Identification Of The Combine Of Green Tea Polyphenol EGCG And Liver Nuclear Receptor FXR?/RXR?

Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B(HBV),which spreads widely in the world highly-infectious and leads to serious damage to liver and others organs.In recent years,the main treatment of Hepatitis B is anti-viral therapy through the inhibition of viral replication.The replication of hepatitis B virus in vivo depends on the hepatocyte-enriched transcriptional factors including FXR and RXR.The heterodimer of FXR and RXR binds to core promoter and enhancer region?of the DNA of HBV and regulates t HBV transcription.Thus,FXR and RXR are expected to be the new targets of inhibiting transcription and replication of HBV.The major catechins in green tea are(-)-epicatechin-3-gallate,(-)-epigallocatech--in(EGCG)accounting for 60%.In recent years,studies have shown that EGCG had anti-viral activities.In our study,the inhibition of HBV replication and transcription may be dependent on FXR and RXR.In this thesis,the open reading frame(ORF)sequence for FXR? was amplified by RT-PCR form Hep G2 cell line.The sequence was cloned into vector p ET-28 a for expressed in E.coli BL21(DE3).The expression of recombinant fusion FXR? with 6×His-tag was induced by isopropylthio-?-D-galactoside(IPTG).Expression of the target protein was detected by SDS-PAGE and then purified by Ni NTA chelating agarose.SDS-PAGE analysis showed that FXR? protein was success fully expressed in E.coli BL21(DE3).A rabbit polyclonal antibody against recombinant FXR? was obtained by immunization rabbit with the purified FXR? protein.It was showed that the anti-sera could bind to the expressed protein specifically by dot blot analysis.Meanwell,the sequences of FXRa?RXRa and HBV enhancer region ?and core promoter(EN?&CP)were cloned respectively into the vector pc DNA3.1 and p GL-3.293 T cell lines were cotransfected with pc DNA3.1-FXRa,pc DNA3.1-RXRa and Pgl-3-HBV CP&EN?.The effect of FXR/RXR on EN?&CP transcription was analyzed through Luciferase activity according to the interaction of FXR/RXR with their ligands and EGCG.Our data demonstrates that HBV EN?&CP expression was up-regulated if both FXR and RXR were activated by their ligands and this regulation was inhibited by EGCG.To further study the mechanism of EGCG,EGCG binding protein was purified by agarose affinity chromatography with PAB and then identified by Western blot.Our data shows that the EGCG binding protein is FXR.We performed drug affinity responsive target stability(DARTS)assay to confirm that FXR is EGCG binding protein.The whole cell protein treated with EGCG or not was digested with heat resistant Thermolysin and protein(s)binding with EGCG cannot be digested by Thermolysin.Western blot shows that FXR was the only Thermolysin resistant protein.These data suggested that the mechanism of inhibitory effect of EGCG on EN?&CP transcription and expression is via binding to FXR and then interfering its functions.