title: [Construction and expression of recombinant prokaryotic expression vector for disulfide-stabilized anti-TeNT single-chain Fv antibody].
abstract: OBJECTIVE
  To construct the prokaryotic expression vector for disulfide-stabilized anti-TeNT single-chain Fv antibody (27G-scdsFv), express the 27G-scdsFv and characterize its bioactivity.
  
  
  METHODS
  27G-scdsFv gene was constructed by PCR-based point mutagenesis strategy and cloned into plasmid pET22b(+). The plasmid was then transformed into E.coli BL21(DE3) competent cells. The target protein was expressed under the induction of IPTG. SDS-PAGE and Western blotting were used to identify the expression products. The specific binding activity to TeNT-Hc and relative stability in vitro of 27G-scdsFv were assayed by ELISA, and the affinity of the antibody was measured by non-competitive enzyme-linked immunosorbentassay. The method of immunofluorescence was employed to determine whether or not the 27G-scdsFv kept the neutralizing activity in vitro.
  
  
  RESULTS
  Sequencing analysis proved that 27G-scdsFv gene was correctly constructed. The target protein was expressed in the form of inclusion body, accounting for about 50% of total bacterial proteins. After renaturated, the 27G-scdsFv protein still maintained specific binding activity to TeNT-Hc, with a higher affinity constant (K(D);) of 0.93×10(-7); mol/L as compared with that of 27G-scFv, and the yield of the protein was about 5 mg/L induced culture. The relative stability of 27G-scdsFv was improved obviously as compared with the scFv form. 27G-scdsFv strongly inhibited the binding of TeNT-Hc to PC-12 in vitro.
  
  
  CONCLUSIONS
  The porkaryotic expression vector of 27G-scdsFv has been successfully constructed and the activated target protein has been obtained, which lays a foundation for the further study on the biological function of 27G-scdsFv.
authors.affiliation: Vaccine and Antibody Engineering Research Laboratory, Army Key Laboratory of Biological Weapon Injury Prevention Pharmaceuticals, Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China. wanghan129@sina.com
authors.forename: Han
authors.initials: H
authors.lastname: Wang
authors.forename: Rui
authors.initials: R
authors.lastname: Yu
authors.forename: Xiaopeng
authors.initials: X
authors.lastname: Zhang
authors.forename: Jun
authors.initials: J
authors.lastname: Ren
authors.forename: Na
authors.initials: N
authors.lastname: Xie
authors.forename: Hongyan
authors.initials: H
authors.lastname: Fan
authors.forename: Jinlong
authors.initials: J
authors.lastname: Zhang
authors.forename: Ting
authors.initials: T
authors.lastname: Fang
authors.forename: Changming
authors.initials: C
authors.lastname: Yu
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
mesh_terms: D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D001483:Base Sequence; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D017354:Point Mutation; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D013744:Tetanus Toxin / Q000276:immunology* / Q000378:metabolism
chemical_list: D004220:Disulfides; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D013744:Tetanus Toxin