title: [Construction and expression of human anti-HER2 scFv-9R fusion protein and identification of its activity].
abstract: OBJECTIVE
  To construct,  express and purify a human fusion protein,  which is composed of a single-chain antibody fragment scFv that recognizes HER2 protein and an oligo-9-arginine,  and to analyze the binding activity of the expressed fusion protein.
  
  
  METHODS
  Pairs of oligonucleotide primers were designed and used to amplify the scFv-9R. The fusion protein gene scFv-9R was then cloned into expression vector pQE30 and expressed in E.coli M15.Expressed protein was detected by SDS-PAGE and Western blot and purified by Ni-NTA chelating agarose. Then, the purified protein was refolded by dialysis and concentrated by ultrafiltration. The antigen-binding activity of the scFv-9R fusion protein was confirmed by ELISA,  and the siRNA binding ability was confirmed by electrophoretic mobility shift assay (EMSA).
  
  
  RESULTS
  HER2 scFv-9R encoding sequence was correctly cloned into the expression vector.  The recombinant protein was insolubly expressed in E.coli M15 induced by IPTG.  ELISA confirmed that it had specific antigen binding activity; EMSA assured that it had siRNA binding activity.
  
  
  CONCLUSIONS
  The scFv-9R fusion protein can specially bind with both HER2 antigen and siRNA.
authors.affiliation: Orthopedics Oncology Institute of Chinese PLA, Tangdu Hospital, Xi'an, China. jiangkuo520@gmail.com
authors.forename: Kuo
authors.initials: K
authors.lastname: Jiang
authors.forename: Rui
authors.initials: R
authors.lastname: Chen
authors.forename: Li-feng
authors.initials: LF
authors.lastname: Wang
authors.forename: Qiong
authors.initials: Q
authors.lastname: Ma
authors.forename: Yan-ling
authors.initials: YL
authors.lastname: Meng
authors.forename: An-gang
authors.initials: AG
authors.lastname: Yang
authors.forename: Bao-an
authors.initials: BA
authors.lastname: Ma
authors.forename: Xiu-chun
authors.initials: XC
authors.lastname: Qiu
mesh_terms: D000936:Antigen-Antibody Complex / Q000276:immunology; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D009842:Oligopeptides / Q000235:genetics / Q000276:immunology; D010455:Peptides / Q000235:genetics / Q000276:immunology*; D034741:RNA, Small Interfering / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*
chemical_list: D000936:Antigen-Antibody Complex; D009842:Oligopeptides; D010455:Peptides; D034741:RNA, Small Interfering; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C015462:polyarginine