Polypyrimidine Tract Binding Protein Nnegatively Regulates The Expression Of HBV Surface Antigen By Interacting With HBV Posttranscriptional Regulatory Element

HPRE is a regulatory element within hepatitis B virus genome. As regulating expression of HBV gene at posttranscriptional level, it is named HBV posttranscriptional regulatory element (HPRE). It is required for high-level HBV gene expression. Since it was found by Huang J et al in 1993, functional mechanism of HPRE and protein factors, which interacting with it, have being research focous. Although its role is currently unknown, it has been considered that the HPRE has its function just by binding with cell proteins. For this reason, it is helpful to elucidate the function of HPRE in HBV life cycle by studying its binding proteins.Polypyrimidine tract binding protein(PTB) is a nuclear protein,which bound with HPRE.Objective: To verify the binding ability of the protein PTB with HPRE RNA in vitro, recombinant PTB-His fusion protein was purificated largely ; CAT expression plasmid pDM138-HPRE system was adopted to identify the role of PTB in HPRE function. HepG2.2.15 cell line and HBs-HPRE transient expression cells were adopted for identifying PTB function in HBV life cycle.Methods: 1. The plasmid pGEM-HPRE was linearization with XhoI. It was used as the template for transcription in vitro by T7 RNA polymerase to generate Biotin-HPRE labeled RNA. The fusion protein PTB-His, expressed by BL21 and purified via affinity chromatography were mixed with Biotin-HPRE RNA, the His protein was used as a negative control.2. M-280 Streptavidin specially binding to Biotin, complexes were isolated on magnet subsequently. Binding proteins in the pull-down complex were analyzed with Western Blot; Nickel column specially binding to His protein, the eluant was done with RT-PCR analysis.3. Plasmid pDM138-HPRE and protein PTB eukaryotic expression plasmid PCMV-SPORT6-PTB were cotransfected into Hela cells in different group and different proportion manner. CAT activity detection assay was performed to identify the function of interaction of PTB and HPRE.4. PTB eukaryotic expression plasmid PCMV-SPORT6-PTB was transfected into HepG2.2.15 cells. PCMV-SPORT6-PTB was cotran- sfected with HBsAg expressing plasmids, pcDNA3.1-HBs and pcDNA3.1 -HBs-HPRE. The expression of S antigen were detected with ELISA analysis.Results: 1. Western Blot: After detection with Ni-NTA HRP conjugate, a strap was shown in the sample of PTB-His+Biotin-HPRE RNA , however, no strap was detected in the control His+HPRE-biotin RNA . RT-PCR: electrophoresis was displayed that sample PTB-His+Biotin-HPRE RNA had HPRE DNA, the control His+Biotin-HPRE RNA had not.2. The expression of reporter gene CAT in cells transfected with pDM138-HPRE was high, meanwhile, the expression of CAT was inhibited obviously in the cells cotransfected with pDM138-HPRE and PCMV-SPORT6-PTB, and the inbibition was independent on upstream splice donor site.3. ELISA assay results showed that the expression of HBsAg was decreased clearly in HepG2.2.15 cells, which was transfected PCMV-SPORT6-PTB. When two HBsAg expressing plasmids, pcDNA3.1-HBs and pcDNA3.1-HBs-HPRE transfected Hela cells, pcDNA3.1-HBs-HPRE expressed HBs 3 times as much as pcDNA3.1-HBs. Comparing to merely transfected with pcDNA3-HBs-HPRE, cotransfection of PCMV-SPORT6-PTB lead to the expression of HBsAg decreased more than 2 times, cotransfection of PCMV-SPORT6 had not any affection. The result desmonated that PTB negatively regulated expression of HBsAg by interaction with HPRE. Conclusion: The protein PTB could bind with HPRE RNA. The PTB protein inhibited HPRE function in pDM138-HPRE assay system. PTB negatively regulated expression of HBsAg.