title: [Construction of pSG5/TRIF and its expression in Huh7 cells].
abstract: OBJECTIVE
  To construct the plasmid pSG5/TRIF and investigate its expression in Huh7 cells.
  
  
  METHODS
  The plasmid pCX4pur/Myc-TRIF was digested with Not I and the digestion product was blunted followed by further digestion with EcoR I to obtain the insert Myc-TRIF. pSG5 was digested sequentially with Sma I and EcoR I. All the digested products were analyzed with agarose gel electrophoresis. The products with the expected size were extracted and ligated, and the positive clones were screened by ampicillin and amplified. The recombinant pSG5/TRIF was extracted, purified, and identified by restriction endonuclease BamH I and agarose gel electrophoresis. The recombinant plasmids were transfected into Huh7 cells with FuGene 6 reagents and into Huh7 cells previously infected with recombinant vaccinia virus (rVV) via Lipofectin. Immunofluorescence and Western blotting were performed to detect the expression of the recombinant plasmids, and the transfection efficiency with different transfection reagents was compared.
  
  
  RESULTS
  BamH I digestion resulted in a fragment with the expected size. Immunofluorescence staining showed successful expression of Myc-TRIF protein in Huh7 cells, and the transfection efficiency was enhanced in Huh7 cells previously infected with rVV. SDS-PAGE analysis showed that the relative molecular mass of the expressed product by pSG5/Myc-TRIF was about 100 ku, and prior infection of the cells with rVV obviously increased transfection efficiency, as was consistent with the results of immunofluorescence.
  
  
  CONCLUSIONS
  pSG5/Myc-TRIF is successfully constructed and expressed in Huh7 cells. The expression efficiency can be increased by prior infection of the cells with rVV.
authors.affiliation: Institute of Geriatric Cardiology, General Hospital of PLA, Beijing 100853, China. xueping127@hotmail.com
authors.forename: Xue-ping
authors.initials: XP
authors.lastname: Wang
authors.forename: Shi-wen
authors.initials: SW
authors.lastname: Wang
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.forename: Tong
authors.initials: T
authors.lastname: Yin
authors.forename: Hong-juan
authors.initials: HJ
authors.lastname: Wang
authors.forename: Bin
authors.initials: B
authors.lastname: Xu
authors.forename: Qing-lei
authors.initials: QL
authors.lastname: Zhu
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics*; D045744:Cell Line, Tumor; D015870:Gene Expression; D005822:Genetic Vectors; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014162:Transfection
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D011993:Recombinant Fusion Proteins; C470409:TICAM1 protein, human