[Cloning, expression and cytotoxin to human lens epithelial cells of targeting toxin DT389-hbFGF].
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.


CONCLUSIONS
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.
Yin
Lian-rong
LR
Tianjin Medical University Eye Center, Tianjin 300070, China. yinlianrong@sina.com
Hao
Yan-sheng
YS
Yuan
Jia-qin
JQ
Li
Xiao-rong
XR
D002478:Cells, Cultured; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic; D004167:Diphtheria Toxin / Q000096:biosynthesis* / Q000235:genetics / Q000633:toxicity; D004847:Epithelial Cells / Q000473:pathology*; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000633:toxicity
D004167:Diphtheria Toxin; D011993:Recombinant Fusion Proteins; D016222:Fibroblast Growth Factor 2