Interacting Of Hepatitis B Spliced Protein (HBSP) With Hepatocyte Ubiquitously Expressed Transcript

HBSP (Hepatitis B spliced protein) is a protein encoded by2.2kb singly splicedvariant of hepatitis B virus genome which spliced at the nucleotides position2447nt-489nt. Previous studies showed that HBSP could induce liver apoptosis andpositively correlated with liver cirrhosis; it could also induce specific CTL responsewhich could lead to liver damage, however, the mechanisms involved are far frombeen elucidated. To study the pathogenic mechanism of HBSP, yeast two hybridsystem was used in our previous study and some hepatocellular proteins includingUbiquitously expressed transcript(UXT) were identified which may interact withHBSP. In the present study, recombinant vector pCMVTNT-UXT was constructed bywhich TNT rabbit reticulocyte lysate transcription/translation coupling system wasused to produce UXT protein in vitro. And fusion protein of GST-HBSP wasexpressed in E.Coli and purified. GST-pull down assay indicated that HBSP and UXTcan interact with each other in vitro. The interaction between HBSP and UXT in vivowas further identified by mammalian two-hybrid system by which co-transfectingliver hepatoma cell line Huh7with recombinant plasmid pBIND-HBSP, pACT-UXTand reporting gene vector pG5luc. Furthermore, recombinant adenovirus expressingHBSP were packaged, which laid the foundation for further studies exploring theinteraction between HBSP and UXT and the pathogenicity of HBSP in thehepatocytes.