论文部分内容阅读
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p ET-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。
OBJECTIVE: To optimize the expression and purification of hirudin fusion protein SA-H-RGD and test its biological activity to obtain a conjugate capable of binding to biotin-labeled fibrin aptamer G81-2. Methods: Prokaryotic expression of plasmid p ET-44b-SA-H-RGD was carried out by using different concentrations of IPTG and time. The fusion protein was purified by nickel affinity gel chromatography and identified by Western-blot protein. The fusion protein activity was tested by prothrombin time (PT) and anti-platelet aggregation experiments; fibrin-specific conjugates were then prepared at a 4: 1 biotin-G81-2: SA-H-RGD molar ratio, Gel-Migration Assay (EMSA) was used to verify the coupling between the two. Results: The fusion protein SA-H-RGD was highly expressed in E. coli at IPTG 0.9 mmol / L for 5 h. The fusion protein SA-H-RGD could prolong the activity of PT and inhibit platelet aggregation. EMSA showed that SA- RGD has the function of binding to the biotinylated G81-2 aptamer. Conclusion: The fusion protein SA-H-RGD with anti-coagulation and anti-platelet aggregation has been successfully optimized and expressed. The targeted conjugate consisting of hirudin fusion protein and biotin-G81-2 aptamer .