title: [Establishment of bacteria display technology for Fab antibody library screening].
abstract: OBJECTIVE
  To establish bacterial display technology for the purpose of Fab antibody library screening,  by using six amino acids (CDQSSS) of the amino termimus of NlpA protein to anchore antibodies to the periplasmic side of the bacterial inner membrane.
  
  
  METHODS
  The NlpA Leader sequences (encoding CDQSSS) was amplified from pNAD plasmid. The PCR product was subcloned into pComb3 expression vector to generate Fab display vector pBFD. The heavy chains of the Fab gene fragments and the light chains of the anti-human IL-1β (hIL-1β) antibody were inserted downstream of the NlpA leader and pelB leader respectively to construct the pBFD-Fab for Fab antibody display. Then pBFD-Fab transformed E.coli DH5α was induced by IPTG to express the Fab antibodies,  as detected by flow cytometry (FCM),  and positive populations were sorted. Instead of PCR,  plasmids were extracted for rescue purpose. The rescue plasmids were retransformed to E.coli DH5α and FCM was performed again.
  
  
  RESULTS
  The pBFD-Fab-transformed bacteria were incubated with antigen and antigen specific FITC-antibody,  and showed strong fluorescence as detected by FCM in a dose-dependent manner. The rescued pBFD-Fab displayed similar fluorescence intensity,  indicating the reliability of this technology.
  
  
  CONCLUSIONS
  The Fab expressed by the bacterial display system folds efficiently and binds to hIL-1β specifically. The plasmid rescue works well and it can avoid mutation and mis-pairing chains. This bacterial display technology has the stability of antibody expression. This study has used the technology to screen anti-hIL-1β Fab antibody Library successfully.
authors.affiliation: Northeast Agricultural University, Harbin, China. lmxu0917@163.com
authors.forename: Li-ming
authors.initials: LM
authors.lastname: Xu
authors.forename: Cheng-kai
authors.initials: CK
authors.lastname: Yin
authors.forename: Gui-ping
authors.initials: GP
authors.lastname: Ren
authors.forename: Hui
authors.initials: H
authors.lastname: Tian
authors.forename: Xue-qin
authors.initials: XQ
authors.lastname: Wang
authors.forename: Liang-jun
authors.initials: LJ
authors.lastname: Ding
authors.forename: De-shan
authors.initials: DS
authors.lastname: Li
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology*; D053583:Interleukin-1beta / Q000235:genetics / Q000276:immunology; D019151:Peptide Library
chemical_list: D007140:Immunoglobulin Fab Fragments; D053583:Interleukin-1beta; D019151:Peptide Library