Expression of scFv SA3 against hepatoma fused with enhanced green fluorescent protein and its targeted ability in vivo.

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.

CONCLUSION

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.