本文關(guān)鍵詞： 纖溶酶QK 畢赤酵母 疏水層析 酶聯(lián)免疫吸附試驗(yàn) 藥代動(dòng)力學(xué)　出處：《武漢大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：在生活水平日益提高的現(xiàn)在,血栓類疾病已經(jīng)成為對人類生命健康威脅最嚴(yán)重的疾病之一。血栓類疾病具有極高的致死致殘率,且發(fā)生形式多樣,對血栓類疾病的預(yù)防和治療越來越引起人們的關(guān)注。目前針對血栓類疾病的治療主要主要有手術(shù)和藥物治療兩種。本文主要研究一種具有良好應(yīng)用前景的高纖溶活性的溶栓酶。纖溶酶QK是一種來從天然菌株Bacillus subtilis QK02發(fā)酵產(chǎn)物中分離得到的絲氨酸蛋白酶。其與納豆激酶高度同源,在體內(nèi)和體外都具有高纖溶活性,并且能抵抗胰蛋白酶分解,可以通過口服發(fā)揮抗血栓作用,同時(shí)其副作用小,安全性高,使其具有良好的潛力應(yīng)用于血栓類疾病的預(yù)防和治療。本文通過畢赤酵母表達(dá)系統(tǒng)成功表達(dá)重組QK,并對其大規(guī)模發(fā)酵、純化和凍干體系進(jìn)行了研究,研發(fā)了針對重組QK的雙抗夾心ELISA檢測方法并對其藥代動(dòng)力學(xué)進(jìn)行了初步研究,研究結(jié)果分為以下三個(gè)部分:1、針對畢赤酵母偏愛密碼子對QK基因序列進(jìn)行了優(yōu)化,并將優(yōu)化后的基因序列連接到穿梭質(zhì)粒pPPICZαA上,轉(zhuǎn)化入GS115中,成功獲得了重組表達(dá)菌株GS115/pPPICZαA-QK。構(gòu)建的菌株能高效分泌表達(dá)具有良好纖溶活性的重組QK蛋白。2、在搖瓶發(fā)酵的基礎(chǔ)上確立了 GS115/pPPICZαA-QK重組菌株大規(guī)模發(fā)酵條件。甘油誘導(dǎo)菌體擴(kuò)張和甘油補(bǔ)料階段,控制溫度在28℃,控制pH在5.0左右,溶氧不低于20%。在甘油補(bǔ)料階段,控制甘油補(bǔ)料時(shí)間,使細(xì)胞濕重達(dá)到190-200g/L。甲醇誘導(dǎo)表達(dá)階段開始后,發(fā)酵溫度下降到26℃,繼續(xù)控制pH在5.0左右,整個(gè)誘導(dǎo)過程控制發(fā)酵中溶氧不低于20%。在甲醇誘導(dǎo)發(fā)酵92h后,重組QK酶活最高可達(dá)到112,000IU/mL,發(fā)酵液中總蛋白濃度可達(dá)7.63 g/L。發(fā)酵液經(jīng)過疏水層析(HIC)和凝膠過濾層析(GFC),后,91.73%的酶活性得到回收,同時(shí)重組QK蛋白純度達(dá)到95%以上。此外對多種凍干保護(hù)劑在重組QK凍干過程中的保護(hù)效果進(jìn)行了研究,5%的海藻糖和1%的脫脂奶粉的保護(hù)效果最好,可保持重組QK經(jīng)過凍干后依然有94.2%和96.2%的活性,而在保存過程中海藻糖可以使重組QK凍干粉末在室溫下一周活性基本無變化。3、通過對兔和小鼠使用純化的重組QK蛋白進(jìn)行免疫,獲取了特異性針對重組QK蛋白的多克隆抗體和4株單克隆抗體,并在此基礎(chǔ)上對抗體配對篩選,構(gòu)建了以單抗為包被抗體,多抗為檢測抗體的雙抗夾心ELISA檢測方法。通過對ELISA方法進(jìn)行優(yōu)化,成功建立了具有較高的靈敏度,高度的準(zhǔn)確度、精密度,良好的重復(fù)性的針對重組QK蛋白的雙抗夾心ELISA方法,檢測靈敏度可達(dá)1.207ng/mL,組間和組內(nèi)變異系數(shù)小于5%,回收率在95%-105%之間。成功運(yùn)用ELISA方法初步分析了重組QK在大鼠中靜脈注射藥代動(dòng)力學(xué),確定其為二室房室模型,并獲得了其主要?jiǎng)恿W(xué)參數(shù),T為后續(xù)更進(jìn)一步的研究打下了基礎(chǔ)。本論文中利用畢赤酵母表達(dá)系統(tǒng)表達(dá)分泌表達(dá)了具有纖溶活性的重組QK,并為其工業(yè)化大規(guī)模生產(chǎn)提供了參考方法。同時(shí)其檢測方法和藥代動(dòng)力學(xué)的研究,也為溶栓酶QK的進(jìn)一步推廣和應(yīng)用,以及其代謝機(jī)理的闡明打下樂基礎(chǔ)。
[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.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TQ925.2;Q78

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