title: [Expression of Dengue virus type 2 nonstructural protein 3 and isolation of host proteins interacting with it].
abstract: OBJECTIVE
  To construct the plasmid expressing the fusion protein of Dengue virus type 2 (DENV2) nonstructural protein 3 (NS3) with affinity tag, and isolate the cellular proteins interacting with NS3 protein using tandem affinity purification (TAP) assay.
  
  
  METHODS
  Primers for amplifying NS3 gene were designed according to the sequence of DENV2 genome and chemically synthesized. The NS3 fragments, after amplified by PCR with DENV2 cDNA as template, were digested and cloned into the mammalian eukaryotic expression vector pCI-SF with the tandem affinity tag (FLAG-StrepII). The recombinant pCI-NS3-SF was transiently transformed by Lipofectamine(TM) 2000 into HEK293T cells, and the expression of the fusion protein was confirmed by Western blotting. Cellular proteins that interacted with NS3 were isolated and purified by TAP assay.
  
  
  RESULTS
  The eukaryotic expression vector expressing NS3 protein was successfully constructed. The host proteins interacting with NS3 protein were isolated by TAP system.
  
  
  CONCLUSIONS
  TAP is an efficient method to isolate the cellular proteins interacting with DENV2 NS3.
authors.affiliation: Department of Microbiology, School of Preclinical Medicine, Fourth Military Medical University, Xi' an 710032; Department of Clinical Laboratory, Second Artillery General Hospital, Beijing 100088, China.
authors.forename: Daihui
authors.initials: D
authors.lastname: Weng
authors.affiliation: Department of Microbiology, School of Preclinical Medicine, Fourth Military Medical University, Xi' an 710032, China.
authors.forename: Yingfeng
authors.initials: Y
authors.lastname: Lei
authors.affiliation: Department of Microbiology, School of Preclinical Medicine, Fourth Military Medical University, Xi' an 710032, China.
authors.forename: Yangchao
authors.initials: Y
authors.lastname: Dong
authors.affiliation: Department of Microbiology, School of Preclinical Medicine, Fourth Military Medical University, Xi' an 710032, China.
authors.forename: Peijun
authors.initials: P
authors.lastname: Han
authors.affiliation: Department of Microbiology, School of Preclinical Medicine, Fourth Military Medical University, Xi' an 710032, China.
authors.forename: Chuantao
authors.initials: C
authors.lastname: Ye
authors.affiliation: Department of Microbiology, School of Preclinical Medicine, Fourth Military Medical University, Xi' an 710032, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Microbiology, School of Preclinical Medicine, Fourth Military Medical University, Xi' an 710032, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: [1]Department of Microbiology, School of Preclinical Medicine, Fourth Military Medical University, Xi'an 710032; [3]Department of Blood Transfusion, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. *Corresponding author, E-mail: yinwen@fmmu.edu.cn.
authors.forename: Wen
authors.initials: W
authors.lastname: Yin
mesh_terms: D003715:Dengue / Q000378:metabolism* / Q000821:virology; D003716:Dengue Virus / Q000235:genetics* / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000378:metabolism*; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D011506:Proteins; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases