| 屈园利,刘家云,何林全,刘西涛,李晓东,龙 铟.水蛭素融合蛋白的表达及其与靶向适配子的偶联[J].现代生物医学进展英文版,2017,17(19):3610-3614. |
| 水蛭素融合蛋白的表达及其与靶向适配子的偶联 |
| Expression of Hirudin Fusion Protein and Its Conjugation with Targeted Aptamer |
| Received:March 29, 2017 Revised:April 25, 2017 |
| DOI：10.13241/j.cnki.pmb.2017.19.003 |
| 中文关键词: 血栓性疾病 水蛭素 融合蛋白 RGD 适配子 |
| 英文关键词: Thrombotic diseases Hirudin Fusion protein RGD Aptamer |
| 基金项目:国家自然科学基金项目（81073095） |
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| 中文摘要: |
| 摘要 目的：优化表达并纯化水蛭素融合蛋白SA-H-RGD，检测其生物学活性，获得能够与生物素标记的纤维蛋白适配子G81-2结合的偶联物。方法：将序列正确的质粒pET-44b-SA-H-RGD进行原核表达，采用不同浓度的IPTG及时间优化融合蛋白表达条件，镍亲和凝胶层析柱纯化融合蛋白，Western-blot鉴定蛋白。通过凝血酶原时间(PT)和抗血小板聚集实验检测融合蛋白活性；之后按照生物素-G81-2:SA-H-RGD摩尔比为4:1的比例制备纤维蛋白特异性的偶联物，用凝胶迁移阻滞实验(EMSA)验证二者的偶联。结果：融合蛋白SA-H-RGD在IPTG 0.9 mmol/L、5 h时在大肠杆菌中获得可溶性高效表达，纯化的融合蛋白具有延长PT的作用和抑制血小板聚集的活性，EMSA表明SA-H-RGD具有结合生物素标记的G81-2适配子的功能。结论：本研究成功地优化表达了具有抗凝血和抗血小板聚集功能的融合蛋白SA-H-RGD，获得了水蛭素融合蛋白与生物素-G81-2适配子组成的靶向偶联物。 |
| 英文摘要: |
| ABSTRACT Objective: To optimize the expression of fusion protein streptavidin (SA)-hirudin (H)-RGD, determine its biological activities and obtain its conjugates with fibrin aptamer. Methods: The pET-44b-SA-H-RGD plasmid was sequenced and the isopropy- lthio-β-D-galactoside (IPTG) inducted conditions was optimized. Then it was purified with Ni affinity gel chromatography column and identified with Western blotting. The function of SA-H-RGD was identified by prothrombin time (PT) and the experiment of anti-platelet aggregation. The conjugates that targeted to fibrin was prepared by mixing the biotin-labeledG81-2 aptamer and the SA-H-RGD at the molar ratio of 4:1, and validated by electrophoretic mobility shift assay (EMSA). Results: The sequence of pET-44b-SA-H-RGD plasmid was absolutely correct, the expression of fusion protein SA-H-RGD in Escherichia coli was optimized with 0.9mmol/L IPTG for 5 hours and purified. The activity of anticoagulant and anti-platelet aggregation of SA-H-RGD was identified. The activity to combine with the biotin-labeled G81-2 aptamer was showed by EMSA. Conclusion: The expression and purification of hirudin fusion protein SA-H-RGD were successfully performed. The expressed protein possessed anticoagulant and anti-platelet aggregation functionandits conjugates with biotin-labeled G81-2 aptamer was obtained ultimately. |
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