Screening Of Binding Protein To The Core Promoter DNA Of Hepatitis B Virus By Phage Display

Objective: Hepatitis B virus (HBV) is the primarily hepatotrophic member of the Hepadnavirus family, which causes acute and chronic liver diseases, as well as cirrhosis and hepatocellular carcinoma in our country. The HBV genome is a circular, partly double-stranded molecule of DNA. The core promoter (CP) of hepatitis B virus plays a central role in HBV replication and morphogenesis, directing the transcription of pregenomic (pg) RNA and precore (pre-C) mRNA. The expression and regulation of virual gene is most important for the replication of HBV. We intend to investigate to those possible DNA-protein which interact with HBV CP. It can furtherly explain the mechanism of CP in HBV infection. Methods: To explore the possible function of HBV CP, the CP gene sequence was cloned from the plasmid of HBV pCP10. We design the sense primer and antisense primer according the reports. Then we construct the chloramphenicol acetyltransferase (CAT) reporter vector and and HepG2 cells were transfected to investigate the activity of CAT. At the same time, we transfect the other mutations of HBV CP. Afterwards, we use the biotinylated CP promoter DNA as the selective molecule, the T7 select phage human liver cDNA library was amplified and positive clones were selected. After screening, plaques were performed to amplify for inserted DNA fragment and cloned into pGEM-Teasy vector. The binding protein of HBV CP DNA was identified by phage display screening. We construct the expression plasmid pcDNA3.1 (-)-β₂-M and pcDNA3.1(-)-NPIP, then HepG2 cells were transfected respectively. Results: 1.The HBV CP DNA was successfully cloned. We co -nstruct the recombined vector pCAT-CP, then the HepG2 was tra -nsfected. The activity of CP was confirmed and the CAT could b -e upregulated. The activity of mutants of HBV CP is different. 2. The binding protein of HBV CP was identified by phage display screening. They are β₂-microglobulin, nuclear pore complex inter -acting protein and homo sapiens PI-3-kinase-related kinase SM -G-1-like. The β₂-microglobulin and nuclear pore complex intera -cting proteins downregulate the activity of the CP. Conclusion: We clone the HBV CP DNA and we construct the expression plasmid pcDNA3.1(-)-β₂-M and pcDNA3.1(-)-NPIP. We identify that the β₂-M and NPIP can bind with the HBV CP DNA through the coinfection. The results are very important. Be -cause we know these two proteins can downregulate the activity of the HBV CP through binding with HBV CP. The HBV CP dire -ct the transcription of pregenomic (pg) RNA and precore (pre-C) mRNA. But the mechanism of the regulationis to also unknown, we should further reaserch it. We have the new acknowledgment about the function the CP after the binding protein of HBV CP w...