本文選題：中介蝮 + 纖溶酶��； 參考：《哈爾濱師范大學(xué)》2011年碩士論文

【摘要】：血栓性疾病是目前危害人類健康的首要疾病之一,而利用溶栓類藥物治療血栓性疾病被認(rèn)為是最為有效的方法。蛇毒纖維蛋白(原)溶解酶(fibrin(ogen)olytic enzyme,FLE簡稱纖溶酶)是一種新型的強(qiáng)力溶栓劑,能直接溶解纖維蛋白或纖維蛋白原。利用蛋白質(zhì)分離技術(shù)從蛇毒中高度純化而得的蛇毒纖溶酶在心腦血管疾病的治療方面具有潛在的應(yīng)用價(jià)值。然而天然蛇毒資源有限,不易分離、保存和運(yùn)輸,因此通過分子生物學(xué)的方法和基因工程技術(shù)獲得高效、安全的蛇毒纖溶酶,對于更有效地治療血栓性疾病具有十分重要的意義。 本實(shí)驗(yàn)利用雙酶切技術(shù)從pMD18T-FLE I克隆載體上獲得中介蝮(Gloydius intermedius)蛇毒纖溶酶基因片段,再將其亞克隆到真核表達(dá)載體pFASTBACHTa上,經(jīng)轉(zhuǎn)化、篩選和鑒定獲得重組真核表達(dá)質(zhì)粒pFASTBACHTa-FLE。將純化后的重組質(zhì)粒(約20μg)經(jīng)小鼠尾靜脈快速(5s內(nèi))注入小鼠體內(nèi),進(jìn)行瞬時(shí)表達(dá),8h后取小鼠肝臟組織制備實(shí)驗(yàn)樣本。 經(jīng)SDS-PAGE分析顯示,在小鼠肝臟中有重組蛋白表達(dá),其分子量大小約為30kDa;再經(jīng)Western blot檢測,在30kDa處得到一條特異性的雜交條帶,表明小鼠肝臟中表達(dá)的重組蛋白為FLE蛋白。免疫組織化學(xué)方法檢測結(jié)果顯示,重組FLE蛋白在小鼠肝臟組織中大量表達(dá)。纖維蛋白平板法鑒定纖溶活性結(jié)果表明,小鼠肝臟中表達(dá)的重組FLE蛋白具有較高的纖溶活性,且其活性呈現(xiàn)出劑量相關(guān)性和時(shí)間依賴性。 本實(shí)驗(yàn)在真核表達(dá)體系中成功表達(dá)出中介蝮蛇毒纖溶酶重組蛋白,為進(jìn)一步對蛇毒纖溶酶的生物學(xué)特性及其應(yīng)用的研究奠定了堅(jiān)實(shí)的基礎(chǔ)。
[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.
【學(xué)位授予單位】：哈爾濱師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346;Q78

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