CN1062016C - 重组人糖基化尿激酶原的制备方法 - Google Patents
重组人糖基化尿激酶原的制备方法 Download PDFInfo
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Abstract
本发明是利用生物工程的方法，通过基因工程技术构建了一种有双重放大功能的表达载体，并获得一株高效表达的CHO工程细胞。经微载体和低血清培基高密度灌流培养和纯化，制备一种治疗冠状动脉梗塞，脑血栓，中央视网膜动静脉血栓和其他血栓病的药物一重组人糖基化尿激酶原。
Description
本发明是利用生物工程技术，对以下六种载体如pUC19,pMM-UK,pSV2-dhfr,pSV2-pro-UK,pXMT,pMTSV-dhfr上的有用元件，经过重组，构建一个适合在中国仓鼠卵巢(CHO)母细胞中高效表达外源基因的新型载体(pMTSV-du)，然后将转染筛选获得的CHO工程细胞株CL-11G用微载体(Biosilon,SH-2,Cytopore)灌流培养技术和低血清培基进行高密度培养，并对产品进行四步或一步纯化，制备一种治疗冠状动脉梗塞，脑血栓，中央视网膜动静脉血栓及其他血栓病的药物-重组人尿激酶原(prourokinase,简称pro-UK)，此属于生物工程技术。
尿激酶原是尿激酶的前体，是一种糖蛋白，由411个氨基酸组成，其本身无活性，经纤维蛋白溶解酶和激肽酶的激活，使其Lys158-Ile159之间的肽链打开形成尿激酶，才能发挥其溶血拴的效能。
自Bernit1973年发现pro-UK以来，已有人相继从人的血液，尿液和各种重组宿主细胞培养液中获得，如用大肠杆菌培养液中获得的有Holmes,W.E. 1985以及安内.布兰法兹等(中国专利，CN 1042181A,1990-05-16)，用酵母表达成功的如Melnick,L.M.等(1990)用中国仓鼠卵巢(CHO)母细胞表达成功的有Nells,L.等(1987)；Avgerinos,G.C.等(1990)。
本发明包括：1．人尿激酶原全长cDNA基因的克隆，2．构建在CHO细胞高效表达的转移载体和工程细胞株，3．对CHO工程细胞的大量培养，获得大量尿激酶原原料，并加以纯化，其目的在于利用生物工程技术制备一种适于临床应用的糖基化尿激酶原产品。
本发明的技术要点是：
1．通过对Detroit 562细胞的诱导和提取细胞总RNA，经反转录和dG·dC接尾构建了cDNA文库，再通过筛选和人工合成寡核苷酸等DNA重组技术，构建了尿激酶原全长cDNA基因并克隆至pUC19质粒DNA，获得pMM-UK重组质粒。
2．将几种表达载体如pUC19，pMM-UK，pSV2-dhfr，pSV2-proUK,pXMT,pMTSV-dhfr，等载体上的有用元件，经多次基因操作和重组，构建了一个适合在CHO细胞中高效表达外源基因的新型载体(pMTSV-du)。
3．采用微载体(Biosilon,SH-2,Cytopore)灌流培养技术和低血清培养基高密度培养CL-11G工程细胞，获得大量含高活性尿激酶原的原料。
4．采用四步法，即CM阳离子亲和层析-MPG吸附层析-Sephacryl S-200凝胶过滤-对氨基苯甲脒色谱，或采用一步法，即用抗体亲和层析法纯化产品。
实施例：
1．人尿激酶原全长cDNA基因的构建和克隆：采用肉豆寇酯(PMA)诱导Detroit 562细胞，使细胞内尿激酶mRNA含量提高8倍以上，用酸性硫氰胍-酚-氯仿法提取细胞总RNA，oligo(dT)-纤维素柱层析法分离poly(A+)RNA,通过反转录和dG·dC接尾重组技术构建了Detroit 562细胞的cDNA文库，通过筛选获得了含有编码尿激酶基因片段的阳性克隆株。再采用人工合成寡核苷酸片段和DNA重组技术，构建了人尿激酶原全长cDNA基因并克隆至pUC19质粒DNA，获得了pMM-UK重组质粒。全序列测定结果表明，cDNA基因全长为1565bp，含有60bp(20aa)信号肽序列，1233bp编码序列(1-411aa)和272bp 3'非编码序列。该基因5'端含有HindⅢ和EcoR 1单一酶切位点，3'端含有SalⅠ,XbaⅠ,SmaⅠ,KpnⅠ和SacⅠ 5个单一酶切位点，便于尿激原基因的克隆和表达。
2．中间载体1 pSV2-pro-UK的构建：将pSV2-dhfr载体用BglⅡ酶切后用Klenow F酶补平，再用HindⅢ酶切，分离回收载体大片段；从pMM-UK质粒DNA中，用HindⅢ和SmaⅠ双酶切，分离pro-UK cDNA；将载体大片段与pro-UK cDNA连接形成可表达pro-UK的重组质粒pSV2-pro-UK。
3．中间载体2 pMTSV-dhfr的构建：将pSV2-dhfr表达质粒用BglⅡ酶切，并用Klenow F酶将末端补平，再通过T4DNA连接酶将质粒重新环化并消除了质粒中的BglⅡ酶切位点。然后将该质粒经BamH 1和pVUⅡ双酶切及Klenow F酶将末端补平，通过电泳回收1.9Kb DNA片段(内含SV40增强子和二氢叶酸还原酶基因)。将此片段插入经EcoR1酶切，Klenow F酶末端补平的pXMT载体中，从而获得了长度为8.25Kb的中间载体pMTSV-dhfr。
4．新型高效表达载体pMTSV-du的构建：将pSV2-pro-UK重组质粒经SalⅠ酶切，Klenow F酶末端补平，再通过T4 DNA连接酶将质粒重新环化并消除了质粒中的SalⅠ酶切位点。然后将该质粒经pVUⅠ和EcoRⅤ双酶切及Klenow F酶将末端补平，通过电泳回收3.8Kb片段(含全长尿激酶原cDNA)，并将该片段插入经Bgl Ⅱ酶切，Klenow F酶末端补平的pMTSV-dhfr中间载体，从而获得长度为12Kb，可高效表达尿激酶原的载体pMTSV-du.该载体的特点是：
A．将表达尿激酶原和二氢叶酸还原酶基因的两个转录单位置于同一载体，分别受金属硫蛋白(MT)和SV40早期启动子控制，使之具有用氨甲喋呤(MTX)使基因扩增和用金属Zn++诱导的双重放大功能，这有利于尿激酶原的高表达。
B．将SV40增强子置于两个启动子之间，为两个启动子公用。由于SV40增强子的增强作用广谱性强，有利于尿激酶原基因在多种细胞中获得高效表达。
C．在载体的非必需区，有-SalⅠ单一酶切位点，可方便地进行重组载体的线性化，有利于通过电击介导法获得多考贝尿激酶原基因，并整合在细胞染色体上使稳定表达。
D．使表达尿激酶原和二氢叶酸还原酶的两个转录单位转录方向相反，这可减少dhfr基因转录时对尿激酶原基因转录的影响，有利于尿激酶原的高表达。
5．转染和筛选高效表达的CHO工程细胞：将20-40μgpMTSV-du质粒DNA通过磷酸钙共沉淀法转染CHO-dhfr细胞，先用HAT选择培基筛选，10天后换成含1-3x10-8M MTX的选择培基进行dhfr和MTX双重筛选，并对33株表达有尿激酶原活性的阳性转化细胞经多次亚克隆和MTX加压扩增基因，锌离子诱导，最终筛选到三株能高效表达尿激酶原的细胞株，其中一株CL-11G表达的活性可达500-800 IU/106细胞/天。该株细胞经长期液氮保存和一百多次转代培养，表达水平基本不变。表达产物经抗原性和Western印迹分析，其分子量为52Kd，较大肠杆菌表达的分子量(47Kd)大，表明它确与天然的相似，是糖基化了的尿激酶原。
6．CHO工程细胞的高密度培养：采用固体的聚苯乙烯微载体如Biosilon,SH-2，和多孔纤维素微载体如Cytopore，在我们改进了的灌流培养控制系统(已获中国国家专利，专利号：ZL91208893.1,1994-08-24)控制下，用搅拌式生物反应器(如Celligen)培养，灌流量自0.5个工作体积/天逐渐增加至2个工作体积/天，细胞密度可达1-2x107/ml，尿激酶原产量可高达8096 IU/ml。培养时间可长达53天。
7．放大生产规模：是利用该细胞能在微载体之间自动转移的特点，直接将已长有细胞的微载体从小反应器转入加有3倍，5倍或7倍量培养基和新微载体的大反应器内。
8．研制和采用低血清培养基：通过对培养过程中培养基成份的氨基酸分析，以及通过正交试验，确定以DMEM/F12(1∶1)为基础培基的低血清培基，佐以天冬氨酸，胱氨酸，亮氨酸，甲硫氨酸，丝氨酸，谷氨酰胺，蛋白胨，叶酸以及抑肽酶(aprotinin),Hepes,葡萄糖等。血清量从原来的5％减少到0.5％。
9．四步法纯化尿激酶原：
第一步，CM-径向离子交换法：该色谱柱是由聚乙烯与纤维素共聚膜共价结合羧甲基绕在多孔轴管上并固定在有机玻璃柱内构成。先用0.15mol/L，pH5.7的磷酸缓冲液平衡。尿激酶原原液经3000Xg，4℃离心后用6mol/L HCl调pH至5.7后上柱，流速为12-15ml/分(柱床体积250ml)或25-30ml/分(柱床体积700ml)。当洗液中蛋白吸收峰值低于0.005以下时改用0.025mol/L，pH6.8的醋酸缓冲液(含0.75mol/L硫酸铵)，0.005％Tween-80)洗脱，收集活性蛋白峰流出液。
第二步，MPG吸附色谱法：它是由微孔高硅玻璃珠组成的色谱柱。将第一步的收集液直接上柱，用0.5mol/LTris-HCl，pH9.5的缓冲液洗去杂蛋白，再用0.02mol/L，pH8.0的磷酸缓冲液(含0.1mol/L NaCl)洗至吸收峰值低于0.005以下后，分别用含1mol/L和2mol/L硫酸铵的0.5mol/L，pH9.25的Tris-HCl缓冲液线性梯度洗脱并收集蛋白吸收峰流出液。
第三步，凝胶色谱Sephacryl S-200 HR高效凝胶色谱先用0.092mol/L，pH5.3(含0.1mol/L NaCl)醋酸缓冲液平衡，然后将第二步的收集液上柱，继续用该缓冲液洗柱并收集第二蛋白峰组分。
第四步，对氨基苯甲脒亲和色谱：先用0.092mol/L，pH5.3(含0.2mol/L NaCl)的醋酸缓冲液平衡，然后将第三步收集液上柱，继续用该缓冲液洗柱并收集穿过蛋白流出液。
10．一步抗体亲和层析法纯化：用制备的单克隆抗体和Sepherose 4B偶联并装成层析柱。将收集的尿激酶原原液先与0.002M苯甲醚在室温保温0.5h，然后上柱。用0.1M，pH2.2的Gly-HCl缓冲液洗脱，收集活性峰。
11．纯化产品的理化性能测定：纯化后的产品的活性可被抗尿激酶的抗体中和，但不能被DFP抑制。分子量为52Kd，等电点为8.9，N-末端氨基酸序列为SNELHQVPSNCDCLN，这些结果表明，它与天然的糖基化的尿激酶原完全一致。
附图说明：
图1．pMTSV-du高效表达载体的构建。
Claims (8)
1．一种重组人糖基化尿激酶原的制备方法，包括以下步骤：(1)人尿激酶原全长cDNA基因的构建和克隆，(2)表达载体的构建，(3)CHO工程细胞的转染和筛选，(4)CHO工程细胞的培养和(5)CHO工程细胞分泌产物的纯化，其特征在于CHO工程细胞是以CHO dhfr-为宿主细胞，所述的表达载体是pMTSV-du，它由下图所示的各元件组成，并用微载体灌流培养技术高密度培养和放大培养CHO工程细胞。
2．如权利要求1所述的一种重组人糖基化尿激酶原的制备方法，其特征在于：
(1)人尿激酶原全长cDNA基因的克隆，是采用肉豆寇酯诱导Detroit 562细胞，使细胞中尿激酶mRNA的含量提高8倍以上，用酸性硫氰胍-酚-氯仿法提取细胞总RNA，oligo(dT)-纤维素柱层析法分离poly(A+)RNA，通过反转录和dG·dC接尾重组技术构建成Detroit 562细胞的cDNA文库，通过筛选而获得含有编码尿激酶基因片段的阳性克隆株，采用人工合成寡核苷酸片段和DNA重组技术，构建尿激酶原全长cDNA基因并克隆至pUC19质粒DNA，获得一个pMM-UK重组质粒，经全序列测定证明，cDNA核苷酸序列全长为1565bp，包括编码20个氨基酸信号肽的60bp序列，编码411氨基酸尿激酶原的1233bp序列和272bp3'非编码序列，该基因5'端含有HindⅢ和EcoR 1单一酶切位点，3'端含有SdlⅠ，XbaⅠ，SmaⅠ，KpnⅠ和SacⅠ 5个单一酶切位点，便于尿激酶原的克隆和表达；
(2)中间载体pSV2-pro-UK的构建，是将pSV2-dhfr载体用Bgl Ⅱ酶切后用Klenow F酶末端补平，再用HindⅢ酶切，分离回收载体大片段，从pMM-UK质粒DNA中，用HindⅢ和SmaⅠ双酶切，分离pro-UK cDNA，再将载体大片段与pro-UK cDNA连接形成可表达pro-UK的重组质粒pSV2-pro-UK；
(3)中间载体pMTSV-dhfr的构建，是将pSV2-dhfr表达质粒用BglⅡ酶切，并用Klenow F酶将末端补平，再通过T4 DNA连接酶将质粒重新环化，并消除质粒中的BglⅡ酶切位点，然后将该质粒经BamH 1和pVUⅡ双酶切及KlenowF酶将末端补平，通过电泳回收1.9Kb DNA片段，它含SV40增强子和二氢叶酸还原酶基因，将此片段插入经EcoR1酶切，klenowF酶末端补平的pXMT载体中，从而获得了长度为8.25Kb的中间载体pMTSV-dhfr；
(4)表达载体pMTSV-du的构建，将pSV2-pro-UK重组质粒经SalⅠ酶切，Klenow F酶末端补平，再通过T4 DNA连接酶将质粒重新环化并消除了质粒中的SalⅠ酶切位点，然后将该质粒经pVU1和EcoR Ⅴ双酶切及KlenowF酶将末端补平，通过电泳回收3.8Kb片段，它含全长尿激酶原cDNA，并将该片段插入经BglⅡ酶切，Klenow F酶末端补平的pMTSV-dhfr中间载体，从而获得一个长度为12Kb，能高效表达尿激酶原的载体pMTSV-du。
3．如权利要求1所述的一种重组人糖基化尿激酶原的制备方法，其特征在于转染和筛选高效表达的CHO工程细胞是将20-40微克表达载体pMTSV-du质粒DNA通过磷酸钙共沉淀法转染CHO-dhfr-细胞，先用HAT选择培养基筛选，10天后换成含1-3×10-8MMTX选择培养基进行dhfr和MTX双重筛选，并对33株表达有尿激酶原活性的阳性转化细胞多次亚克隆和MTX加压扩增基因，再经锌离子诱导筛选出高效表达，活性达到500-800IU/10-6细胞/天的尿激酶原工程细胞株。
4．如权利要求1所述的一种重组人糖基化尿激酶原的制备方法，其特征在于CHO工程细胞的高密度培养是用固体聚乙烯微载体Biosilon或SH-2和多孔纤维素微载体Cytopore在一个由灌流培养控制装置的反应器内进行灌流培养，灌流量逐渐增加，使细胞密度达1-2×107/ml，尿激酶原的产量随细胞密度的增加而增加。
5．如权利要求1所述的一种人重组糖基化尿激酶原的制备方法，其特征在于利用该CHO工程细胞在培养中能在微载体之间自动转移的特点，用以放大生产规模，即直接将小反应器内已长有细胞的微载体，转入加有3倍、5倍或7倍量的培养基和新鲜微载体的大反应器内，继续灌流培养。
6．如权利要求1所述的一种人重组糖基化尿激酶原的制备方法，其特征在于低血清培养基是通过对培养过程中培养基成份的氨基酸分析，以及通过正交试验确定以DMEM/F12=1∶1培养基为基础，佐以天门冬氨酸、胱氨酸、亮氨酸、甲硫氨酸、丝氨酸、谷氨酰胺、蛋白胨、叶酸、抑肽酶、Hepes缓冲液和葡萄糖，同时将原来培养基中的小牛血清用量从5％减至0.5％。
7．如权利要求1所述的一种重组人糖基化尿激酶原的制备方法，其特征在于工程细胞分泌产物的纯化是采用四步纯化方法，包括：
第一步：CM-径向离子交换色谱，色谱柱的构成是用聚乙烯与纤维素共聚膜共价结合羧甲基绕在多孔轴管上，并固定在有机玻璃柱内，柱层析时的pH值为5.7，柱床体积700ml时流速为25-30ml/分，
第二步：微孔高硅玻璃珠色谱，用微孔高硅玻璃珠装填层析柱，层析后收集其蛋白吸收峰流出液，
第三步：凝胶色谱，收集其第二蛋白峰组份，
第四步：对氨基苯甲脒亲和色谱，层析其第三步的收集液，并继续收集穿过蛋白的流出液，
8．如权利要求1所述的一种重组人糖基化尿激酶原的制备方法，其特征在于工程细胞分泌产物的纯化是采用一步法，即抗体亲和层析法，将尿激酶原先与苯甲脒在室温保温0.5小时，再上单抗与Sepherose 4B偶联的层析柱，然后收集活性峰。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005074979A1 (fr) * | 2004-02-04 | 2005-08-18 | Shanghai Tasly Pharmaceutical Co., Ltd. | Utilisation de la prourokinase pour le traitement du syndrome pulmonaire thrombo-embolique ou de l'occlusion de l'artere centrale de la retine |
| WO2006012791A1 (fr) * | 2004-08-06 | 2006-02-09 | Shanghai Tasly Pharmaceutical Co., Ltd. | Composition contenant de la pro-urokinase active, son procede de lyophilisation et sa preparation lyophilisee |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100420745C (zh) * | 2003-06-05 | 2008-09-24 | 上海实业科华生物药业有限公司 | 杂交瘤细胞表达基因工程尿激酶原的制备方法 |
| CN101880655B (zh) * | 2009-05-06 | 2013-12-04 | 中国人民解放军军事医学科学院生物工程研究所 | 一种纯化cho细胞表达尿激酶原的方法 |
| CN103789291B (zh) * | 2014-02-24 | 2016-08-17 | 东北制药集团股份有限公司 | 一种重组大肠杆菌发酵液中分离纯化重组人尿激酶原的制备工艺 |
| CN106867985A (zh) * | 2015-12-11 | 2017-06-20 | 上海天士力药业有限公司 | 一种重组人尿激酶原的纯化及去除病毒方法 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1033289A (zh) * | 1987-08-10 | 1989-06-07 | 格鲁普莱皮蒂特股份有限公司 | 制备单链尿激酶的方法 |
| CN1075165A (zh) * | 1993-03-04 | 1993-08-11 | 北京大学 | 利用昆虫细胞高效表达人尿激酶原及其它有用蛋白质的方法 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1033289A (zh) * | 1987-08-10 | 1989-06-07 | 格鲁普莱皮蒂特股份有限公司 | 制备单链尿激酶的方法 |
| CN1075165A (zh) * | 1993-03-04 | 1993-08-11 | 北京大学 | 利用昆虫细胞高效表达人尿激酶原及其它有用蛋白质的方法 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005074979A1 (fr) * | 2004-02-04 | 2005-08-18 | Shanghai Tasly Pharmaceutical Co., Ltd. | Utilisation de la prourokinase pour le traitement du syndrome pulmonaire thrombo-embolique ou de l'occlusion de l'artere centrale de la retine |
| WO2006012791A1 (fr) * | 2004-08-06 | 2006-02-09 | Shanghai Tasly Pharmaceutical Co., Ltd. | Composition contenant de la pro-urokinase active, son procede de lyophilisation et sa preparation lyophilisee |
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