Coloning, Expression And Cytotoxicity To Human Lens Epithelial Cells Of Targeting Toxin DT₃₈₉-hbFGF

PCO is the most frequent complication associated with decreased vision after cataract surgery. Previous methods of preventing PCO haven't proven to be effective, practical and safe for routine clinical procedure. Nd:YAG laser posterior capsulotomy may lead to severe complications and increase the cost for patient. So prevention is more improtant than treatment.The opinion that PCO results from the proliferation, migration and producing extracellular matrix of residual LECs on the capsule has been accepted widely. So we can prevent PCO only by eliminating LECs. LECs can highly express high-affinity FGFR. bFGF can induce cell proliferation by binding to FGFR. DT consists of catalytic domain, transmembrane domain and receptor binding domain which are independent from each other on structure. So the structure of DT suits gene recombination and keeping toxicity.To construct pGEX-DT389-hbFGF expression plasmid, the fragment of truncated DT [encoding 389 ammo acids of N-terminus, DT₃₈₉] and full length hbFGF gene (encoding 18KD protein) were amplified by PCR technique respectively, then the two fragments were inserted into prokaryotic expression vector pGEX-4T-1. After testing sequence, the expression plasmid was transformed into E.Coli BL21 strain and induced expression under IPTG. The expressed fusion protein was purified and identified. We identified the fusion protein by 10%SDS-PAGE and Western blot. MTT colorimetry tested cytotoxicity of the fusion protein to HLECs in vitro under...