Screening Of Proteins Binding With The HBV Surface Antigen Promoter Ⅰ And Functional Study Of SBP1

Hepatitis B virus (HBV) is the prototypic member of the hepadnavirus family, which causes acute and chronic liver diseases, including chronic active hepatitis, as well as cirrhosis and hepatocellular carcinoma. HBV infects an estimated 200 million persons worldwide and thus represents a viral pandemic. However, so far, the patho genesis and transcriptional regulation of HBV is not clear, the c/s-acting elements of which includes 4 promoters(CP, XP, SP I , SPII) ,2 enhancers(Enh I and Enh II) and GRE, which contribute to regulate the replication, transcription and translation of viral polypeptides. Among them, SP I regulates the transcription of 2.4 kb mRNA and encodes the large surface antigen. In the previous research, the large surface antigen is strictly limited in liver, which plays a important role in tropism and replication cycle of HBV. To investigate the mechanism of transcriptional regulation and find unknown liver-specific transcriptional factor. We employed yeast one hybrid system developed by Li in 1993 to screen the human liver cDNA library to fish the proteins binding with the HBV large surface antigen promoter DNA.The full-length HBV DNA sequence was used to design 3 copies of core sequence of HBV- SP I as a bait, and bait sequence has been cloned into the pHISi, pHISi-1 and pLacZi vectors, respectively. After being verified it accurate by digestion and sequencing, the linearized vectors were transformed into the yeast strain YM4271, then integrated into its genome, and resulted in the reporter strain YMSP1-HIS and YMSP1-LacZ. The human liver cDNA library was transformed into the reporter strain YMSP1-HIS, plated the entire transformation mixture on SD/-Leu/-His +30 mmol/L 3-AT plates. The positive plasmids were isolated from the yeast colonies by their larger size (>2 mm) , then transformed into another reporter strain YMSPl-LacZ, plated the entire transformation mixture on SD/-Leu/-Ura. The colony lift filter assay was employed to test the P -galactosidase activity. To sequence the dual-positive plasmids, and to analyze the known genesand one unknown gene by bioinformatics.The coding sequence of this unknown gene which be named as the large surface antigen promoter specific DNA-binding protein 1 (SBP1) was cloned by bioinformatics methods and polymerase chain reaction (PCR) from the genomic DNA of human hepatoma cell line. This sequence was ligated into the eukaryotic expression vector pcDNA3.1(-), and was transfected human hepatoma cell line HepG2 with choloraphenical acetyltransferase (CAT) reporter vector pCAT3-SP I . We found that the expression of SBP1 can inhibit the transcriptional activity of SP I . This result suggested that SBP1 can down-regulate the expression of large surface antigen of hepatitis B virus. Furthermore, microarray was used for studying the change of gene expression profiles of hepatocyte in addition of SBP1 .The screening of proteins binding with the HBV large surface antigen promoter DNA may be provide some clues for mechanism of transcriptional regulation and life cycle of HBV. Interestingly, SBP1 can inhibit the expression of the large surface antigen, which may indicate a new way to treat the infection of HBV.