title: Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase.
abstract: OBJECTIVE
  To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase (AP).
  
  
  METHODS
  The VH-linker-VL, namely scFv gene, was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by Sfi I and Not I, it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1. The positive colonies were screened by colony PCR and their expressions were induced by IPTG. ScFv gene was gained by digesting ScFv expression vector pUC19/119 with Sfi I and Not I restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1. The positive colonies were selected by bacterial colony PCR. The expression of fusion protein (scFv-AP) was induced by IPTG. Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
  
  
  RESULTS
  The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku. Immunofluorescent assay (IFA) demonstrated its reactivity with TfR. The molecular weight of scFv-AP was 75 ku. Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity.
  
  
  CONCLUSIONS
  We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP. It is promising for immunological experiments.
authors.affiliation: Division of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
authors.forename: Dao-Feng
authors.initials: DF
authors.lastname: Yang
authors.forename: Hui-Fen
authors.initials: HF
authors.lastname: Zhu
authors.forename: Zhi-Hua
authors.initials: ZH
authors.lastname: Wang
authors.forename: Guan-Xin
authors.initials: GX
authors.lastname: Shen
authors.forename: De-Ying
authors.initials: DY
authors.lastname: Tian
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics* / Q000276:immunology*; D000906:Antibodies / Q000235:genetics; D003001:Cloning, Molecular; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D011990:Receptors, Transferrin / Q000235:genetics* / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics
chemical_list: D000906:Antibodies; D007135:Immunoglobulin Variable Region; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase