Transactivating capacity of the novel protein encoded by the 2.2 kb doubly spliced variant of hepatitis B virus genome
Objective To investigate the function of the novel protein encoded by the 2.2 kb doubly spliced variant of hepatitis B virus genome.Methods The splicing-specific gene generated by the 2.2 kb doubly spliced variant of HBV genome was amplified by means of PCR and cloned into the mammalian expression vector pcDNA3,1/HisC.The recombinant vector,i.e.,pcDNA3.1/HisC-TPds,was transfected into Huh7 hepatocytes by FuGENE6 transfection reagent and the expressed protein was detected by Western blot.HUb7 hepatocytes were co-transfected by pcDNA3,1/HisC-TPds together with theβ-galactosidase reporter plasmid pCMVβand pSV-β-gal- actosidase,cells were lysed 48 hours post transfection,the intracellularβ-galatosidase activities were monitored and calculated by SPSS11.5 software.Results The splicing-specific gene with the size of around 420 bp was success- fully cloned,the protein by which encoded expressed well in Huh7 cells with the highest level in the point of 48 h after transfection.The intracellularβ-galatosidase activities of HUb7 hepatocytes co-transfected with the reporter plasmids and pcDNA3,1/HisC-TPds were higher than those of empty vector pcDNA3.1/HisC with the ratio ranging from 1:10 to 1:50 of reporter plasmid to pcDNA3.1/HisC-TPds,and demonstrated the dose-effects manner within the ratio ranging from 1:10 to 1:40 of reporter plasmid to pcDNA3.1/HisC-TPds.Conclusion The splicing-spe- cific protein encoded by the 2.2 kb doubly spliced variant of hepatitis B virus genome could transactivate the CMV immediate-early promoter and the SV40 promoter and enhancer,which suggested a possible role in the pathogenesis of HBV.