作者：Lian-Rong Yin 1,2，Jia-Qin Yuan2   

    作者单位：1Eye Hospital, China Academy of Traditional Chinese Medicine, Beijing 100040, China; 2Eye Center, Tianjin Medical University, Tianjin 300070, China

    【摘要】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.

    【关键词】  immunotoxin; DT; hbFGF

    INTRODUCTION

    Posterior capsular opacification (PCO), the most frequent complication associated with decreased vision after extra capsular cataract surgery, is a result of lens epithelial cell (LEC) proliferation and migration on the posterior capsular. Therefore, prevention of PCO is an important step towards improving the quality of the surgical outcome. At this time, there is no method of preventing PCO that has proven to be effective, practical and safe for routine clinical procedures. The most convenient method for treating PCO is a neodymium: YAG (Nd:YAG) laser posterior capsulotomy. When an Nd:YAG laser is not available, conventional capsulotomy surgery is performed.

    Within the wide range of recent therapeutic targets, cytokines and receptors may play important roles because they are expressed by LECs and can influence postoperative proliferation of LECs in the capsular bag. LECs can express high-affinity fibroblast growth factor (FGFR) and secrete the basic fibroblast growth factor (bFGF) which can induce cell proliferation by binding to FGFR[1]. Diphtheria toxin (DT) is a ribosome-inactivating protein, which consists of the catalytic, transmembrane and cell-binding domains. The 389 amino acids of N-terminus of diphtheria toxin (DT389) includes only the catalytic and transmembrane domains. The cell-binding domain of DT was replaced by hbFGF, rendering the fusion protein to specifically bind to human LECs. This report describes how immunotoxin DT389-hbFGF is cloned and expressed.

    MATERIALS AND METHODS

    The primers used in these studies are given in Table 1.

    Cloning DT389 Fragment The genomic DNA of inactivated diphtheria bacillus was extracted and amplified to obtain the full-length genomic sequence (1.6 kb) by PCR. The DT389 fragment was amplified from the full-length genomic sequence. After being separated and purified by 10g/L agarose electrophoresis and QIAquick Gel Extraction Kit (Promega), the DT389 fragment was inserted into pGEM-T Easy plasmid by A-T cloning using T-4 ligase. The Escherichia coli (E. coli ) strain JM109 was transformed by the plasmid and inoculated on an LB plate with ampicillin (Amp 0.1g/L), and incubated overnight at 37℃. The inserted fragment was sequenced by the Chinese National Human Genome Center, Beijing (CHGB).

    Cloning hbFGF Fragment The total RNA of embryonic 12-week cortex was extracted and amplified to obtain 1,000 bp bFGF by one-step RT-PCR Kit (Promega). 500 bp fragment encoding 18KD hbFGF was amplified and Eco RI, Xho I restriction sites were inserted by PCR using a 1,000 bp bFGF template. The amplified products were separated, harvested and purified as described above.

    Construction of Hybrid Gene and Expression Plasmid The DT389-pGEM-T Easy plasmid was extracted using a mini plasmid extracting kit. The Bam HI and Eco RI restriction sites were introduced to the bi-side of DT389 by PCR. The DT389 containing the restriction sites and pGEX-4T-1 were incubated with Bam HI and Eco RI, respectively, and were linked using T4 ligase to construct pGEX-DT389 plasmid. The pGEX-DT389 plasmid and hbFGF fragment were incubated with Eco RI and Xho I, respectively, and were linked using T4 ligase to obtain pGEX-DT389-hbFGF expression plasmid. The E. coli JM109 was transformed by pGEX- DT389- hbFGF plasmid and cultured in an LB plate with Amp (0.1g/L) at 37℃. The sequence was identified using gene sequencer.

    Expression of Fusion Protein Plasmid pGEX-DT389- hbFGF was transformed into the E. coli strain BL21(DE3), which were then inoculated. in an LB plate with Amp (0.1g/L) and incubated overnight at 37℃. One clone was transferred into 5mL LB medium with 0.1g/L Amp and cultured at 37℃ until the OD600 of the culture reached 1.0. The bacteria were transferred to 50mL fresh LB medium and continued to be cultured at 37℃. When the OD600 reached 0.6, expression of the hybrid gene was induced by the addition of isopropyl-P-D-thiogalactopyranoside (IPTG, 0.1 mmol/L; GIBCO BRL). Bacteria (1mL) were harvested at IPTG induction 0, 1, 2, and 3 hours, and sonicated 5 minutes in an ice-bath, mixed with the same volume of protein electrophoresis loading buffer and boiled 7 minutes for SDS-PAGE. Remaining bacteria were harvested at 3hours by centrifugation at 5 000g for 10 minutes. The bacterial pellet was resuspended in ice-cold 1 mmol/L EDTA/PBS, sonicated 5 minutes, and centrifuged 30 minutes at 15 000g. To determine the localization of expressed protein, the supernatant and pellet, respectively, were loaded for SDS-PAGE. The expression was analyzed on 125g/L SDS-PAGE using a Mini-Protein I1 gel apparatus (Bio-Rad). Proteins were stained with Coomassie brilliant blue.

    Purification and Identification of Fusion Protein Several clones were selected and pre-cultured as described above. The bacteria were transferred to 1L LB medium and cultured under IPTG induction 3 hours. The bacteria were harvested and sonicated as described above. The supernatant was collected and added into two Glutathione Sephrose 4B columns. Column I was incubated with GSH (glutamyl cysteinyl glycine) overnight, and the liquid phase was collected (Sample 1). Sample 1 was incubated with thrombin (20U/mL) overnight (Sample 2). Column II was incubated with thrombin overnight and the liquid phase was collected (Sample 3), and then the column II was incubated with GSH overnight, the liquid phase was collected (Sample 4). The four samples were mixed respectively with loading buffer and boiled 7 min at 100°C. The expression was analyzed as described above. For immunoblotting, the separated proteins (Sample 3 and bFGF) were transferred to nitrocellulose membranes. Membranes were blocked with 50mL/L milk-containing TBS (20mmol/L Tris, 500mmol/L NaCI, pH 7.5) and washed with TTBS (TBS. 0.5g/L Tween-20, pH 7.5). Membranes were incubated with rabbit anti-bFGF sera (Santa Cruz), and HRP-conjugated goat anti-rabbit IgG at 1∶2000 dilution was applied with 2 hours incubation. ECL blotting reagents (Amersham Biosciences) was used for protein visualization.

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