可口革囊星蟲纖溶酶基因的表達及優(yōu)化
發(fā)布時間：2018-07-29 08:50
【摘要】：血栓性疾病對人類生活的影響越來越大,嚴重危害著人類的健康,開發(fā)新型有效的溶栓試劑迫在眉睫。而纖溶酶是溶栓藥物的主要來源。關于溶栓藥物的開發(fā)已經(jīng)歷了四代,各有其優(yōu)缺點。海洋源纖溶酶的來源非常廣,包括動物、植物、微生物均可見報道。本實驗室前期從海洋星蟲-可口革囊星蟲腸道中提取得到一種具有纖溶活性較強的酶,并且測得其N端的氨基酸序列,結合5’RACE和3’RACE技術得到了兩條纖溶酶序列,分別命名為PFE-1和PFE-2。本課題就是對PFE-2進行外源的表達。成功構建了原核表達載體pET-22b(+)-PFE-2,乳糖誘導表達,但是重組蛋白無活性;隨后又構建了帶組氨酸標簽的表達載體pET-22b(+)-PFE-2(his)和分子伴侶共表達,雖然相較于之前,重組蛋白的可溶性表達提高了,但是依舊無活性。成功構建構建了真核表達載體pPic9-PFE-2和pPic9-PFE-2(his),隨即電轉于畢赤酵母GS115之后,篩選出了兩株工程菌pPic9-PFE-2-GS115和pPic9-PFE-2(his)-GS115,甲醇誘導表達,纖維平板實驗結果出現(xiàn)了透明圈,說明重組蛋白纖溶活性良好。所以采用真核表達系統(tǒng)要優(yōu)于原核表達系統(tǒng)。研究結果說明采用真核表達系統(tǒng)可以表達出有纖溶活性的重組纖溶酶,活性良好,這也拓寬了海洋源的纖溶酶庫,并且也尋找出了一條采用外源表達系統(tǒng)表達PFE-2的途徑,從而為新型溶栓藥物的研發(fā)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.
【學位授予單位】：華僑大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q78;Q55
，
本文編號：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.
【學位授予單位】：華僑大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q78;Q55
，
本文編號：2152146
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