重組人組織因子途徑抑制因子在畢赤酵母中的表達、純化及其抗血清的制備
發(fā)布時間：2018-11-01 15:42
【摘要】：體內凝血過程主要有兩種：內源性凝血途徑和外源性凝血途徑,內源性凝血途徑是由凝血因子Ⅻ啟動,外源性凝血途徑起始于組織因子(Tissue Factor)暴露于血液。外源性凝血途徑又稱組織因子途徑由組織因子作為啟動因子。組織因子途徑抑制因子(TFPI)是具有庫尼茲結構的絲氨酸蛋白酶抑制劑,是外源性凝血途徑的主要抑制因子之一,在動脈粥樣硬化、彌散性血管內凝血(DIC)、血管再狹窄、血栓形成、腫瘤、炎癥及感染性休克等疾病中具有潛在的治療及應用前景。因此制備重組人組織因子途徑抑制因子具有一定的臨床價值。 在眾多表達系統(tǒng)中,利用釀酒酵母表達體系表達TFPI產(chǎn)量較低,發(fā)酵培養(yǎng)時產(chǎn)量約0.02 mg/L；昆蟲細胞表達系統(tǒng)表達TFPI重組蛋白,其表達量只有0.21 mg/L；哺乳動物細胞表達體系所獲得的重組蛋白,產(chǎn)量達2-6 mg/L,但成本昂貴,操作繁瑣,周期長。本研究采用巴斯德畢赤酵母表達重組人組織因子途徑抑制因子,于72小時表達量最高達到7.623lmg/L,重組TFPI對FXa的抑制率也在72小時最高為0.8918。不僅大大提高了重組蛋白的表達量,而且節(jié)約了生產(chǎn)成本。應用超濾透析,陰離子交換、凝膠過濾層析法分離純化重組蛋白,純度達90%,收率達55%,重組TFPI對FXa的抑制率為0.5341。本研究探索出低成本,高純度,適用于工業(yè)化規(guī)模生產(chǎn)的純化技術。將純化的重組蛋白與免疫佐劑結合免疫日本大耳白兔并成功制備效價為1：32的多克隆抗體,為重組人組織因子途徑抑制因子的功能研究及在相關疾病防治中的應用提供了良好的實驗室基礎。
[Abstract]:There are two main coagulation processes in vivo: endogenous coagulation pathway and exogenous coagulation pathway. Endogenous coagulation pathway is initiated by coagulation factor XII, and exogenous coagulation pathway starts from tissue factor (Tissue Factor) exposure to blood. Exogenous coagulation pathway, also known as tissue factor pathway, is initiated by tissue factor. Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor with Kuniz structure and is one of the main inhibitors of exogenous coagulation pathway. It is found in atherosclerosis and disseminated intravascular coagulation (DIC),) vascular restenosis. Thrombosis, tumor, inflammation and septic shock and other diseases have potential treatment and application prospects. Therefore, the preparation of recombinant human tissue factor pathway inhibitor has certain clinical value. In many expression systems, the expression of TFPI by Saccharomyces cerevisiae was relatively low, and about 0.02 mg/L; insect cell expression system expressed TFPI recombinant protein in fermentation culture, and its expression amount was only 0.21 mg/L;. The recombinant protein obtained from mammalian cell expression system can produce 2-6 mg/L, but its cost is high, the operation is cumbersome and the cycle is long. In this study, Pichia pastoris was used to express recombinant human tissue factor pathway inhibitor. The highest expression level of recombinant human tissue factor pathway was 7.623 mg / L at 72 hours, and the inhibition rate of recombinant TFPI on FXa was 0.8918 at 72 hours. It not only improves the expression of recombinant protein, but also saves the production cost. The recombinant protein was purified by ultrafiltration dialysis, anion exchange and gel filtration chromatography. The purity and yield of recombinant TFPI were 90 and 55, respectively. The inhibition rate of recombinant TFPI on FXa was 0.5341. In this study, a low-cost, high-purity purification technology suitable for industrial scale production was explored. The purified recombinant protein was combined with the immune adjuvant to immunize Japanese white rabbits and the polyclonal antibody with the titer of 1:32 was prepared successfully. It provides a good laboratory basis for the functional study of recombinant human tissue factor pathway inhibitor and its application in the prevention and treatment of related diseases.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R346
[Abstract]:There are two main coagulation processes in vivo: endogenous coagulation pathway and exogenous coagulation pathway. Endogenous coagulation pathway is initiated by coagulation factor XII, and exogenous coagulation pathway starts from tissue factor (Tissue Factor) exposure to blood. Exogenous coagulation pathway, also known as tissue factor pathway, is initiated by tissue factor. Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor with Kuniz structure and is one of the main inhibitors of exogenous coagulation pathway. It is found in atherosclerosis and disseminated intravascular coagulation (DIC),) vascular restenosis. Thrombosis, tumor, inflammation and septic shock and other diseases have potential treatment and application prospects. Therefore, the preparation of recombinant human tissue factor pathway inhibitor has certain clinical value. In many expression systems, the expression of TFPI by Saccharomyces cerevisiae was relatively low, and about 0.02 mg/L; insect cell expression system expressed TFPI recombinant protein in fermentation culture, and its expression amount was only 0.21 mg/L;. The recombinant protein obtained from mammalian cell expression system can produce 2-6 mg/L, but its cost is high, the operation is cumbersome and the cycle is long. In this study, Pichia pastoris was used to express recombinant human tissue factor pathway inhibitor. The highest expression level of recombinant human tissue factor pathway was 7.623 mg / L at 72 hours, and the inhibition rate of recombinant TFPI on FXa was 0.8918 at 72 hours. It not only improves the expression of recombinant protein, but also saves the production cost. The recombinant protein was purified by ultrafiltration dialysis, anion exchange and gel filtration chromatography. The purity and yield of recombinant TFPI were 90 and 55, respectively. The inhibition rate of recombinant TFPI on FXa was 0.5341. In this study, a low-cost, high-purity purification technology suitable for industrial scale production was explored. The purified recombinant protein was combined with the immune adjuvant to immunize Japanese white rabbits and the polyclonal antibody with the titer of 1:32 was prepared successfully. It provides a good laboratory basis for the functional study of recombinant human tissue factor pathway inhibitor and its application in the prevention and treatment of related diseases.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R346
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