纤溶酶QK在毕赤酵母内的表达和发酵研究以及针对重组QK夹心酶联免疫吸附法的研发与运用

发布时间：2018-02-15 13:08

本文关键词： 纤溶酶QK 毕赤酵母疏水层析酶联免疫吸附试验药代动力学　出处：《武汉大学》2017年博士论文　论文类型：学位论文

【摘要】：在生活水平日益提高的现在,血栓类疾病已经成为对人类生命健康威胁最严重的疾病之一。血栓类疾病具有极高的致死致残率,且发生形式多样,对血栓类疾病的预防和治疗越来越引起人们的关注。目前针对血栓类疾病的治疗主要主要有手术和药物治疗两种。本文主要研究一种具有良好应用前景的高纤溶活性的溶栓酶。纤溶酶QK是一种来从天然菌株Bacillus subtilis QK02发酵产物中分离得到的丝氨酸蛋白酶。其与纳豆激酶高度同源,在体内和体外都具有高纤溶活性,并且能抵抗胰蛋白酶分解,可以通过口服发挥抗血栓作用,同时其副作用小,安全性高,使其具有良好的潜力应用于血栓类疾病的预防和治疗。本文通过毕赤酵母表达系统成功表达重组QK,并对其大规模发酵、纯化和冻干体系进行了研究,研发了针对重组QK的双抗夹心ELISA检测方法并对其药代动力学进行了初步研究,研究结果分为以下三个部分:1、针对毕赤酵母偏爱密码子对QK基因序列进行了优化,并将优化后的基因序列连接到穿梭质粒pPPICZαA上,转化入GS115中,成功获得了重组表达菌株GS115/pPPICZαA-QK。构建的菌株能高效分泌表达具有良好纤溶活性的重组QK蛋白。2、在摇瓶发酵的基础上确立了 GS115/pPPICZαA-QK重组菌株大规模发酵条件。甘油诱导菌体扩张和甘油补料阶段,控制温度在28℃,控制pH在5.0左右,溶氧不低于20%。在甘油补料阶段,控制甘油补料时间,使细胞湿重达到190-200g/L。甲醇诱导表达阶段开始后,发酵温度下降到26℃,继续控制pH在5.0左右,整个诱导过程控制发酵中溶氧不低于20%。在甲醇诱导发酵92h后,重组QK酶活最高可达到112,000IU/mL,发酵液中总蛋白浓度可达7.63 g/L。发酵液经过疏水层析(HIC)和凝胶过滤层析(GFC),后,91.73%的酶活性得到回收,同时重组QK蛋白纯度达到95%以上。此外对多种冻干保护剂在重组QK冻干过程中的保护效果进行了研究,5%的海藻糖和1%的脱脂奶粉的保护效果最好,可保持重组QK经过冻干后依然有94.2%和96.2%的活性,而在保存过程中海藻糖可以使重组QK冻干粉末在室温下一周活性基本无变化。3、通过对兔和小鼠使用纯化的重组QK蛋白进行免疫,获取了特异性针对重组QK蛋白的多克隆抗体和4株单克隆抗体,并在此基础上对抗体配对筛选,构建了以单抗为包被抗体,多抗为检测抗体的双抗夹心ELISA检测方法。通过对ELISA方法进行优化,成功建立了具有较高的灵敏度,高度的准确度、精密度,良好的重复性的针对重组QK蛋白的双抗夹心ELISA方法,检测灵敏度可达1.207ng/mL,组间和组内变异系数小于5%,回收率在95%-105%之间。成功运用ELISA方法初步分析了重组QK在大鼠中静脉注射药代动力学,确定其为二室房室模型,并获得了其主要动力学参数,T为后续更进一步的研究打下了基础。本论文中利用毕赤酵母表达系统表达分泌表达了具有纤溶活性的重组QK,并为其工业化大规模生产提供了参考方法。同时其检测方法和药代动力学的研究,也为溶栓酶QK的进一步推广和应用,以及其代谢机理的阐明打下乐基础。
[Abstract]:Improvement in the standard of living now, thrombotic diseases have become one of the most serious threats to human health diseases. Thrombotic diseases with high morbidity and mortality, occurrence forms, prevention and treatment of thrombotic diseases has attracted people's attention. The current treatment for thrombotic diseases mainly there are two kinds of surgery and drug treatment. This paper mainly studies a promising high fibrinolytic activity of the fibrinolytic enzyme. Enzyme QK is a separate serine protease obtained from natural strain Bacillus subtilis QK02 fermentation products. It is highly homologous with nattokinase, both in vitro and in vivo with high fiber fibrinolytic activity, and can resist trypsin decomposition, can play the antithrombotic effect by oral administration, while the small side effect, high safety, so it has good potential for application in thromboembolic disease The prevention and treatment of disease. The successful expression of recombinant QK in Pichia pastoris, and its large scale fermentation, purification and freeze drying system were studied, developed the double antibody sandwich ELISA method to detect the recombinant QK and its pharmacokinetics were studied. The research results are divided into the following three part: 1, according to Pichia pastoris preferred codons optimized for QK gene sequence and gene sequence optimization after connected to the shuttle plasmid pPPICZ was transformed into a A, GS115, successfully obtained the recombinant strains GS115/pPPICZ alpha A-QK. strains can construct efficient secretory expression has good fibrinolytic activity of recombinant QK protein.2, on the basis of shake flask fermentation was established on a large scale fermentation conditions of GS115/pPPICZ alpha A-QK recombinant strain. Glycerol induced cell expansion and glycerol feeding stage, temperature controlled at 28 DEG C, pH control in about 5, do not Below the 20%. feeding stage in glycerol, glycerol feeding time, the cell wet weight reached 190-200g/L. methanol to induce expression stage, fermentation temperature dropped to 26 degrees Celsius, continue to control the pH at around 5, the whole process control of dissolved oxygen in fermentation by not less than 20%. by fermentation of 92h in methanol, the recombinant QK enzyme activity can be the highest at 112000IU/mL, the total protein concentration is up to 7.63 g/L. in the fermentation liquid fermentation broth by hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC), after 91.73% of the enzyme activity was recovered, and the recombinant QK protein reached a purity of more than 95%. In addition to a variety of cryoprotector in recombinant QK freeze protection effect in the process of doing is studied, the best protective effect of 5% trehalose and 1% skim milk powder, can be maintained after freeze-dried recombinant QK still has 94.2% and 96.2% activity, and in the preservation process of trehalose can make the recombinant QK freeze-dried powder in the room Temperature next week basically no change of.3 activity, immunization of rabbit and mice by using purified recombinant QK protein, the specificity of the polyclonal antibody against the recombinant QK protein and 4 monoclonal antibodies, and on the basis of antibody screening, constructed by monoclonal antibody as coating antibody and polyclonal antibody as testing antibody the sandwich ELISA method. The ELISA method was optimized, was successfully established with high sensitivity, high accuracy, precision, double antibody sandwich ELISA method with good reproducibility for the recombinant QK protein, the detection sensitivity can reach 1.207ng/mL, inter group and intra group variation coefficient is less than 5%, recovery the success rate of 95%-105%. A preliminary analysis of the recombinant QK vein in rats of phaemacokinetics using ELISA method to determine the room two compartment model, and the kinetic parameters obtained for further follow-up T The study of the foundation. The expression of secretory expression with fibrinolytic activity of recombinant QK in the Pichia pastoris expression system in this thesis, and the reference method for the large-scale industrialized production. At the same time the study of detection method and pharmacokinetics, but also for the further promotion and application of thrombolytic enzyme QK, and the the metabolic mechanism lay music foundation.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ925.2;Q78

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