title: [Single chain antibody against β-site of amyloid precursor protein inhibits the generation of β-amyloid peptide].
abstract: OBJECTIVE
  To construct and express a single chain fragment of variety region (scFv) against β-site of amyloid precursor protein (APP), and evaluate the effect of scFv on α- and β-processing of APP.
  
  
  METHODS
  The β-site specific scFv 2H10 was amplified and fused with signal sequence Igκ and myc tag by PCR to create the expression cassette, which was then subcloned into expression vector pcDNA3.1hyg⁺. The plasmid was transferred into CHO cells over-expressing Swedish mutation type human APP695 (APP695sw/CHO). The positive clones were identified by hygromycin B. The concentrations of Aβ40 and soluble APPα (sAPPα) in the conditioned media supernatant were measured by sandwich ELISA.
  
  
  RESULTS
  The recombinant eukaryotic expression plasmids were identified by PCR, restriction endonuclease digestion and DNA sequencing. The expression of scFv was confirmed by Western blotting. When scFv 2H10 was stably expressed in APP695sw/CHO, the extracellular level of Aβ40 decreased by 30.4%, while sAPPα increased by 23.1%.
  
  
  CONCLUSIONS
  The APP β-site scFv 2H10 could inhibit the secretion of Aβ. Meanwhile, it might be favorable for the α-processing of APP. Hence it can be explored as an inhibitor of β-site APP-cleaving enzyme (BACE).
authors.affiliation: Deparment of Preclinical Medicine, Wuhan Institute of Medical Sciences, Wuhan 430014, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.affiliation: Deparment of Preclinical Medicine, Wuhan Institute of Medical Sciences, Wuhan 430014, China.
authors.forename: Lirong
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Deparment of Pediatrics, People's Hospital, Wuhan University, Wuhan 430060, China.
authors.forename: Xiaoou
authors.initials: X
authors.lastname: Li
authors.affiliation: Deparment of Preclinical Medicine, Wuhan Institute of Medical Sciences, Wuhan 430014, China.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Sun
authors.affiliation: Deparment of Preclinical Medicine, Wuhan Institute of Medical Sciences, Wuhan 430014, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Zhou
authors.affiliation: Deparment of Preclinical Medicine, Wuhan Institute of Medical Sciences, Wuhan 430014, China.
authors.forename: Xiaogang
authors.initials: X
authors.lastname: Huang
authors.affiliation: Deparment of Preclinical Medicine, Wuhan Institute of Medical Sciences, Wuhan 430014, China.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Liu
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors; D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D016564:Amyloid beta-Protein Precursor / Q000276:immunology*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors; D016466:CHO Cells; D003412:Cricetulus; D010957:Plasmids; D057127:Single-Chain Antibodies / Q000494:pharmacology*
chemical_list: D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D057127:Single-Chain Antibodies; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human