title: [Establishment of a stable cell line replicating hepatitis B virus DNA carrying the reverse transcriptase region derived from a clinical isolate].
abstract: OBJECTIVE
  To establish a stable cell line that can replicate hepatitis B virus (HBV) DNA carrying the reverse transcriptase sequence derived from a clinical isolate.
  
  
  METHODS
  Nested PCR was used to amplify the HBV DNA fragment from the serum. The fragment was cloned into a plasmid that can support HBV replication in vitro by fragment substitution reaction (FSR), followed by the cloning of the neomycin expressing fragment downstream from HBV DNA. G418 selection was conducted after the transfection of HepG2 cells with the recombinant DNA. Real-time PCR and enzyme linked immunosorbent assay (ELISA) were used to screen stable cell lines that can replicate HBV DNA, and the replication of HBV DNA by the cell line was confirmed by using Southern blot analysis.
  
  
  RESULTS
  Fragment nt55-1654 amplified from the serum DNA was substituted to the plasmid pLL, generating the plasmid p11. The neomycin fragment was cloned into p11, leading to the plasmid p11-neo, and p11-neo was confirmed to be HBV-replication-competent. A stable cell line named 3-10 that can replicate HBV DNA was obtained.
  
  
  CONCLUSIONS
  A stable cell line was established that can replicate HBV DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Real-time PCR plus ELISA may help to rapidly screen out stable cell lines replicating HBV DNA.
authors.affiliation: Department of Infectious Diseases, Chongqing Medical University, Chongqing, China.
authors.forename: Ming-que
authors.initials: MQ
authors.lastname: Xiang
authors.forename: Xue-fei
authors.initials: XF
authors.lastname: Cai
authors.forename: Wen-lu
authors.initials: WL
authors.lastname: Zhang
authors.forename: Ai-long
authors.initials: AL
authors.lastname: Huang
authors.forename: Jie-li
authors.initials: JL
authors.lastname: Hu
mesh_terms: D002460:Cell Line; D003001:Cloning, Molecular; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D005822:Genetic Vectors; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000235:genetics*; D022781:Hepatocytes / Q000166:cytology* / Q000821:virology; D006801:Humans; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D014779:Virus Replication / Q000235:genetics
chemical_list: D004279:DNA, Viral; D012194:RNA-Directed DNA Polymerase