title: The anti-HBV effect mediated by a novel recombinant eukaryotic expression vector for IFN-α.
abstract: BACKGROUND
  Chronic hepatitis B is a primary cause of liver-related death. Interferon alpha (IFN-α) is able to inhibit the replication of hepadnavirus, and the sustained and stable expression of IFN-α at appropriate level may be beneficial to HBV clearance. With the development of molecular cloning technology, gene therapy plays a more and more important role in clinical practice. In light of the findings, an attempt to investigate the anti-HBV effects mediated by a eukaryotic expression plasmid (pSecTagB-IFN-α) in vitro was carried out.
  
  
  METHODS
  HBV positive cell line HepG2.2.15 and its parental cell HepG2 were transfected with pSecTagB-IFN-α or empty plasmid by using Lipofectamine™ 2000 reagent. The expression levels of IFN-α were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA methods. The effects of pSecTagB-IFN-α on HBV mRNA, DNA and antigens were analyzed by real-time fluorescence quantitative PCR (qRT-PCR) and ELISA assays. RT-PCR, qRT-PCR and western blot were employed to investigate the influence of pSecTagB-IFN-α on IFN-α-induced signal pathway. Furthermore, through qRT-PCR and ELISA assays, the suppressive effects of endogenously expressed IFN-α and the combination with lamivudine on HBV were also examined.
  
  
  RESULTS
  pSecTagB-IFN-α could express efficiently in hepatoma cells, and then inhibited HBV replication, characterized by the decrease of HBV S gene (HBs) and HBV C gene (HBc) mRNA, the reduction of HBV DNA load, and the low contents of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Mechanism research showed that the activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal pathway, the up-regulation of IFN-α-induced antiviral effectors and double-stranded (ds) RNA sensing receptors by delivering pSecTagB-IFN-α, could be responsible for these phenomena. Furthermore, pSecTagB-IFN-α vector revealed effectively anti-HBV effect than exogenously added IFN-α. Moreover, lamivudine combined with endogenously expressed IFN-α exhibited stronger anti-HBV effect than with exogenous IFN-α.
  
  
  CONCLUSIONS
  Our results showed that endogenously expressed IFN-α can effectively and persistently inhibit HBV replication in HBV infected cells. These observations opened a promising way to design new antiviral genetic engineering drugs based on IFN-α.
authors.affiliation: Institute of Immunopharmacology and Immunotherapy, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan 250012, China. zhangj65@sdu.edu.cn.
authors.forename: Haotian
authors.initials: H
authors.lastname: Yu
authors.forename: Zhaohua
authors.initials: Z
authors.lastname: Hou
authors.forename: Qiuju
authors.initials: Q
authors.lastname: Han
authors.forename: Cai
authors.initials: C
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000956:Antigens, Viral / Q000032:analysis; D000998:Antiviral Agents / Q000378:metabolism*; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors; D006515:Hepatitis B virus / Q000187:drug effects* / Q000502:physiology; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D012367:RNA, Viral / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000956:Antigens, Viral; D000998:Antiviral Agents; D004279:DNA, Viral; D016898:Interferon-alpha; D012367:RNA, Viral