title: Expression of scFv SA3 against hepatoma fused with enhanced green fluorescent protein and its targeted ability in vivo.
abstract: OBJECTIVE
  To express and purify the human scFv antibody, SA3, against the hepatoma fused to enhanced green fluorescent protein, and to observe the targeted capacity of fusion protein EGFP-SA3 in vivo.
  
  
  METHODS
  SA3 and EGFP genes were cloned into plasmid pET-25b(+) to construct the recombinant plasmid EGFP-SA3/pET-25b(+), followed by DNA sequencing. Then it was transformed into E.coli BL21(DE3) and induced for fusion expression of EGFP-SA3 with IPTG. The expressed fusion protein EGFP-SA3 was purified and detected with SDS-PAGE. HepG2 cells were incubated with the fusion protein EGFP-SA3 in vitro, and the binding bioactivity was observed under the fluorescent microscope. Further more, we injected the EGFP-SA3 by caudal vein into nude mice planted by hepatoma and observed the whole body fluorescence image of EGFP.
  
  
  RESULTS
  SA3 and EGFP genes were successfully cloned into pET-25b(+), which was confirmed by restriction enzyme NcoI-XhoI or NcoI-EcoRI. A band migrated at the position 750 bp, same to EGFP gene, emerged when recombinant plasmid was digested by restriction enzyme NcoI-EcoRI. Similarly, a band, about 1 500 bp, emerged when digested by NcoI-XhoI. The open-reading frame was confirmed by DNA sequencing. Fusion protein EGFP-SA3 was expressed as inclusion body. After purification and refolding, the result of immunofluorescence detection verified that EGFP-SA3 could specifically bind to HepG2 cells and maximum tumor penetration was at 24 h after the injection.
  
  
  CONCLUSIONS
  The purified fusion protein EGFP-SA3 has strong binding capacity to HepG2 cells, indicating the scFv SA3 has a potential value as a targeting molecule for diagnosis and targeted therapy for liver cancer.
authors.affiliation: Cancer Research Institution, Central South University, Changsha 410078, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Huang
authors.forename: Yuehui
authors.initials: Y
authors.lastname: Li
authors.forename: Fengjie
authors.initials: F
authors.lastname: Guo
authors.forename: Yongqing
authors.initials: Y
authors.lastname: Tong
authors.forename: Jiajia
authors.initials: J
authors.lastname: Wang
authors.forename: Jinyue
authors.initials: J
authors.lastname: Hu
authors.forename: Guancheng
authors.initials: G
authors.lastname: Li
mesh_terms: D000818:Animals; D000912:Antibodies, Neoplasm / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D006528:Carcinoma, Hepatocellular / Q000276:immunology / Q000628:therapy*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000276:immunology* / Q000494:pharmacology; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000276:immunology / Q000628:therapy*; D051379:Mice; D008819:Mice, Nude; D058990:Molecular Targeted Therapy; D009368:Neoplasm Transplantation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology* / Q000494:pharmacology; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000276:immunology* / Q000494:pharmacology
chemical_list: D000912:Antibodies, Neoplasm; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C410726:scFV green fluorescent protein, recombinant; D049452:Green Fluorescent Proteins