Construction Of PEYFP/HBVX Recombinant Plasmid And Preliminary Investigate HBx' Effect On The Activity Of P4 Promoter Of IGF-Ⅱ

Objective:To clone the HBV X and P4 promoter gene of human IGF-â…¡,and then construct HBVX-EYFP eukaryotic expression plasmid and pGL3-Basic eukaryotic expression vector under P4 promoter,preliminary investigate HBx' effect on the activity of P4 promoter,to provide the groundwork for exploring the HBx' effect on the methylation of P4 promoter of IGF-â…¡and its cellular signal transduction access.Methods:1.Adding restriction enzyme sites EcoRI and SalI,HBVX gene according to the pCMV-tag2B-X was amplified by polymerase chain reaction and was digested by EcoRI and SalI as well as pEYFP-C1.The purified HBVX gene fragment was inserted into the EcoRI-SalI site of EYFP expression vector, that is pEYFP-C1,to construct the eukaryotic expression plasmid, pEYFP-C1-X,which was further confirmed by double restricted enzyme digestion and DNA sequencing.2.Adding restricted enzyme sites KpnI and Hindâ…¢,P4 promoter gene according to the genosome of L-02 cell lines was amplified by polymerase chain reaction and was digested by KpnI and Hindâ…¢as well as pGL3-Basic. Then P4 promoter gene fragment was inserted into the KpnI-Hindâ…¢site of pGL3-Basic vector,to construct pGL3-Basic eukaryotic expression vector bearing P4 promoter gene,pGL3-P4,which was further confirmed by double enzyme digestion and DNA sequencing.3.To make it methylation,pGL3-P4 was incubated in methyltransferases SssICpG..4.HepG2 and Hela cells were transfected with pEYFP-C1-X,or pEYFP-C1, and resistant cell clones were selected with G418.Then pGL3-P4 was transiently transfected into the cells above,and the activity of P4 gene promoter was determined by adding luciferase substrate into transfectd cells.Result:1.The pEYFP-C1-X eukaryotic expression vector was confirmed by restricted enzyme digestion and sequencing.2.The pGL3-P4 eukaryotic expression vector was confirmed by restricted enzyme digestion and sequencing3.Dual luciferase reporter assay system preliminary showed that HBx could enhance the activation of the P4 promoter.Conclusion:1.About 465bp HBVX gene frament was successfully amplified by PCR method and pEYFP-C1-X eukaryotic expression vetor was successfully constructed.2.P4 promoter gene frament was successfully amplified by PCR method and P4 promoter- pGL3-Basic eukaryotic expression vetor was successfully constructed.3.Dual luciferase reporter assay system had preliminary displayed that HBx could active the P4 promoter.4.This study established a foundation for explorig the mechanism of hypomethylation of P4 promoter in the future.