| Sepsis and septic shock are prime cause of death in intensive care unit (ICU). However, no effective therapeutic drugs are available. Lipopolysaccharide (LPS),a cell wall component of gram-negative bacteria, is one of the main causes of sepsis and septic shock. Identification of molecules involving LPS signaling transduction pathways may lead to discovery of new drug targets. It has been proved that interferon regulatory factor-3 (IRF-3) plays a key role in the MyD88-independent signaling pathway, and IRF-3 deficient mice were resistant to LPS-induced endotoxin shock. Although some molecules involving MyD88-independent signaling pathway, including TRIF, TRAM, TBK1 and IKKε, have been reported recently, our understanding of the molecular mechanism is still very limited. In order to identify molecules involving LPS-induced IRF-3 signaling pathway, the regulatory elements of IFN-βgene promoter and IRF-3 binding sequence were analyzed, and IRF-3-Luciferase and IRF-3-EGFP reporter vectors were constructed.The main theory of this reseach:IRF-3 can regulate the expression of IFN-βgene by binding to the conservative DNA sequence of IFN-βpromoter, called IFN stimulated response elemen (ISRE), through its DNA binding domain. LPS binds toll like recepter 4 (TLR4) and induces production of IFN-βvia IRF-3 pathway. Based on these findings, an IRF-3 reporter vector was developed by using IFN-βpromoter sequence.The main content and results of this reseach:1. Construction of promoter-less EGFP reporter vectors:The promoter-less EGFP reporter vectors pGF3basic (without promoter and enhancer), pGF3enhancer (without promoter, with enhancer) and pGF3control (with both promoter and enhancer) were constructed by replacing the luciferase gene of pGL3-basic/enhancer/control vectors (purchased from Promega) with EGFP gene. In order to test whether the EGFP reporter gene could be expressed, pGF3control vector was transfected into HeLa cells. Strong...
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