Selection Of The Functional Domains Of Daxx And Its Effect On Cholesterol Contents In HepG₂ Cells

Object: Our study is to select the functional domain of Fas death domain-associated protein (Daxx),which can interact with androgen receptor(AR).The eukaryotic vector of the functional domain mutant of Daxx was constructed to be stably transfected into HepG2 cells.We observed the change of cholesterol contents in HepG2 cells.As all in above, we looks for a new way to prevent and cure Hyperlipidemia.Methods: Four functional domain mutants of Daxx were respectively amplified by Polymerase chain reaction(PCR) and were subcloned to a prokaryotic expression vector (pGEX-6p-1) to construct four expression vectors.The recombinant gene was indentified by agarose gel electrophoresis and sequencing.All recombinant genes were stably transfected into E Coli. DH5αBL21DE3 using the method of Calcium chloride.After screening with ampicillin,the functional domain mutants of Daxx were expressed induced by IPTG in E Coli. DH5αBL21DE3.Four recombinant protens were respectively detected to interact with AR by using GST pull-down in vitro.Eukaryotic vector of the domain corresponded to the protein Daxx(aa626-740) that could interact with AR was constructed.The eukaryotic vector was stably transfected into HepG2 cells to detect the cholesterol contents by using oil red O stain and HPLC.Result: The results of pGEX-6p-1/Daxx/DM1-240,pGEX-6p-1/Daxx/DM241-501,pGEX-6p-1/Daxx/DM502-625 and pGEX-6p-1/Daxx/DM626-740 from sequencing experiment indicated that there were no reading frame shifts and mutations in recombinant.The protein expressing from pGEX-6p-1/Daxx/DM626-740 could interact with AR by using GST pull-down in vitro.Oil red O and HPLC analysis showed that cholesterol contents decreased in HepG2 cells after pCDNA3.1(+)/ Daxx/DM626-740 transfection stably. Conclusion: Daxx(aa626-740) can combine AR and decrease the cholesterol contents in HepG2 cells.