A Study On Binding Proteins Of HBV EnhⅠ, SPⅠ And SP Ⅱ

Objective: To investigate binding proteins potential role in the regulation of replication and expression of HBV DNA, and may provide some new clues for the prevention and cure of hepatitis B.Methods: (1) Using HBV Enhl biotinylated promoter DNA as the selective molecule, the T7 select human liver cDNA library was biopanned and positive clones were selected. After screening, positive plaques were performed to amplify for inserted DNA fragment and cloned into pGEM-Teasy vector. (2) Using HBV SPI biotinylated promoter DNA as the selective molecule, the positive plaques were selected as mentioned above methods. (3) Using HBV SP II biotinylated promoter DNA as the selective molecule, the T7 select human liver cDNA library was biopanned and positive clones were selected. After screening, positive plaques were performed to amplify for inserted DNA fragment and cloned into pGEM-Teasy vector. (4) HBV SP II and NADHDH4 sequences were amplified from PCP10 plasmid and human hepatoblastoma cell line-HepG2 cell genomic DNA by polymerase chain reaction (PCR). The amplified products were cloned into pCAT3 and pcDNA3.1(-)vector,respectively. The HepG2 cell line was tranfected by pCAT3-SPII and pcDNA3.1(-)-NADHDH4. The CAT activity was detected by an enzyme-linked immunosorbent assay (ELISA) kit.Results: (1) Twenty positive plaques were obtained for Enhl DNA sequencing. They encode 3 proteins,including homo sapiens similar to nuclear pore complex interacting protein,human serum albumin (ALB) gene, and homo sapiens PI-3-kinase-related kinase SMG-1-like.The binding protein of HBV Enhl DNA was identified by phage display screening. (2) Fourteen positive plaques were obtained for HBV SPI DNA sequencing. They encodes 7 proteins, three of which have not found in the GenBank,the others include 4-aminobutyrate aminotransferase, haptoglobin, and RF-C/activator 1 homolog. The binding proteins of HBV SP I DNA were identified by phage display screening. (3) Twenty positive plaques were obtained for DNA sequencing. They encode 7 proteins, two of whichhave unknown functions,the others include PCTAIRE protein kinase 2, nicotinamide adenine dinucleotide dehydrogenase dehydrogenase4, homo sapiens albumin, alpha-1-microglobulin/bikunin precursor, homo BAC clone RP11-815N9,and homo sapiens mitochondrion.The binding proteins of HBV SP II DNA were identified by phage display screening. (4) The expression of pCAT3-SPII was 1.4 times as higher as the negative control plasmid pCAT3-basic.The expression of pCAT-SPII+pcDNA3.1(-)-NADHDH4 was nearly 2.8 times as higher as the pCAT3-SP plasmid.Conclusions: (1) Our results suggested that these proteins can bind to Enhl DNA, and maybe are the transactivating factors of Enhl. However whether these proteins regulate the transcription of Enhl DNA,its need further study. The studies implied that this approach provides a new search tool for the study of replication and expression mechanism of HBV DNA. (2) These results suggested that these proteins can bind to SP I DNA, and maybe are the transactivating factors of SP I. (3) Our studies implied that these proteins can bind to SP II DNA, and maybe are the transactivating factors of SP II. However whether these proteins regulate the transcription of SP II DNA need further study. (4) The finding suggested that NADHDH4 could bind the HBV SP II DNA in vivo,and upregulated the expression of HBV SP,which pave the way for further studying of the regulated mechanism of HBV DNA.