Investigation On Function Of The Novel Protein Encoded By The 2.2Kb Spliced Variant Of Hepatitis B Virus Genome

Spliced variants of hepatitis B virus genomes are a group of subgenomic length DNA generated from 3.5Kb pregenomic RNA by splicing and reverse transcription. More than 80% of these spliced variants are 2.2Kb doubly or singly spliced variants of hepatitis B virus genome, which could encode splicing-specific proteins and closely related with persistent infection and pathogenicity of HBV. The aim of this study was to explore potential pathogenicity of splicing-specific proteins encoded by the 2.2Kb doubly or singly spliced variants of HBV by yeast two-hybrid system.In the first part of this study, 2.2Kb doubly or singly spliced variants of hepatitis B virus genome were separated from the serum of liver carcinoma patients and cloned into pUC18 vectors. Splicing-specific genes of TPds and TPss that generated from the 2.2Kb doubly and singly spliced variants of hepatitis B virus genome were respectively obtained by the means of PCR. TPds and TPss were separately inserted into the pGBKT7 vector to construct the bait plasmids of yeast two-hybrid system. During the qualification-check stage of yeast two-hybrid assay, TPds was found to be able to transactivate GAL4 responsive element, while TPss could not .The second part of this study was aimed to elucidate furtherly the transactivation capacity of TPds. A set of fragments of TPds gene were subcloned into pGBKT7 and the recombinants were transformed into yeast AH109. The intracellularβ-galactosidase activities of the transformants were measured qualitatively and quantitatively. It was demonstrated that the transactivating domain of TPds was located within the central region of 28 amino acids, which was overlapped with the transactivating domain of preS1 protein . To verify transactivation capacity of TPds in human liver cells, eukaryotic expression vector of pcDNA3.1/HisC-TPds was constructed and cotransfected into Huh7 cells with reporter plasmids ; and it was verified that TPds could transactivate the CMV immediate-early promoter and the SV40 promter and enhancer. Moreover, TPds also could transactivate HBV promters and enhancers.In the third part of this study, a two-hybrid library screening was performed using a pre-transformed human liver cDNA library to seek for the cellular protein which could interact with TPss. Four candidates were screened as followed : cathepsin B; epoxide hydrolase 1, microsomal (xenobiotic); cathepsin D and fibrinogen gamma chain. And mammalian two-hybrid assay between TPss and fragments of those four protein were done to primarily confirm their interactions in mammalian cells. It was suggested that TPss may effect multifarious on cancer metastasis, apoptosis of liver cells,detoxication of liver tissue,and the blood clotting as well.