重组人组织因子途径抑制因子在毕赤酵母中的表达、纯化及其抗血清的制备

发布时间：2018-11-01 15:42

【摘要】：体内凝血过程主要有两种：内源性凝血途径和外源性凝血途径,内源性凝血途径是由凝血因子Ⅻ启动,外源性凝血途径起始于组织因子(Tissue Factor)暴露于血液。外源性凝血途径又称组织因子途径由组织因子作为启动因子。组织因子途径抑制因子(TFPI)是具有库尼兹结构的丝氨酸蛋白酶抑制剂,是外源性凝血途径的主要抑制因子之一,在动脉粥样硬化、弥散性血管内凝血(DIC)、血管再狭窄、血栓形成、肿瘤、炎症及感染性休克等疾病中具有潜在的治疗及应用前景。因此制备重组人组织因子途径抑制因子具有一定的临床价值。在众多表达系统中,利用酿酒酵母表达体系表达TFPI产量较低,发酵培养时产量约0.02 mg/L；昆虫细胞表达系统表达TFPI重组蛋白,其表达量只有0.21 mg/L；哺乳动物细胞表达体系所获得的重组蛋白,产量达2-6 mg/L,但成本昂贵,操作繁琐,周期长。本研究采用巴斯德毕赤酵母表达重组人组织因子途径抑制因子,于72小时表达量最高达到7.623lmg/L,重组TFPI对FXa的抑制率也在72小时最高为0.8918。不仅大大提高了重组蛋白的表达量,而且节约了生产成本。应用超滤透析,阴离子交换、凝胶过滤层析法分离纯化重组蛋白,纯度达90%,收率达55%,重组TFPI对FXa的抑制率为0.5341。本研究探索出低成本,高纯度,适用于工业化规模生产的纯化技术。将纯化的重组蛋白与免疫佐剂结合免疫日本大耳白兔并成功制备效价为1：32的多克隆抗体,为重组人组织因子途径抑制因子的功能研究及在相关疾病防治中的应用提供了良好的实验室基础。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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