ABSTRACT
AIM: 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.CONCLUSIONS: The successful cloning and expression of DT389-hbFGF immunotoxin provides a foundation for targeting therapy towards posterior capsule opacification.
ABSTRACT
AIM: To explore the feasibility of combining small pieces of allo-conjunctiva transplantation (ACT) with amniotic membrane transplantation (AMT) to reconstruct ocular surface.METHODS: Bilateral superior bulbar conjunctiva of New Zealand white rabbits were burned by NaOH (1mol/L).After 24h, the right eye (experimental groups Exps) was performed AMT and ACT;the left eye (control groups Cons)was done merely ACT. The viability and function of allografts were identified by PAS staining and scanning electron microscope (SEM) of cytology impression (CI) every week after the operation.RESULTS: In Exps, no obvious rejection was observed.All grafts survived, extended and covered the conjunctival defect. The appearance of grafts, and the shape, number,and function of goblet cells recovered perfectly four weeks after the operation;however, in Cons, obvious rejection was found. Seven grafts were rejected and. symblepharon was observed in those eyes. The appearance of survival allografts were similar to the normal conjunctiva, with similar shape, number and function of goblet cells till the fifth week after the operation. The survival ratet was significantly higher in Exps than in Cons (P ＜0.05).CONCLUSION: ACT based on AMT can be considered a good way to reconstruct ocular surface for large area conjunctiva alkali burns in early-stage.