Cloning And Identification Of Different HBV PreS DNA Fragments And Screening Of Presl-Interacting Proteins In The Yeast Two-Hybrid System

The initial event in the life cycle of a virus is its interaction with receptors present on the surface of a susceptible host cell. The term "virus receptor" here is used to mean a host surface component (usually proteins) that participates in virus binding, facilitates viral infection, and also one determinant of virus host range and tissue tropism. Understanding these interactions is important to our understanding of viral tropism, spread, and pathogenesis. It has been showed that Hepatitis B virus(HBV) surface antigen preS, an important protective antigen, possesses many biological functions, and thus is considered to be closely associated with establishment of HBV infection, HBV replication and assembly in hepatocytes. For this reason, the work pertaining to HBV preS antigen (mainly preS; and preS: antigens) and their corresponding binding receptor is becoming a hotspot in HBV research area at present. Some experiment results indicated that preSi(21-47aa) can directly bind to human hepatoma HepGi cells with specificity, whereas preS: bind to hepatocytes through polymerized human serum albumin(pHSA) normally present on the hepatocyte surface. These work have provided us important clues to understanding biological functions of preS and HBV pathogenesis. Yeast two-hybrid technique is a contemporary new method for detection ofdirect protein-protein interactions in a eukaryotic cell. In our work, different HBV preS gene fragments were amplified by PCR, cloned into pGBKT? vector in yeast two-hybrid system 3, and then transformed AH 109 yeast cells. The results showed that these preS expression products were nontoxic to yeast cells, but all self-activated the yeast two-hybrid system. Even so, by truncating HBV preS gene, we finally obtained some useful ''bailors", either nontoxic or self-activating, and used them to fish DNA fragments of HBV preS interacting protein(s) from an AD vector constructed human embryonic cDNA library. After high stringent screening and further identification, we got several meaningful clones and finished sequencing for some of them, supplementing some experimental evidence for further research on HBV hepatocyte tropism and pathogenesis.