Cloning And Expression Of Hepatitis B Virus E Antigen Binding Protein 2, And Its Partial Functions

The infection of Hepatitis B virus (HBV) can lead to cirrhosis and hepatocellular carcinoma. However, there is no effectively therapeutic technique and means for HBV infection at present. Insights into the viral hepatitis pathogenesis of HBV should be sure to obtain an exciting advance in its treatment. In recent years, the HBV pathogenetic mechanism was researched deeply. The complicated interaction mechanism involved in the course of HBV with hepatocytes, which related possibly with HBV pathogenesis mechanism.So, our lab had screened the hepatocyte proteins interacting with HBeAg,and one new gene with unknown functions was named Hepatitis B Virus e Antigen binding protein 2 (HBEBP2).Based on this,we study to explore its function as followings:1. A DNA fragment of HBEBP2 with BamHI and EcoRⅠcut sites was extracted and amplified by reverse transcription polymerase chain reaction (RT-PCR), totle RNA from HepG2 cells as the template, and ligated into pGEM-T cloning vector. After sequencing, the correct DNA fragment was inserted into green fluorescent protein expressive vector pEGFP-C1. HepG2 cells were transfected, and observed by fluorescent inverted microscope after 24hr. The result suggests that HBEBP2 subcellularly locates in cell nuclear for visible green fluorescent signal.2. The DNA fragment of HBEBP2 with EcoRⅠand SacⅠcut sites was inserted into inducible prokaryotic expressive vector pET-32a(+). The competent BL21 (DE3) E. coli was transformed, and then cultured andinduced by 1mM/ml IPTG at 37℃for 4.5hr. The recombinant HBEBP2 was expressed optimistically and confirmed by Western Blotting.3. The DNA fragment of HBEBP2 with EcoRⅠand BamHⅠcut sites was constructed into yeast expressive vector pGBKT7, and transformed into AH109 yeast strains. The yeast protein was isolated and detected by Western blotting analysis.Yeast two-hybrid screening was performed by mating AH109 with Y187 containing liver cDNA library plasmid. There were four kinds of hepatocyte proteins interacting with HBEBP2: human sapiens lactate dehydrogenase D, human sapiens mitochondrion, human sapiens mannose, human sapiens aldehyde oxidase,human sapiens serpin peptidase inhibitor etc. These results by screening and analyzing indicated that HBEBP2 protein was correlated with glycosylation, lipid metabolism, cell proliferation,ect. In addition, two new genes with unknown functions was named HBEBP2-binding protein B ( HBEBP2BPB )( GenBank Accession:DQ499598) and binding protein C (HBEBP2BPC) (GenBank Accession:DQ499599).4. Then,we clone the new gene, binding protein B of HBEBP2, and explore its function and structure by bioinformatics analysis.These results brought some new clues for studying the biological functions of HBEBP2 further.