WO2018176683A1 - 一种制备尿激酶的方法 - Google Patents

一种制备尿激酶的方法 Download PDF

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WO2018176683A1
WO2018176683A1 PCT/CN2017/092581 CN2017092581W WO2018176683A1 WO 2018176683 A1 WO2018176683 A1 WO 2018176683A1 CN 2017092581 W CN2017092581 W CN 2017092581W WO 2018176683 A1 WO2018176683 A1 WO 2018176683A1
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urokinase
cells
drosophila
puromycin
gene
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王海瑶
靳海宁
熊祖应
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深圳市伯劳特生物制品有限公司
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21031—Urokinase (3.4.21.31)
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/103—Plasmid DNA for invertebrates
    • C12N2800/105—Plasmid DNA for invertebrates for insects

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  • the invention belongs to the field of biotechnology, and in particular relates to a method for preparing urokinase.
  • Venous thrombosis is one of the major diseases that endanger human health. In China, there are more than 10 million cases of cardiovascular and cerebrovascular diseases, and the cost of treatment is over 10 billion yuan. More than one million patients die of thrombotic diseases each year. Currently, most of the clinical use of thrombolytic drugs for venous thrombosis, the sales of thrombolytic drugs market has reached more than 100 billion US dollars.
  • Urokinase has been used as a thrombolytic drug for the treatment of venous thrombosis for decades.
  • the urokinase currently used for clinical treatment is directly extracted from urine, and the second is the expression of recombinant urokinase in E. coli.
  • Urokinase which is widely used in China, is extracted from fresh human urine. It has limited sources, severely inhibits productivity, and has low inefficiency and low purity.
  • the FDA requires that organs of tissue-derived drugs must be examined for the pathogens of infectious diseases (HBV, HIV, etc.) and may be phased out.
  • Urokinase which is currently widely used in the treatment of venous thrombosis in the European and American markets, is isolated and purified in E. coli.
  • the advantage is that it can be produced in large quantities without the pathogen of infectious diseases, but E. coli is a prokaryotic cell, and the expressed protein is not glycosylated, and is structurally different from the human urokinase, and has its own function and its own urokinase. the difference.
  • many steps are required to obtain a relatively purified urokinase.
  • the present invention provides a method for preparing urokinase for solving the problem of inefficient and low purity extraction of urokinase from urine, and urokinase-free glycosylation extracted from Escherichia coli, The problem of purification is difficult.
  • the invention provides a method for preparing urokinase, comprising:
  • step b) transfecting the cloning plasmid obtained in step a) into Drosophila S2 cells to obtain Drosophila S2 cells expressing urokinase;
  • urokinase-expressing Drosophila S2 cells obtained in the step b) are cultured, and the cultured cells are treated to obtain urokinase.
  • the gene in step a) is a human urokinase whole gene cDNA, and the cloning sites are NcoI and XhoI.
  • the human urokinase whole gene cDNA end is added with a cDNA which can express 6 histidines.
  • the carrier in step a) is pMT/Bip/V5-A.
  • the N-terminus of the plasmid in step a) contains a Bip-signal sequence, and the plasmid further contains an ampicillin and a puromycin resistance gene.
  • the treatment is specifically to remove the cell culture supernatant, add the expression solution, shake the culture flask, collect the supernatant, pass the supernatant through the column, elute, and collect urokinase.
  • the expression solution is a serum-free medium containing 500 ⁇ M copper sulfate.
  • the shaking condition time is 72 h and the rotation speed is 225 r/min.
  • the column is a nickel column.
  • the eluent is an imidazole-containing TBS having a pH of 7.2.
  • the column is preferably washed with TBS containing 10-25 mM imidazole, pH 7.2, then the protein is eluted with TBS buffer containing 250 mM imidazole, pH 7.2, and the eluate is collected.
  • the urokinase-expressing Drosophila S2 cells obtained in the step b) were screened to obtain stable Drosophila S2 cells which highly express urokinase.
  • the screening culture is a Snyder broth containing 10% fetal calf serum.
  • the culture solution is supplemented with ampicillin and puromycin, the concentration of ampicillin is 50 ug/ml, and the concentration of puromycin is 2 ⁇ g/ml.
  • the urokinase obtained in the step c) is dialyzed and purified to obtain a high-purity urokinase;
  • the dialysis bag has a molecular weight cut-off of 10 KD
  • the dialysate is TBS having a pH of 7.2
  • the dialysis time is 24 h.
  • the method expresses high yield and easy purification of urokinase, and the expressed urokinase protein is glycosylated, and has the same structure and function as the human body's self-expressed urokinase.
  • the method is not limited by natural resources, and can produce unlimited amounts of urokinase with the same structure according to market demand, with low input, high output, good thrombolytic effect, and no toxic and side effects.
  • Example 1 and 2 are plasmid information in Example 1;
  • Example 3 is a graph showing the purity analysis of urokinase obtained in Example 2.
  • the left side is an electrophoresis pattern before purification, and the right side is an electrophoresis pattern after purification;
  • FIG. 4 is a graph showing the results of binding of urokinase to its receptor using the surface plasmon resonance method in Example 3, wherein a is an interaction diagram of uPA and uPAR at different concentrations, and FIG. b is a negative control diagram;
  • Figure 5 is a graph showing SDS/PAGE results of urokinase-activated plasminogen producing plasmin in Example 4, wherein lane "+” is a SDS/PAGE result map of urokinase added, and lane "-" is a urokinase not added.
  • the SDS/PAGE results are shown in the figure, complex is complex, plasminogen is plasminogen, and anti-plasmin is anti-plasmin.
  • Cloning human urokinase whole gene cDNA into NcoI and XhoI sites can be Drosophila S2 cells were expressed in the vector pMT/Bip/V5-A, and a cDNA encoding 6 histidine (CAT CAT CAC CAT CAC CAT) was added to the end of the urokinase cDNA for purification.
  • the N-terminus of the plasmid contains a Bip-signal sequence, so that the expressed urokinase can be exported to the cells for purification; the ampicillin and puromycin resistance genes are included for easy screening.
  • the plasmid information is shown in Figures 1 and 2.
  • the plasmid obtained in Example 1 was transfected into Drosophila S2 cells by placing Drosophila S2 cells in a 70-90% transfection mixture and diluting Reagent's transfection reagent Medium (1:1 dilution), dilute the plasmid Medium. Add the diluted plasmid to the diluted The 2000 reagent was then incubated for 45 min and the mixture was added to the cells.
  • Drosophila S2 cells expressing urokinase were subcultured and expanded. After one week of expansion, the cells reached 1 L at a concentration of 2 ⁇ 10 6 /ml. The cell culture supernatant was removed and the cells were placed in 1 L of 500 ⁇ M copper sulfate. In the expression solution of the serum-free medium, the flask was shaken at 225 r/min for 72 h. Thereafter, the supernatant containing urokinase was collected by centrifugation, and the supernatant containing urokinase was slowly passed through a column equipped with an effective ni-NTA resin to pass the urokinase through the end.
  • the colloidal binding of the six histidine and the specific histidine peptide chains remains in the column, and other impurities cannot be bound to remain in the liquid because there are no six histidines.
  • the column was washed with 1 liter of washing solution (Tris-buffer saline (TBS) pH 7.2, containing 10-25 mM imidazole), and the low concentration of imidazole was washed away with non-specific protein bound to the micelle, and then with a high concentration of imidazole (250 mM).
  • TBS Tris-buffer saline
  • the eluate was dialyzed against a dialysate (Tris-buffer saline (TBS) pH 7.2) for 24 h, and the dialyzed eluate was collected to obtain purified urokinase.
  • the purity of the obtained urokinase was analyzed by SDS gel, and the results are shown in Fig. 3.
  • uPA The interaction between uPA obtained in Example 2 and its receptor uPAR was analyzed by surface plasmon resonance (SPR) in BIAcore 2000 (BIAcore AB, Uppsala, Sweden).
  • SPR surface plasmon resonance
  • the uPAR was immobilized on the surface of the carboxymethyl dextran of the CM5 chip (BIAcore AB) using amine coupling according to the manufacturer's instructions.
  • uPAR was diluted to a final concentration of 2.5 ⁇ g/ml using a pH 5.5 sodium acetate buffer.
  • the uPAR was fixed to the surface in a separate flow cell at a flow rate of 10 ⁇ l/min for 7 minutes, activated in the remaining two flow cells by amine coupling and then immediately deactivated to prepare a reference surface.
  • 6 different concentrations of uPA were injected for 3 minutes followed by a 10 minute dissociation phase.
  • the surface was regenerated by injecting 1 M NaCl for 1 minute, then 1 minute with 0.02 M 6-aminocaproic acid, and a brief washing step with phosphate/saline buffer.
  • the experiment was carried out in a 0.04 M phosphate buffer (pH 7.4) containing 0.1 M NaCl at a flow rate of 30 ⁇ l/min.
  • the activity of urokinase obtained in Example 2 was analyzed using kinetics of plasminogen activation.
  • the catalyzed concentration of plasminogen was 3.0 times its estimated activity index and was incubated in the presence of 0.05 M lysine.
  • Activation was initiated by the addition of urokinase and the change in absorbance at 405 nm was followed in a spectrophotometer at 37 °C. After 1 minute, the reaction was stopped by the addition of 50% glacial acetic acid and the absorbance at 405 nm was measured relative to the reference blank.
  • the ability of anti-plasmin to form a stable complex with uPA-activated plasmin was investigated by SDS/PAGE. The test results are shown in Figure 5.
  • plasminogen by urokinase forms plasmin, plasmin and antifibrinolytic
  • the enzyme binds to form a complex, and a macromolecular complex can be seen on the SDS gel, indicating that the urokinase obtained in Example 2 can activate plasminogen to produce plasmin.

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Abstract

一种制备尿激酶的方法，该方法表达尿激酶的产量高、易纯化，表达的尿激酶蛋白糖基化和人体自身表达的尿激酶结构相似，功能相同。并且，该方法不受自然资源的限制，可根据市场需求不限量生产和人体相同结构的尿激酶，投入少、产出高，溶栓效果好，无毒副作用。

Description

一种制备尿激酶的方法
本申请要求于2017年3月29日提交中国专利局、申请号为201710197822.7、发明名称为“一种制备尿激酶的方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物技术领域，具体涉及一种制备尿激酶的方法。
背景技术
静脉血栓疾病是危害人类健康的主要疾病之一，我国每年心脑血管病例逾千万，治疗费用上百亿元。每年死于血栓性疾病病人达百万以上，目前临床上大多使用溶栓药治疗静脉血栓疾病，溶栓药市场的销售额已达1000多亿美元。
尿激酶作为溶栓药用于治疗静脉血栓已有几十年的历史。目前用于临床治疗的尿激酶一是直接从尿液中提取，二是在大肠杆菌中表达重组的尿激酶。国内广泛应用的尿激酶是从新鲜人尿液中提取的，不仅来源有限，严重抑制产能，存在低效纯度低等问题。而且FDA要求组织器官来源的药品必须检查传染性疾病的病原(HBV，HIV等)，并有逐步取消的可能。
欧美市场上目前广泛用于治疗静脉血栓的尿激酶是在大肠杆菌中表达分离纯化。其优点是可以大量生产且没有传染疾病的病原，但大肠杆菌是原核细胞，表达出的蛋白没有糖基化，和人体自身的尿激酶有结构上的不同，功能上也和自身的尿激酶有区别。并且，表达后从大肠杆菌的裂解液中提取纯化，需要许多步骤才能得到较为纯化的尿激酶。
发明内容
有鉴于此，本发明提供了一种制备尿激酶的方法，用于解决从尿中提取尿激酶存在低效、低纯度，大肠杆菌中提取的尿激酶无糖基化、 纯化困难的问题。
本发明的具体技术方案如下：
本发明提供了一种制备尿激酶的方法，包括：
a)将尿激酶基因克隆到载体中，得到克隆质粒；
b)将步骤a)得到的克隆质粒转染进Drosophila S2细胞，得到表达尿激酶的Drosophila S2细胞；
c)对步骤b)得到的表达尿激酶的Drosophila S2细胞进行培养，对培养后的细胞进行处理，得到尿激酶。
优选的，步骤a)中所述基因为人尿激酶全基因cDNA，克隆位点为NcoI和XhoI。
所述人尿激酶全基因cDNA末端加上可表达6个组氨酸的cDNA。
优选的，步骤a)中所述载体为pMT/Bip/V5-A。
优选的，步骤a)中所述质粒N-端含有Bip信号序列(Bip-signal sequence)、所述质粒还含有氨苄青霉素和嘌呤霉素抗性基因。
优选的，处理具体为除去细胞培养上清液，加入表达液，对培养瓶进行摇晃，收集上清液，将上清液过柱，洗脱，收集尿激酶。
优选的，所述表达液为含500μM硫酸铜的无血清培养液。
优选的，摇晃条件时间为72h，转速为225r/min。
优选的，所述柱为镍柱。
优选的，所述洗脱液为含咪唑的TBS，pH为7.2。
在一些实施方案中，优选用含10-25mM咪唑、pH为7.2的TBS洗柱，然后用含250mM咪唑、pH为7.2的TBS缓冲液洗脱蛋白，并收集洗脱液。
进一步的，对步骤b)得到的表达尿激酶的Drosophila S2细胞进行筛选，得到高表达尿激酶的稳定的Drosophila S2细胞。
优选的，筛选培养液为含有10％的胎牛血清的Snyder培养液。
优选的，所述培养液添加了氨苄青霉素和嘌呤霉素，氨苄青霉素浓度为50ug/ml，嘌呤霉素浓度为2μg/ml。
进一步的，将步骤c)得到的尿激酶透析进行纯化，得到高纯度的尿激酶；
优选的，透析袋的截留分子量为10KD，透析液为pH7.2的TBS，透析时间为24h。
与现有技术相比，该方法表达尿激酶的产量高、易纯化，表达的尿激酶蛋白糖基化，和人体自身表达的尿激酶结构相似，功能相同。并且，该方法不受自然资源的限制，可根据市场需求不限量生产和人体相同结构的尿激酶，投入少、产出高，溶栓效果好，无毒副作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1和图2为实施例1中质粒信息；
图3为实施例2中得到的尿激酶纯度分析图，图左是纯化前的电泳图，图右是纯化后的电泳图；
图4为实施例3中使用表面等离子体共振方法测试尿激酶和其受体结合的结果图，图a是不同浓度的uPA与uPAR的相互作用图，图b是阴性对照图；
图5为实施例4中尿激酶激活纤溶酶原产生纤溶酶的SDS/PAGE结果图，其中泳道“+”为加入尿激酶的SDS/PAGE结果图，泳道“-”为未加入尿激酶的SDS/PAGE结果图，complex为复合物，plasminogen为纤溶酶原，anti-plasmin为抗纤溶酶。
具体实施方式
实施例1
将人尿激酶全基因cDNA，以NcoI和XhoI的位点克隆到可以在 Drosophila S2细胞表达的载体pMT/Bip/V5-A中，并在尿激酶cDNA的末端加上可表达6个组氨酸的cDNA(CAT CAT CAC CAT CAC CAT)便于纯化。质粒N-端含有Bip-signal sequence，使得表达后的尿激酶可以出细胞便于纯化；含有氨苄青霉素和嘌呤霉素抗性基因，便于筛选。质粒信息如图1、图2所示。
实施例2
将实施例1得到的质粒转染进Drosophila S2细胞中，具体操作为：将Drosophila S2细胞置于70-90％的转染混合液，稀释
Figure PCTCN2017092581-appb-000001
Reagent的转染试剂于
Figure PCTCN2017092581-appb-000002
培养基(1:1比例稀释)，稀释质粒于
Figure PCTCN2017092581-appb-000003
培养基。将稀释的质粒加入稀释的
Figure PCTCN2017092581-appb-000004
2000试剂，然后孵育45min，将该混合液加入细胞中。
转染48小时后用含2μg/ml嘌呤霉素和50μg/ml氨苄青霉素的10％胎牛血清的Snyder培养液进行筛选。没有转染成功细胞在该培养液中1-2d内凋亡裂解，转染成功的细胞在该培养液中能够存活并传代。每四天更换一次培养基，经过3周的筛选，得到可以表达尿激酶的Drosophila S2细胞。
将可以表达尿激酶的Drosophila S2细胞传代扩增，经过一周扩增，细胞达到1L、浓度为2×106个/ml时，去掉细胞培养上清液，将细胞置于1L含500μM硫酸铜的无血清培养液的表达液中，将培养瓶以225r/min的转速摇晃72h。之后，离心收取含有尿激酶的上清液，将含有尿激酶的上清液缓慢通过用特异组氨酸肽链的胶粒(effective Ni-NTA resin)装备的柱中，使尿激酶通过末端的6个组氨酸和特异组氨酸肽链的胶粒结合留在柱子中，其它杂质因没有6个组氨酸不能结合留在液体中。用1升洗液(Tris-buffer saline(TBS)pH 7.2,含10-25mM咪唑)洗柱，低浓度的咪唑洗去没有和胶粒结合的非特异性蛋白，然后用含有高浓度的咪唑(250mM)洗脱液将蛋白从胶粒上分离到洗脱液中。将洗脱液置于透析袋中在透析液(Tris-buffer saline (TBS)pH 7.2)中透析24h，收集透析后的洗脱液得到纯化的尿激酶。用SDS胶分析得到的尿激酶纯度，结果如图3所示。
实施例3
在BIAcore 2000(BIAcore AB，Uppsala，Sweden)中通过表面等离子体共振(SPR)分析实施例2得到的uPA与其受体uPAR之间的相互作用。根据制造商的说明书，使用胺偶联将uPAR固定在CM5芯片(BIAcore AB)的羧甲基葡聚糖表面。对于固定程序，使用pH 5.5的乙酸钠缓冲液将uPAR稀释至2.5μg/ml的终浓度。将uPAR在10μl/min的流速下在分开的流动池中固定在表面7分钟，在剩余的两个流动池中通过胺偶联活化然后立即失活制备参考表面。动力学测量时，注射6种不同浓度的uPA 3分钟，然后是10分钟解离相。在每个循环的末端，通过1分钟注射1M NaCl，然后用0.02M 6-氨基己酸注射1分钟，以及用磷酸盐/盐水缓冲液短暂洗涤步骤，使表面再生。实验在含有0.1M NaCl的0.04M磷酸盐缓冲液(pH 7.4)中以30μl/min的流速进行。假设在双分子反应模型中的1:1化学计量，使用BIA评估软件(版本3.1)通过全局曲线拟合分析所获得的uPA浓度系列的传感图的结合和解离相。Chi2分析支持该模型，在不同时间的uPA分析揭示约±20％的变异性。试验结果如图4所示，和阴性对照相比，反应值超过100，说明实施例2得到的尿激酶可以和其受体相结合。
实施例4
使用纤溶酶原激活的动力学分析实施例2得到的尿激酶的活性。被催化的纤溶酶原的浓度为其估计的活性指数的3.0倍，在0.05M赖氨酸存在下温育。通过加入尿激酶启动活化，并在37℃下在分光光度计中跟踪405nm处的吸光度变化。1分钟后，通过加入50％冰醋酸终止反应，并相对于参照空白测量405nm处的吸光度。通过SDS/PAGE研究抗纤溶酶与由uPA激活的纤溶酶形成稳定复合物的能力。试验结果如图5所示，纤溶酶原经尿激酶的激活形成纤溶酶，纤溶酶和抗纤溶 酶结合形成复合物，在SDS胶上可以看到大分子复合物，说明实施例2得到的尿激酶可以激活纤溶酶原产生纤溶酶。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种制备尿激酶的方法，包括：
    a)将尿激酶基因克隆到载体中，得到克隆质粒；
    b)将步骤a)得到的克隆质粒转染进Drosophila S2细胞，得到表达尿激酶的Drosophila S2细胞；
    c)对步骤b)得到的表达尿激酶的Drosophila S2细胞进行培养，对培养后的细胞进行处理，得到尿激酶。
  2. 根据权利要求1所述的方法，其特征在于，步骤a)中所述基因为人尿激酶全基因cDNA，克隆位点为NcoI和XhoI；
    所述人尿激酶全基因cDNA末端加上可表达6个组氨酸的cDNA。
  3. 根据权利要求1所述的方法，其特征在于，步骤a)中所述载体为pMT/Bip/V5-A。
  4. 根据权利要求1所述的方法，其特征在于，步骤a)中所述质粒带有Bip信号序列、氨苄青霉素和嘌呤霉素抗性基因。
  5. 根据权利要求1所述的方法，其特征在于，处理具体为除去细胞培养上清液，加入表达液，对培养瓶进行摇晃，收集上清液，将上清液过柱，洗脱液洗脱，收集尿激酶。
  6. 根据权利要求5所述的方法，其特征在于，所述表达液为含500μM硫酸铜的无血清培养液。
  7. 根据权利要求5所述的方法，其特征在于，摇晃条件时间为72h，转速为225r/min。
  8. 根据权利要求5所述的方法，其特征在于，所述柱为镍柱；
    所述洗脱液为含咪唑的TBS，pH为7.2。
  9. 根据权利要求1所述的方法，还包括，对步骤b)得到的表达尿激酶的Drosophila S2细胞进行筛选，得到高表达尿激酶的Drosophila S2细胞；
    筛选培养液为含有10％的胎牛血清的Snyder培养液；
    所述筛选培养液添加了氨苄青霉素和嘌呤霉素，氨苄青霉素浓度 为50μg/ml，嘌呤霉素浓度为2μg/ml。
  10. 根据权利要求1所述的方法，还包括，将步骤c)得到的尿激酶透析进行纯化，得到高纯度的尿激酶；
    所述透析袋为截留分子量为10KD，透析液为pH7.2的TBS，透析时间为24h。
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