可口革囊星虫纤溶酶基因的表达及优化
发布时间：2018-07-29 08:50
【摘要】：血栓性疾病对人类生活的影响越来越大,严重危害着人类的健康,开发新型有效的溶栓试剂迫在眉睫。而纤溶酶是溶栓药物的主要来源。关于溶栓药物的开发已经历了四代,各有其优缺点。海洋源纤溶酶的来源非常广,包括动物、植物、微生物均可见报道。本实验室前期从海洋星虫-可口革囊星虫肠道中提取得到一种具有纤溶活性较强的酶,并且测得其N端的氨基酸序列,结合5’RACE和3’RACE技术得到了两条纤溶酶序列,分别命名为PFE-1和PFE-2。本课题就是对PFE-2进行外源的表达。成功构建了原核表达载体pET-22b(+)-PFE-2,乳糖诱导表达,但是重组蛋白无活性;随后又构建了带组氨酸标签的表达载体pET-22b(+)-PFE-2(his)和分子伴侣共表达,虽然相较于之前,重组蛋白的可溶性表达提高了,但是依旧无活性。成功构建构建了真核表达载体pPic9-PFE-2和pPic9-PFE-2(his),随即电转于毕赤酵母GS115之后,筛选出了两株工程菌pPic9-PFE-2-GS115和pPic9-PFE-2(his)-GS115,甲醇诱导表达,纤维平板实验结果出现了透明圈,说明重组蛋白纤溶活性良好。所以采用真核表达系统要优于原核表达系统。研究结果说明采用真核表达系统可以表达出有纤溶活性的重组纤溶酶,活性良好,这也拓宽了海洋源的纤溶酶库,并且也寻找出了一条采用外源表达系统表达PFE-2的途径,从而为新型溶栓药物的研发提供了新的渠道。
[Abstract]:Thrombotic diseases have more and more influence on human life and seriously harm human health. It is urgent to develop new effective thrombolytic agents. Fibrinolytic enzyme is the main source of thrombolytic drugs. The development of thrombolytic drugs has gone through four generations, each has its own advantages and disadvantages. The sources of marine plasmin are very wide, including animals, plants and microbes. An enzyme with strong fibrinolytic activity was obtained from the intestinal tract of marine mammary mammoth, and its N-terminal amino acid sequence was determined. Two plasminolytic enzyme sequences were obtained by 5'RACE and 3'RACE techniques. They were named PFE-1 and PFE-2respectively. This subject is the exogenous expression of PFE-2. The prokaryotic expression vector pET-22b () -PFE-2 was successfully constructed, which was induced by lactose, but the recombinant protein was not active. Subsequently, the expression vector pET-22b () -PFE-2 (his) with histidine label was constructed, which was coexpressed with the molecular chaperone, although compared with before, Soluble expression of recombinant protein increased, but remained inactive. The eukaryotic expression vectors pPic9-PFE-2 and pPic9-PFE-2 (his), were successfully constructed and electroporated into Pichia pastoris GS115. Two engineering strains, pPic9-PFE-2-GS115 and pPic9-PFE-2 (his) GS115, were screened out. The methanol induced expression was induced by methanol. The results of fiber plate experiment showed that the recombinant protein had good fibrinolytic activity. Therefore, the eukaryotic expression system is superior to the prokaryotic expression system. The results showed that the recombinant plasminase with fibrinolytic activity could be expressed by eukaryotic expression system, and its activity was good, which also widened the plasminogen library of marine source and found a way to express PFE-2 with exogenous expression system. It provides a new channel for the research and development of new thrombolytic drugs.
【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78;Q55
，
本文编号：2152146
[Abstract]:Thrombotic diseases have more and more influence on human life and seriously harm human health. It is urgent to develop new effective thrombolytic agents. Fibrinolytic enzyme is the main source of thrombolytic drugs. The development of thrombolytic drugs has gone through four generations, each has its own advantages and disadvantages. The sources of marine plasmin are very wide, including animals, plants and microbes. An enzyme with strong fibrinolytic activity was obtained from the intestinal tract of marine mammary mammoth, and its N-terminal amino acid sequence was determined. Two plasminolytic enzyme sequences were obtained by 5'RACE and 3'RACE techniques. They were named PFE-1 and PFE-2respectively. This subject is the exogenous expression of PFE-2. The prokaryotic expression vector pET-22b () -PFE-2 was successfully constructed, which was induced by lactose, but the recombinant protein was not active. Subsequently, the expression vector pET-22b () -PFE-2 (his) with histidine label was constructed, which was coexpressed with the molecular chaperone, although compared with before, Soluble expression of recombinant protein increased, but remained inactive. The eukaryotic expression vectors pPic9-PFE-2 and pPic9-PFE-2 (his), were successfully constructed and electroporated into Pichia pastoris GS115. Two engineering strains, pPic9-PFE-2-GS115 and pPic9-PFE-2 (his) GS115, were screened out. The methanol induced expression was induced by methanol. The results of fiber plate experiment showed that the recombinant protein had good fibrinolytic activity. Therefore, the eukaryotic expression system is superior to the prokaryotic expression system. The results showed that the recombinant plasminase with fibrinolytic activity could be expressed by eukaryotic expression system, and its activity was good, which also widened the plasminogen library of marine source and found a way to express PFE-2 with exogenous expression system. It provides a new channel for the research and development of new thrombolytic drugs.
【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78;Q55
，
本文编号：2152146
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2152146.html
最近更新
教材专著
