The Related Research Of NIRF Protein And HBV Core Protein

Objective: 1.To detect the expression of NIRF protein and HBV-CP in human normal liver tissues and HBV-infected liver tissues ; To detect the expression of NIRF in human normal liver tissues and liver cancer tissues; 2. To construct NIRF eukaryotic expression plasmid and transfect into human embryonic kidney epithelial cells HEK-293; 3.To transfect the recombinant plasmid into HepG2.2.15 cells that can secrete HBV DNA and detect the position of NIRF protein and HBV-CP in cells.Methods: 1.Immunohistochemistry was performed to detect the expression of NIRF protein and HBV-CP in human normal liver tissues(n=25) and HBV-infected liver tissues(n=25); Immunohistochemistry was performed to detect the expression of NIRF in human normal liver tissues(n=25) and human liver cancer tissues(n=25); 2. The gene fragment coding for NIRF was obtained from human liver cancer tissue using RT-PCR,and insert into the eukaryotic expression plasmid pIRES2-EGFP to construct the recombinant plasmid pIRES2-EGFP-NIRF,which was transformed into E.coli DH5α,followed by selection of the positive clones containing the target inserts. The eukaryotic expression plasmid was analysed by PCR,restriction endonucleases digestion and DNA sequencing. The recombinant plasmid pIRES2-EGFP-NIRF was transfected into HEK-293 cells by liposome protocol.The experimental cells were classified into three groups: HEK-293-NIRF, HEK-293-Null and HEK-293.The expression of NIRF mRNA and its protein were examined by RT-PCR and Western blotting methods.3.The recombinant plasmid pIRES2-EGFP-NIRF was transfected into HepG2.2.15 cells that can secrete HBV DNA to detect the position of NIRF protein and HBV core protein in cells by using Immunohistochemistry.Results: 1.To detect de expression of NIRF protein and HBVcore protein by using Immunohistochemistry, we found that NIRF protein content with HBV core protein (HBV-CP) content was positively correlated. NIRF protein in human liver cancer tissues was significantly higher than in human normal liver tissues; 2. The gene fragment of exogenous NIRF was correctly inserted into the eukaryotic expression plasmid pIRES2-EGFP.An eukaryotic expression vector of pIRES2-EGFP-NIRF was constructed successfully and verified by PCR,restriction endonucleases digestion and DNA sequencing. The recombinant expression plasmid pIRES2-EGFP-NIRF has been transfected into HEK-293 cells and obtained stable expression verified by RT-PCR and Western blotting methods. 3. The recombinant expression plasmid pIRES2-EGFP-NIRF was transfected into HepG2.2.15 cells that can secrete HBV DNA and it can be found that NIRF protein and HBV core protein (HBV-CP) has a common subcellular localization by using Immunohistochemistry.Conclusions: NIRF has ubiquitination capacity; The 7th and 96th Lys residues of HBV core protein (HBV-CP) have the molecular basis of ubiquitination; NIRF protein content with HBV core protein (HBV-CP) content was positively correlated in human liver tissues; NIRF protein in human liver cancer tissues was significantly higher than in human normal liver tissues; NIRF protein and HBV core protein (HBV-CP) has a common subcellular localization. Accordingly, we can speculate that NIRF protein may be a E3 ligase of HBV core protein (HBV-CP), thus to explore the impact of NIRF protein to HBV virus in suprincipal cells within the assembly ,sophisticated and external secretion mechanism. Finally, a new way for the clinical treatment of hepatitis B will be found.