title: [Secreted expression of dengue virus type 2 envelope glycoprotein in eukaryotic cells].
abstract: OBJECTIVE
  To secreted express envelope glycoprotein (E) of dengue virus type 2 extracellularly.
  
  
  METHODS
  The entire prM/E gene was amplified by RT-PCR. An optimized signal sequence gene from Japanese encephalits virus (JEV, SA14-14-2 strain) was introduced using fusion PCR. The impact of E protein transmembrane and cytoplasmatic domains was compared by amplifying prM and E with full length of E gene, with 20% truncation of the E gene at 3' terminus and one chimeric gene, which was generated by replacing the 3' terminal 20% region of E gene with the corresponding sequence of JEV (SA14-14-2 strain). The PCR segments were inserted into the NheI and NotI sites of pcDNA5/FRT vector or into the NheI and XhoI sites of pAcUW51-M. Then they were transfected into 293T cells or Sf9 cells respectively. The expression and secretion of E protein were detected by immunofluorescence assay (IFA) and Western Blot.
  
  
  RESULTS
  After transected into 293T cells or Sf9 cells, all constructs expressed E protein intracellularly indentified by IFA while only two plasmids could secret detectable E protein into tissue culture using Western Blot analysis.
  
  
  CONCLUSIONS
  Signal peptide as well as the transmembrane and cytoplasmatic domains is crucial for the secretion of dengue E protein.
authors.affiliation: State Key Laboratory for Molecular Virology & Genetic Engineering, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Zhang
authors.forename: Wen
authors.initials: W
authors.lastname: Gu
authors.forename: Chuan
authors.initials: C
authors.lastname: Li
authors.forename: Fang
authors.initials: F
authors.lastname: Miao
authors.forename: Peng
authors.initials: P
authors.lastname: Lu
authors.forename: Jing
authors.initials: J
authors.lastname: Qu
authors.forename: Yan
authors.initials: Y
authors.lastname: Wei
authors.forename: Quan-fu
authors.initials: QF
authors.lastname: Zhang
authors.forename: Qin-zhi
authors.initials: QZ
authors.lastname: Liu
authors.forename: Jian-dong
authors.initials: JD
authors.lastname: Li
authors.forename: Mi-fang
authors.initials: MF
authors.lastname: Liang
authors.forename: De-xin
authors.initials: DX
authors.lastname: Li
mesh_terms: D000818:Animals; D002460:Cell Line; D003715:Dengue / Q000378:metabolism / Q000821:virology*; D003716:Dengue Virus / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018411:Spodoptera; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: C051821:E-glycoprotein, Dengue virus type 2; D014759:Viral Envelope Proteins