of LPS for cells, and serum factors enhance this effect. Conclusion The extracellular domain of TLR4 was succeeded amplified in from primary human umbilical vein endothelial cells, and the roles of TLR4 in lipopolysaccharide signaling in HUVEC was demonstrated as in macrophages and monocytes...