title: [Preparation and identification of a single chain antibody against the shedding site of N-cadherin cleaved by ADAM10].
abstract: OBJECTIVE
  To construct and express a specific single chain Fv antibody (scFv) against the shedding site of N-cadherin cleaved by ADAM10 and identify its biological activity.
  
  
  METHODS
  The VH; and VL; genes were amplified by RT-PCR from a hybridoma cell line 2B3 which produced the monoclonal antibody (mAb) against the shedding site of N-cadherin cleaved by ADAM10. SOE-PCR was used to splice the VH; and VL; genes to construct ScFv, which was subcloned into the prokaryotic expression vector pET-28a. The positive clones were transformed into E.coli BL21 (DE3) and induced with IPTG. The target protein was purified and refolded via Ni-NTA column, and was analyzed by SDS-PAGE, ELISA and Western blotting.
  
  
  RESULTS
  The VH; and VL; genes of mAb were cloned successfully and the prokaryotic expression vector of scFv was constructed. The analysis of DNA sequencing showed that the full-length of the constructed scFv gene was 744 bp, and coded 248 amino acids. ScFv was expressed in E.coli BL21 (DE3) as inclusion body under the induction of IPTG, and showed relative molecular mass (Mr;) of 29 000 as analyzed by SDS-PAGE and Western blotting. About 90% purity of scFv was obtained following denaturing, purifying and renaturing via Ni-NTA, and ELISA and Western blotting revealed that the soluble scFv exhibited the binding activity to the shedding site of N-cadherin cleaved by ADAM10.
  
  
  CONCLUSIONS
  The scFv against the shedding site of N-cadherin cleaved by ADAM10 has been successfully constructed, which lays the foundation for its diagnostic and therapeutic application.
authors.affiliation: Deparetment of Pediatrics, People's Hospital, Wuhan University, Wuhan, China. lxou27@163.com
authors.forename: Xiaoou
authors.initials: X
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Lirong
authors.initials: L
authors.lastname: Zhou
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000072197:ADAM10 Protein; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D015820:Cadherins / Q000737:chemistry / Q000276:immunology*; D002460:Cell Line; D003001:Cloning, Molecular; D008565:Membrane Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D015820:Cadherins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human