The Influence Of GAPDH On HBs Expression

For most virus, which replicate in the nuclei of host cells, their RNAs export is one of the important steps in posttranscriptional regulation of vrial genes. HBV post-transcriptional regulatory element (HPRE) had been identified in the 1151nt-1684nt sites of HBV genome sequence in 1993. Recent studies have reported that HPRE helps HBs gene trancripts export from nucleus to cytoplasm. Zang et al. have found two cellular proteins that bind to the HPRE RNA by electrophoretic mobility shift assay (EMSA), and demonstrated that the 35 kDa in molecular mass one is glyceraldehyde phosphate dehydrogenase (GAPDH). However, the founction of this binding is still unclear. Our purpose is to verify GAPDH can bind to HPRE RNA in vitro with Dynabeads method. In order to assess the functional significance of this binding, a pDM138-HPRE reporter assay was performed. Overexpression of GAPDH both in cells transiently expressing HBs-HPRE and in HepG2 2.2.15 cells, and the expression of HBs was detected to provide the mechanism of GAPDH involving in HBV posttranscriptional regulation.Methods and results are as follows:1. The plasmid pGEM-HPRE was linearization with XhoI.It was used as the template for transcription in biotin RNA labeling mix system by T7 RNA polymerase to product labeled HPRE RNA. pET32a(+)-GAPDH expressing GAPDH-His fusion protein was transfected into BL21 E.coli bacteria to generate GAPDH-His protein after IPTG induction. After purified by Ni2+ affinity column, protein samples were separated on a 12% SDS-polyacrylamide gel and analyzed by Western blotting with antibodies recognizing 6×His tags and GAPDH.2. M-280 Streptavidin beads specially binding with Biotin, the adsorbate was analyzed by Western blot. After detection with anti-GAPDH and anti-His monoclonal antibody, there is a strap in the mixture of GAPDH-His+HPRE-Biotin RNA. However, there is not any in the control His+HPRE-Biotin RNA. It proved that GAPDH-His protein can bind to HPRE RNA.3. Plasmid pEGFP-N1, pDM138, pDM138-HPRE and proteinGAPDH eukaryotic expression plasmid pcDNA3.1(+)-GAPDH were co-transfected into 293T cells. Plasmid pEGFP-N1 containing a green fluorescent protein (GFP) gene was served as a control for normalizing transfection effectiveness and was verified by flow cytometry. The interaction between the HPRE RNA and protein GAPDH was identified by detecting the expression of the reporter gene CAT by ELISA analysis two days after cotransfection. It was revealed that the expression of reporter gene CAT was high in cells transfected with pDM138-HPRE. However, the expression of CAT was inhibited obviously in the cells co-transfected with pDM138-HPRE and pcDNA3.1(+)-GAPDH (p<0.05).4. GAPDH eukaryotic expression plasmid pcDNA3.1(+)-GAPDH was transfected into HepG2 2.2.15 and transiently expressing HBs-HPRE cells. The expression of HBs antigen was detected with ELISA analysis. The HBs expression was obviously inhibitied (p<0.05).Conclusion: The protein GAPDH could bind with HPRE RNA in vitro, and GAPDH is an inhibitiory protein that bind to HPRE RNA and this binding can reduce the HBs antigen expression.