HBx蛋白表达载体pEYFP-C1-X的构建及对IGF-II基因P4启动子活性影响的初步研究.pdfVIP

Hepatitis B Virus, HBV HBx pEYFP-C1-X IGF-II P4 pGL3-P4 HBx IGF-II P4 HBx IGF-II P4 1. pCMV-tag2B-X X EcoRI SalI PCR X , PCR pEYFP-C1 EcoRI-SalI HBx pEYFP-C1-X 2. Genbank 3. pEYFP-C1-X HepG2 Hela G418 1.PCR pEYFP-C1-X 2.P4 1. HBV X HBx pEYFP-C1-X 2. IGF-II P4 P4 I ABSTRACT Objective:To clone the HBV X and P4 promoter gene of human IGF-II , 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-II 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 HindIII, P4 promoter gen