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鉴定

发布时间：2018-01-06 00:31

本文关键词：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鉴定　出处：《第四军医大学》2015年硕士论文　论文类型：学位论文

【摘要】：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,Western blotting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pET44b-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;纯化获得该目的蛋白,相对分子质量经Western blotting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:有着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。
[Abstract]:Objective: to construct a recombinant SA-hirudin-RGD vector to express and purify the fusion protein and to inhibit thrombin. Methods: the core region of streptomycin (SAA) was linked to the hirudin-RGD sequence by gene recombination technique. And cloned into prokaryotic expression vector pET-44b. Western. Blotting was used to identify the purified fusion protein induced by IPTG. The analysis of anti-thrombin and anti-platelet aggregation showed that the fusion protein had both antithrombin and anti-platelet aggregation functions. The recombinant vector pET44b-SA-hirudin-RGD was successfully constructed by restriction enzyme digestion and gene sequencing. SA-hirudin-RGD fusion protein was highly expressed in Escherichia coli induced by IPTG. The target protein was purified and its molecular weight was about 700000.The results showed that the molecular weight of the target protein was about 700000.The results of anti-thrombin and anti-platelet aggregation were proved. The fusion protein SA-hirudin-RGD has both anti-thrombin and anti-platelet aggregation functions. SA-hirudin-RGD fusion protein with dual functions of anti-thrombin and anti-platelet aggregation was successfully expressed and purified. It establishes the foundation for the function research and clinical application of SA-hirudin-RGD in the next step.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R440

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