title: [Construction of a eukaryotic expression plasmid containing gp120 gene of HIV-1 subtype B and its expression in HepG2 cells].
abstract: OBJECTIVE
  To construct an eukaryotic expression plasmid containing gp120 gene of HIV-1 subtype B and obtain gp120 gene expression in HepG2 cells.
  
  
  METHODS
  According to the published gp120 gene sequence in Genbank, a pair of primers was designed and synthesized. The PCR amplification product of gp120 gene was cloned into pMD-18T vector using TA cloning followed by BamHI and XhoI digestion and sequence analysis. The target gene was then subcloned into a highly efficient eukaryotic expression vector pcDNA3.1 (+). The recombinant plasmid was sequenced and identified by restrictive endonuclease digestion, and transfected into HepG2 cells via liposome. The expression of gp120 gene was analyzed by RT-PCR and Western blotting, respectively.
  
  
  RESULTS
  Restriction endonuclease digestion and sequence analysis verified successful construction of the recombinant vector pcDNA3.1(+)/gp120. The target fragment gp120 was identical with U26942 in Genbank, and the expression of gp120 gene was detected in the lysate of the transfected HepG2 cells by RT-PCR and Western blotting.
  
  
  CONCLUSIONS
  The eukaryotic expression plasmid for gp120 has been constructed successfully, which is capable of stable expression in HepG2 cells.
authors.affiliation: Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, China. lidlcn@yahoo.com
authors.forename: De-liang
authors.initials: DL
authors.lastname: Li
authors.forename: Wen-li
authors.initials: WL
authors.lastname: Ma
authors.forename: Yong-xia
authors.initials: YX
authors.lastname: Shi
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.forename: Bao
authors.initials: B
authors.lastname: Zhang
authors.forename: Wen-ling
authors.initials: WL
authors.lastname: Zheng
mesh_terms: D016915:AIDS Vaccines / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000378:metabolism*; D015870:Gene Expression; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D014162:Transfection; D019444:Vaccines, DNA / Q000096:biosynthesis / Q000235:genetics
chemical_list: D016915:AIDS Vaccines; D015699:HIV Envelope Protein gp120; D019444:Vaccines, DNA