中介蝮蛇毒纤溶酶基因表达的研究

发布时间：2018-04-30 12:26

本文选题：中介蝮 + 纤溶酶　；参考：《哈尔滨师范大学》2011年硕士论文

【摘要】：血栓性疾病是目前危害人类健康的首要疾病之一,而利用溶栓类药物治疗血栓性疾病被认为是最为有效的方法。蛇毒纤维蛋白(原)溶解酶(fibrin(ogen)olytic enzyme,FLE简称纤溶酶)是一种新型的强力溶栓剂,能直接溶解纤维蛋白或纤维蛋白原。利用蛋白质分离技术从蛇毒中高度纯化而得的蛇毒纤溶酶在心脑血管疾病的治疗方面具有潜在的应用价值。然而天然蛇毒资源有限,不易分离、保存和运输,因此通过分子生物学的方法和基因工程技术获得高效、安全的蛇毒纤溶酶,对于更有效地治疗血栓性疾病具有十分重要的意义。本实验利用双酶切技术从pMD18T-FLE I克隆载体上获得中介蝮(Gloydius intermedius)蛇毒纤溶酶基因片段,再将其亚克隆到真核表达载体pFASTBACHTa上,经转化、筛选和鉴定获得重组真核表达质粒pFASTBACHTa-FLE。将纯化后的重组质粒(约20μg)经小鼠尾静脉快速(5s内)注入小鼠体内,进行瞬时表达,8h后取小鼠肝脏组织制备实验样本。经SDS-PAGE分析显示,在小鼠肝脏中有重组蛋白表达,其分子量大小约为30kDa;再经Western blot检测,在30kDa处得到一条特异性的杂交条带,表明小鼠肝脏中表达的重组蛋白为FLE蛋白。免疫组织化学方法检测结果显示,重组FLE蛋白在小鼠肝脏组织中大量表达。纤维蛋白平板法鉴定纤溶活性结果表明,小鼠肝脏中表达的重组FLE蛋白具有较高的纤溶活性,且其活性呈现出剂量相关性和时间依赖性。本实验在真核表达体系中成功表达出中介蝮蛇毒纤溶酶重组蛋白,为进一步对蛇毒纤溶酶的生物学特性及其应用的研究奠定了坚实的基础。
[Abstract]:Thrombotic disease is one of the most important diseases that harm human health at present, and thrombolytic drugs are considered as the most effective method in the treatment of thrombotic diseases. Fibrinolytic enzyme fibrinolytic enzyme (fibrinolytic enzyme FLE) from snake venom is a new powerful thrombolytic agent, which can directly dissolve fibrin or fibrinogen. The highly purified plasmin from snake venom by protein separation technique has potential application value in the treatment of cardiovascular and cerebrovascular diseases. However, natural snake venom is limited in resources and is not easy to separate, preserve and transport. Therefore, high efficient and safe snake venom fibrinolytic enzymes can be obtained by molecular biological methods and genetic engineering techniques. For more effective treatment of thrombotic disease has a very important significance. In this experiment, we obtained the gene fragment of venom fibrinolytic enzyme from pMD18T-FLE I clone vector by double enzyme digestion technique, and then subcloned it into eukaryotic expression vector pFASTBACHTa. After transformation, the recombinant eukaryotic expression plasmid pFASTBACHTa-FLE was obtained by screening and identifying the recombinant eukaryotic expression plasmid pFASTBACHTa-FLE. The purified recombinant plasmid (about 20 渭 g) was injected into the mouse body for 5 seconds through the caudal vein of the mouse. After 8 hours of transient expression, the liver tissue of the mice was taken to prepare the experimental sample. SDS-PAGE analysis showed that the recombinant protein was expressed in mouse liver, and its molecular weight was about 30kDa.After the detection of Western blot, a specific hybridization band was obtained at 30kDa, indicating that the recombinant protein expressed in mouse liver was FLE protein. The results of immunohistochemistry showed that the recombinant FLE protein was highly expressed in the liver of mice. The results of fibrinolytic assay showed that the recombinant FLE protein expressed in mouse liver had high fibrinolytic activity, and its activity was dose-dependent and time-dependent. The recombinant protein of fibrinolytic enzyme from Agkistrodon halys venom was successfully expressed in eukaryotic expression system, which laid a solid foundation for further study on the biological characteristics and application of fibrinolytic enzyme in snake venom.
【学位授予单位】：哈尔滨师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346;Q78

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