RESULT

    The PCR Products of DT389 and hbFGF The full-length sequence of DT was amplified by PCR. Using this sequence as a template, about 1.1 kb of the DT389 fragment, including the transmembrane and catalytic domains, was obtained. The sequencing data showed that the sequence of the DT389 fragment was correct. The 1.0 kb bFGF fragment was amplified by RT-PCR from embryonic cortex. The 500bp DNA fragment encoding 18kDa bFGF was amplified and the Eco RI and Xho I restriction sites were introduced.

    Construction of Hybrid Gene and Expression Plasmid The DT389 containing restriction site and pGEX-4T-1 were incubated with Bam HI and Eco RI, respectively. and were linked using T4 ligase to obtain pGEX-DT389 plasmid. The plasmid and hbFGF fragment were incubated with EcoRI and Xho I, respectively, and were linked using T4 ligase to obtain pGEX-DT389-hbFGF expression plasmid (Figure 1). The sequencing data indicated that the construction of expression plasmid was corrected (Figure2).

    Figure1 Agarose electrophoresis Lane 1 was pGEX- DT389 plasmid；Lane 2-7 were the products of pGEX-DT389-hbFGF cut with EcoRI, XhoI；Lane 8 was the hbFGF fragment；Lane 9: Molecular markers (200bp, 500bp, 800bp, 1.2kb, 2 kb, 3 kb, 4.5kb).

    Figure 2 Construction of pGEX-DT389-hbFGF expression plasmid

    Expression of Fusion Proteins Coomassie brilliant blue staining indicated there was a very bulky band about 86 KD after IPTG induction. The molecular weight coincided with GST-DT389-hbFGF fusion protein. Location analysis showed that the fusion protein was expressed in cytosol mainly, but there was also a small quantity of protein in inclusion (Figure 3).

    Figure 3 Expression products after IPTG induction Lane 1: Before IPTG induction; Lane 2, 3, 4: 1h, 2h, and 3h, respectively, after IPTG induction;Lane 5: The supernatants of 3h after IPTG induction; Lane 6: The sonicated product of 3h after IPTG induction; Lane 7: The Pellet of 3h after IPTG induction

    Purification and Identification of Fusion Protein Because the labeling protein GST can bind with Glutathione Sephrose 4B, the fusion protein was absorbed by the Glutathione Sephrose 4B column. GSH can elute the GST from Glutathione Sephrose 4B column, and the thrombin can cut off GST from the fusion protein. So, Sample 1 is GST-DT389-hbFGF, and its molecular weight is about 86kDa; Sample 2 includes GST (26 kDa) and DT389-hbFGF (60 kDa); Sample 3 is purified fusion protein (60kDa); Sample 4 is GST (26 kDa)(Figure 4). Western blot indicated that the fusion protein is able to bind to the hbFGF antibody and the molecular weight is about 60 kDa , implying that the fusion protein is DT389-hbFGF (Figure 5).

    Figure 4 Expression and purification of recombinant toxin DT389-hFGF2 Lane 1: Sample 4(GST); Lane 2: Sample 3 (fusion protein); Lanes 3 and 7: Molecular Weight Markers ;Lane 4: Sample 2 (GST fusion protein); Lane 5: Sample 1 (fusion protein); Lane 6: Sonicated products before purification.

    Figure 5 Western blot

    DISCUSSION

    Only partial sequences of bFGF can be obtained in many kinds of tissues such as kidney, placenta, fetal liver, and fetal heart by RT-PCR because bFGF mRNA is unstable. We made use of the fact that human brain glial cells can highly express bFGF[2] and therefore could easily obtain the full- length sequences of bFGF from 12-week fetal brain,

    which provide a simple way to detect bFGF cDNA.

    Not only can they secrete bFGF, but LECs can also highly express high-affinity FGFR. bFGF has an important role in the formation of PCO because it can induce LEC proliferation, migration, and production of extracellular matrix. So, some studies regarding the use of the bFGF antibody, which is the inhibitor of the bFGF receptor or antisense nucleic acid of FGF, for inhibiting LEC proliferation. Francine [3,4] added bFGF(SAP or rbFGF-SAP, respectively, into culture medium of bovine LECs or rabbit capsules after cataract surgery and proved that this immunotoxin can inhibit the proliferating of LECs.

    Using gene recombinant technique, the receptor binding domain of natural toxin can be replaced by antibodies or cytokines. The recombinant immunotoxin can specifically recognize and kill target cells, but causes little injury to other cells. DT, which has strong cytotoxicity, consists of three independent structure regions: the catalytic domain, transmembrane domain and the receptor binding domain. It is suitable for gene recombination and maintaining the toxin[5] . ONTAK (DAB389-IL2) has been approved by the US FDA for treatment of cutaneous T-cell and B-cell lymphomas (CTCLs)[6].

    Cloning and expression of the DT-bFGF fusion protein has not been reported before. In the present study, we have successfully cloned and expressed a fusion protein - DT389- hbFGF, in which DT389 represents the N-terminal 389 amino acids of DT, including the catalytic domain and transmembrane domain, while the cell-binding domain of DT was replaced by hbFGF. In the next step, we will determine the efficacy and safety of the DT389-hbFGF, and investigate its potential application as an immunotoxin for prevention of PCO.

    【参考文献】

    1 Seghezzi G, Patel S, Ren CJ, Gualandris A, Pintucci G, Robbins ES, Shapiro RL, Galloway AC, Rifkin DB, Mignatti P. Fibroblast growth factor2 (FGF2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: an autocrine mechanism contributing to angiogenesis. J Cell Biol ,1998;141(7):1659-1673

    2 Gomez-Pinilla F, Lee JW, Cotmam CW. Distribution of basic fibroblast growth factor in the developing rat brain. Neuroscience ,1994;61(4):911-923

    3 Behar-Cohen FF, David T, Buechler Y, Nova MP, Houston LL, Pouliquen YM, Courtois Y. Cytotoxic effects of FGF2-saporin on bovine epithelial lens cells in vitro. Invest Ophthalmol Vis Sci, 1995;36(12):2425-2433

    4 Behar-Cohen FF, David T, D'Hermies F, Pouliquen YM, Buechler Y, Nova MP, Houston LL, Courtois Y. In vivo inhibition of lens regrowth by fibroblast growth factor 2-saporin. Invest Ophthalmol Vis Sci ,1995;36(12):2434-2448

    5 Choe S, Bennett MJ, Fujii G, Curmi PM, Kantardjieff KA, Collier RJ, Eisenberg D. The crystal structure of diphtheria toxin. Nature ,1992;357(6375):216-222

    6 Frankel AE, Powell BL, Hall PD, Case LD, Kreitman RJ. Phase I trial of a novel diphtheria toxin/granulocyte macrophage colony-stimulating factor fusion protein (DT388GMCSF) for refractory or relapsed acute myeloid leukemia. Clin Cancer Res ,2002;8(5):1004-1013

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