Different Transactivating Capacities On MDR1 Gene Between X Proteins Of Genotype B And C Hepatitis B Virus

Objective To investigate the differences of transactivating capacity on MDR1 gene between genotype B and C HBV X proteins .Methords Genotype B and C HBV X gene were amplified by means of PCR and cloned to the pcDNA3.1/HisC to construct the recombinant expression vector pcDNA3.1/HisC-XB and pcDNA3.1/HisC-XC separately. The recombinant vectors were separately transfected to HepG2 cells by FuGENE6 transfection reagent, and the expressed protein was detected by Western-blot using the antibody against the fused X-press epitopes. The promoter region of MDR1 gene was amplified and cloned to the firefly luciferase reporter plasmid pGL3-Basic to construct the recombinant vector as pGL-MDR. HepG2 cells were co-transfected by pGL-MDR together with pcDNA3.1/HisC-XB or pcDNA3.1/HisC-XC, cells were lysed 48 hours post transfection, the intracellular firefly luciferase activities were monitored and calculated by paired-samples t test.Results 1. Recombinant HBV X gene expression vectors and the MDR1 reporter plasmid were successfully constructed. HBV X proteins were expressed well in HepG2 cells with the highest level in the point of 48 hours post transfection, as demonstrated by western blot analysis. 2. While the ratio of pGL-MDR reporter plasmid to either HBV X gene construct or pcDNA3.1/HisC ranged from 1:2 to 1:4, the intracellular luciferse activities of transfected HepG2 cells reduced accordingly as pcDNA3.1/HisC-XB+pGL-MDR>pcDNA3.1/HisC-XC+pGL-MDR>pcDNA3.1/hisC+pGL-MDR, and showed in the dose-effects manner.Conclusion Both of genotype B and genotype C HBV X protein could transactivate the MDR1 gene in transcriptional level, and the transactivation competence of genotype B HBx was higher than that of genotype C.