Construction Of HBV Targeted-ribonuclease And Antivirus Research

Despite the availability of efficient vaccines, hepatitis B virus (HBV) infection remains a major public health problem worldwide. Indeed more than 1 billion people have been infected and 350million people are chronic carriers of the virus. Chronic carriers are exposed to a high risk of developing cirrhosis and hepatocellular carcinoma. This indicates that mass vaccination is required to decrease the prevalence and eventually eradicate HBV infection from the planet to prevent hepa- tocellular carcinoma. Current treatments are of limited efficacy.The sustained response rate of the approved high-dose interferon- a (IFN- a ) therapy is about 30%. Nucleoside analogues such as lamivudine> famciclovi^ lobucavin adevfovir markedly reduce viral load but suffer from the emergence of drug resistant virus variants. Other approaches, such as ribozymes, its ability of catalyzing sequence-specific cleavage of RNA was very not strong. This siruationhas spurred interest in alternative approaches to interfere with HBV replication. A conceptually more powerful approach is capsid-targeted viral inactivation (CTVI) or, generally, virion-targeted viral inactivation, which exploits a viral capsid protein or other virion associated protein as carrier to target a degradative enzyme specifically into virus particles. Its principal feasibility has been demonstrated in retroviral model systems but not yet for a medically relevant virus outside the retrovirus family. HBV replicates through reverse transcription of an RNA molecule, the pregenome. In the cytoplasm, assembling C subunits assimilate complexes of P protein and pregenomic RNA into icosahedral nucleocapsids, within which most viral DNA synthesis occurs. Recently, based onthe experience of replication with HBV, we thought to construct that C proximal fusion to the HBV capsid protein of hEDN( human eosinophil -derived neurotoxin) yields a chimeric protein, hEDN-HBVc, which they could bind and cleave HBV pgRNA, thus , inhibit HBV replication.The total RNA were extracted from HL-60 cells and 2.2.15 cells respectively. The gene encoding hEDN and HBVc amplified by RT-PCR were cloned into eukaryotic expression vector pcDNAS.l(-). The gene encoding hEDNalone and HBVcalone amplified by PCR. Point mutation of hEDN gene by overlap extension PCR losed its cleavage function.They were cloned into pcDNA3.1(-), respectively. pcDNA3.1 (-)/hEDNalone,pcDNA3.1 (-)/HB Vcalone,pcDNA3.1 (-)/hEDNmut-HB Vc were constructed as control. The gene encoding hEDN was cloned into pGEX4T-l and was expressed mE.coli.. After EPTG induction, relative molecule mass(Mr) of expressed fusion protein was 16 000. The purified product immunized the mice and liters of preparing specific antibody was 1:400 . After transfection in 2.2.15 cells, pcDNA3.1(-)/HBVc-hEDN expression is identified by indirect immunofluo-rescence staining. pcDNA3.1(-)/HBVc-hEDN was expressed in 2.2.15 cell lines with higher level. After transfection in 2.2.15 cells, expression of pcDNA3.1(-)/ HBVc-hEDN and three kinds of control plasmid and pcDNA3.1(-) were anaylzed with RT-PCR. Supernatant were analyzed with solid phase RIA The cells were analyzed with MTT. The results show all transfection genes were expressed and proliferation was not restrained, HBsAg was declined 58%, above all.pcDNA3.1(-)/HBVc-hEDN with special targeted molecular that could encapsidate HBV pgRNA and degradative enzyme that could degrade HBV pgRNA could inhibit HBV replication. Our data suggest HBV may therefore be particularly vulnerable to the CTVI approach.,which provided a new thread of thought for inhibiting HBV replication .