Cloning and expression of immunotoxin DT389-hbFGF

Abstract:

To express the DT₃₈₉-hbFGF (389 amino acid residues of the N-terminus of diphtheria toxin (human basic fibroblast growth factor) fusion protein for potential targeting therapy towards posterior capsule opacification (PCO) after cataract surgery. METHODS: The DNA of inactivated diphtheria bacillus and RNA of 12-week fetal brain cortex were extracted, respectively. The fragments of truncated diphtheria toxin (containing 389 amino acids of N-terminus, DT₃₈₉) and full-length human basic fibroblast growth factor (hbFGF) sequence (encoding 18kDa protein) were amplified by PCR. The two fragments were inserted into pGEX-4T-1 prokaryotic expression vector to obtain pGEX-DT₃₈₉-hbFGF prokaryotic expression plasmid. After sequence analysis, the expressing plasmid was transformed into Escherichia Coli BL21 strain and expression was induced under IPTG. The expressed fusion protein was purified and identified. RESULTS: The gene fragments encoding DT₃₈₉ and hbFGF were amplified and their gene sequences were confirmed. Hybrid gene expression plasmid pGEX-DT₃₈₉ (hbFGF) was constructed. The fusion protein DT389-hbFGF was expressed and purified. CONCLUSION: The successful cloning and expression of DT₃₈₉-hbFGF immunotoxin provides a foundation for targeting therapy towards posterior capsule opacification.