Objective: To construct pGEX-DT(389)-hbFGF plasmid, express and identify the cytotoxicity to human lens epithelial cells (HLECs).
Methods: Extracting DNA of dead diphtheria bacillus and RNA of 12-week fetal brain cortex. The fragments of truncated diphtheria toxin (containing 389 amino acids of N-terminus, DT(389)) and full length human bFGF gene (encoding 18kd protein) were amplified by PCR technique respectively. The two fragments were inserted into prokaryotic expression vector pGEX-4T-1. After testing sequence, the expressing plasmid was transformed into E.Coli BL21 strain and induced expression under IPTG. The expressed fusion protein was purified and identified. MTT experiment tested cytotoxicity of the fusion protein to HLECs in vitro. The way of HLECs death under different dosage was identified by flow cytometry.
Results: The gene fragments of DT(389) and human bFGF were accurately amplified. The expression vector including DT(389)-hbFGF fused gene was constructed and expressed successfully. DT(389)-hbFGF fusion protein can induce HLECs apoptosis in a dosage dependence manner during certain range. The LD(50) was about 3.8 x 10(-11) mol/L.
Conclusion: The successful cloning and expression of DT(389)-hbFGF immunotoxin lay a foundation for accelerating lens epithelial cells apoptosis and the targeting therapy toward posterior capsule opacification.