Hepatitis B spliced protein enhanced NF-κB activities via interaction with ubiquitously expressed transcript splice variant 1 / 中国人兽共患病学报

We investigated whether hepatitis B spliced protein affect the NF-kappa B activities by interacting with ubiquitously expressed transcript splice variant 1 (UXT-V1).The HBSP-UXT-V1 protein interactions were screened by yeast two hybrid assay,and confirmed by immunoprecipitation,confocal microscopy,mammalian two hybrid assay,and GST-Pulldown assay.The reporter plasmids driven by NF-kappa B promoter were transfected into UXT-knockdown HBSP stably expressed cell lines,and the reporter genes were detected after transfection.Results showed that the interaction between UXT-V1 and HBSP in yeast was demonstrated.Furtherly,HBSP could interact with UXT-V1 in mammalian cells.HBSP could enhance NF-kappa B activities,and this effect was partly achieved by the interaction with UXT-V1.In conclusion,the effect of HBSPUXT-V1 interaction on the NF-kappa B pathway in hepatocytes may have an impact on HBV related liver diseases.

Stable replication and antigen expression of hepatitis B virus in HepG2 cells / 中华传染病杂志

Objective To establish the HepG2 cell lines which can stably express and replicate hepatitis 13 virus (HBV).Methods One point two X unit length of HBV genome was cloned intn SalⅠsite of the eukaryotic expression vector pREP10 to construct the recombinant plasmid pREP-HBV. Human hepatoblastoma cell HepG2 was transfected with pREP-HBV by Lipofectamine 2000 and seh,cted by bygromycin at the concentration of 250?g/mL.HBsAg and HBeAg were monitored by enzyme linked immunosorbent assay (ELISA)kits.H13V particles presemed in supernatant were ex- amincd by electronic microscopy.DNA isolated from intracellular HBV core particles was analyzed by Southbern blot using HBV-specific probe.Results The recombinant vector pREP HBV containing 1.2?unit length of HBV DNA was constructed successfully.After transfection of pREP-HBV to HepG2 cells and consistently cultured in hygromycin selective medium.5 drug-resistant cell lines, RHBV1-RHBV5.were established,and all of them could stably express HBsAgand HBeAg.South ern blot analysis revealed that HBV could replicate in all cell lines,as confirmed by the presence of replicateintermediatc DNA in intracellular HBV core particles.Clustered 42 nm Dane particles as well as 22-26 nm spherical H13sAg particles in condensed cuhure supernatant were visualized by elec tronic microsopic analysis.Conclusion HepG2 ceil lines in which HBV can replicate and express specific antigens are successfully established.Up to now,the cells have been passaged every three days for 50 times.