SA-hirudin-RGD融合蛋白的原核表达及其抗凝血酶与抗血小板聚集功能的研究

Prokaryotic Expression of SA-hirudin-RGD Fusion Proteinand Identification of Its Anticoagulant and Anti-platelet Aggregation Function

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 a recombinant vector SA-hirudin-RGD expressing purified fusion protein, and to preliminary validate its anticoagulant and anti-platelet aggregation function. Methods： The natu streptavidin （SA） ral core sequence and hirudin-RGD gene were cloned into pET-44b to form the recombinant plasmid pET44b-SA-hirudin-RGD. The fusion protein was induced by IPTG, purified by Ni-NTA agarose and detected by Western-blot. The anticoagulant and anti-platelet aggregation assay to prove the activity of SA-hirudin-RGD. Results： Restriction enzyme digestion and gene sequencing showed that the recombinant plasmid pET44b-SA-Himdin-RGD was successfully constructed. The fusion protein induced by IPTG was efficiently expressed in E.coli. Fusion protein was purified by Ni-NTA agarose with a molecular weight of 70000 Da identified by Western blot. The assays proved that SA-hirudin-RGD has anticoagulant and anti-platelet aggregation activity. Conclusion： SA-hirudin-RGD fusion protein with anticoagulant and anti-platelet aggregation fimction is successfully expressed and purified, which lying a good basis for further functional and clinical application of SA-hirudin-RGD.