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目的:构建SA-hirudin-RGD重组载体,表达和纯化融合蛋白,并对其抗凝血酶、抗血小板聚集功能进行初步验证。方法:利用基因重组技术将链霉亲和素(SA)核心区与hirudin-RGD序列连接,并克隆到原核表达载体PET-44b中,Westernblotting鉴定经IPTG诱导后纯化的融合蛋白。抗凝血酶和抗血小板聚集作用分析证明该融合蛋白既有抗凝血酶又有抗血小板聚集的功能。结果:重组载体p ET44b-SA-hirudin-RGD经限制性酶切鉴定和基因测序证实构建成功;经IPTG诱导后SA-hirudin-RGD融合蛋白在大肠杆菌中高效表达;纯化得到该目的蛋白,相对分子质量经Westernblotting鉴定约为70000。抗凝血酶和抗血小板聚集的实验证明,融合蛋白SA-hirudin-RGD既有抗凝血酶又有抗血小板聚集的功能。结论:具有抗凝血酶和抗血小板聚集双重功能的SA-hirudin-RGD融合蛋白被成功表达和纯化,为下一步SA-hirudin-RGD的功能研究及临床应用确立了基础。
OBJECTIVE: To construct recombinant SA-hirudin-RGD vector and express and purify the fusion protein. The anti-thrombin and anti-platelet aggregation function of SA-hirudin-RGD recombinant vector was preliminarily verified. Methods: The core region of streptavidin (SA) was linked to hirudin-RGD by gene recombination technique and cloned into prokaryotic expression vector PET-44b. The fusion protein purified by IPTG was identified by Western blotting. Antithrombin and anti-platelet aggregation analysis proved that the fusion protein has anti-thrombin and anti-platelet aggregation function. Results: The recombinant vector p ET44b-SA-hirudin-RGD was confirmed by restriction enzyme digestion and DNA sequencing. The fusion protein SA-hirudin-RGD was highly expressed in E.coli after IPTG induction. The target protein was purified, The molecular weight identified by Westernblotting is about 70,000. Antithrombin and anti-platelet aggregation experiments show that the fusion protein SA-hirudin-RGD both anti-thrombin and anti-platelet aggregation function. Conclusion: The SA-hirudin-RGD fusion protein with dual functions of anti-thrombin and anti-platelet aggregation was successfully expressed and purified, which laid the foundation for the functional study and clinical application of SA-hirudin-RGD in the next step.