CN1307129A - 一种重组葡激酶的生产方法 - Google Patents
一种重组葡激酶的生产方法 Download PDFInfo
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- CN1307129A CN1307129A CN 00112673 CN00112673A CN1307129A CN 1307129 A CN1307129 A CN 1307129A CN 00112673 CN00112673 CN 00112673 CN 00112673 A CN00112673 A CN 00112673A CN 1307129 A CN1307129 A CN 1307129A
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- lys
- aaa
- glu
- glucokinase
- staphylokinase
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- GPJGFSFYBJGYRX-YUMQZZPRSA-N Lys-Gly-Asp Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(O)=O GPJGFSFYBJGYRX-YUMQZZPRSA-N 0.000 claims description 2
- QBEPTBMRQALPEV-MNXVOIDGSA-N Lys-Ile-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN QBEPTBMRQALPEV-MNXVOIDGSA-N 0.000 claims description 2
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Abstract
一种重组葡激酶的生产方法,属于生物医学基因工程技术领域,其特征是将已构建好的葡激酶基因工程菌菌体经冰冻—解冻—冰冻反复循环数次,加入冻融液,将冻融液离心提取上清液,经两步柱层析纯化,上样经洗脱、收集、超滤脱盐、浓缩,除菌过滤得到葡激酶原液、冻干后得到本发明产品。该方法大大降低生产过程中葡激酶表达量的损失,延长分离介质使用寿命。设备成本低,能保持细胞完整,生产过程简单、方便、稳定。
Description
本发明是一种重组的生产方法，特别是纯化和除热原的方法，属于生物医学领域。
葡激酶(staphylokinase Sak)是90年代诞生的一种新型生物技术治疗药物，是金黄色葡萄球菌分泌的一种胞外蛋白质，它类似尿激酶(Uk)和链激酶(Sk)，具有溶血栓的特性。通常葡激酶的生产方法是利用已构建好的工程菌株，在培养基中培养达到一定OD值，离心收集菌体，然后高压匀浆泵或超声波破碎细胞高速离心获得上清液，再用两步法纯化。这种常规的方法具有如下缺点：一、在破碎细胞的过程中引起葡激酶表达量的损失；二、这种常规方法操作过程需要提供昂贵的超声波或高压匀浆泵设备和高速离心装置；三、细胞打碎后影响除去杂质，杂质含量高；四、杂质含量高的上清液缩短了昂贵的分离介质的使用寿命；五、生产过程复杂、繁琐。中国专利CN1186106A和CN1207414A中也使用上述的超声波破碎、离心收集上清液的葡激酶生产方法。
本发明的目的在于提供一种重组葡激酶的生产方法，能降低生产过程中葡激酶表达量损失，延长分离介质使用寿命，设备成本低，能保持细胞完整，排除压榨法局部高温对葡激酶活性的损害，生产过程简单、方便、稳定。
本发明是通过以下技术方案实现的：葡激酶纯化和除热原杂质采用：将构建好的葡激酶工程菌菌体经冰冻-解冻-冰冻反复循环至少2次；在经过循环后的菌体中加入冻融液；将冻融液离心提取上清液；上清液经两步法纯化；上样经洗脱、收集、超滤脱盐、浓缩、除菌过滤得到葡激酶原液，经冻干后得重组葡激酶产品。
本发明的目的还可以通过以下技术方案实现：葡激酶基因的工程菌菌体经冰冻-解冻-冰冻的反复循环2至10次；冰冻温度为-100℃至-15℃；冻融液为磷酸盐缓冲液(PBS)；离心机的转速为4000至5000rpm；两步纯化过程采用SP.sepharoseFF和Phenyl SepharoseFF柱层析；重组葡激酶具有高表达的工程菌株EcoliH15(中国微生物保藏中心保藏号CGMCC0432)；重组葡激酶具有如下核苷酸全序列及其编码的氨基酸序列：TCA AGT TCA TTC GAC AAA GGA AAA TAT AAA AAA GGC GAT GAC GCG AGTSer Ser Ser Phe Asp Lys Gly Lys Tyr Lys Lys Gly Asp Asp Ala SerTAT TTT GAA CCA ACA GGC CCG TAT TTG ATG GTA AAT GTG ACT GGA GTT GATTyr Phe Glu Pro Thr Gly Pro Tyr Leu Met Val Asn Val Thr Gly Val AspAGT AAA GGA AAT GAA TTG CTA TCC CCT CAT TAT GTC GAG TTT CCT ATT AAA CCTSer Lys Gly Asn Glu Leu Leu Ser Pro His Tyr Val Glu Phe Pro Ile Lys ProGGG ACT ACA CTT ACA AAA GAA AAA ATT GAA TAC TAT GTC GAA TGG GCA TTA GATGly Thr Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val Glu Trp Ala Leu AspGCG ACA GCA TAT AAA GAG TTT AGA GTA GTT GAA TTA GAT CCA AGC GCA AAG ATCAla Thr Ala Tyr Lys Glu Phe Arg Val Val Glu Leu Asp Pro Ser Ala Lys IleGAA GTC ACT TAT TAT GAT AAG AAT AAG AAA AAA GAA GAA ACG AAG TCT TTC CCTGlu Val Thr Tyr Tyr Asp Lys Asn Lys Lys Lys Glu Glu Thr Lys Ser Phe ProATA ACA GAA AAA GGT TTT GTT GTC CCA GAT TTA TCA GAG CAT ATT AAA AAC CCTIle Thr Glu Lys Gly Phe Val Val Pro Asp Leu Ser Glu His Ile Lys Asn ProGGA TTC AAC TTA ATT ACA AAG GTT GTT ATA GAA AAG AAA TAAGly Phe Asn Leu Ile Thr Lys Val Val Ile Glu Lys Lys End
上述的葡激酶基因的高表达工程菌株，具有引物Ⅰ，其核苷酸序列为：
5′ATCCATGGCCATGCTCAAAAGAAGT 3′
引物Ⅱ，其核苷酸序列为：
5′CTTATAGAAAAGAAATAAGGATCCCC 3′
本发明相比已有技术具有如下优点：
1．本发明将已构建的葡激酶工程菌菌株，通过反复冷冻-解冻-冷冻，可瞬时性改变细胞膜壁的通透性，使可溶性的葡激酶(分子量15,000)逸出在冻融液中，冻融液提供合适的离子强度和pH等条件，让葡激酶保持了较高的活性，而此时大部分细胞的细胞壁并未破碎，因此Ecdi细胞内的绝大部分菌体蛋白和细胞器等仍留在胞体内，不需要通过昂贵的高速离心机即可离心除去，获取含高活性的，占可溶性蛋白20％以上的葡激酶的澄清液中，大大降低设备成本和下游纯化压力。
2．本方法采用阳离子交换层析和疏水层析，两步即得纯度达99％以上的高活性葡激酶，在pH值不变的前提下，采用改变盐浓度的梯度洗脱和阶段洗脱相结合的方式，可以获取活性损失很小，低热源及蛋白纯度很高的葡激酶。
3．本发明的生产方法，大大降低生产过程中葡激酶表达量损失，延长分离介质使用寿命，设备成本低，能保持细胞完整，排除压榨法局部高温对葡激酶活性的损害，生产过程简单、方便、稳定。
附图说明：
图1本发明的生产工艺流程图
图2为离子交换层析峰形图
图3为SDS-PAGE(电泳)照片复印图
图4疏水层析峰形图
图5 SDS-PAGE(电泳)照片复印图
本发明下面将结合附图作进一步详述：
如图1所示：葡激酶高表达工程菌株EcoliH15 375g置于-70℃冰冻→解冻→冰冻，循环重复三次，加入冻融液为磷酸盐缓冲液PBS(10mMPB,10mM NaCl,2mMEDTA,2mMEGTA,pH8.0)，用离心机以4000rpm离心取上清液，将沉淀物置于-70℃再冷冻一次，重复上述过程，将两次上清液合并得7000ml，Folin酚法测得3.01mg/ml，总蛋白21000mg，平板溶纤法测得比活性为3.1×104Au/ml，总活性为6.3×108Au/ml。
离子交换层析：
层析柱型号 100×35 上海锦华厂
层析介质 SP Sepharose FF pharmacia Co.
柱床体积 1440ml
以25ml/分的流速，床柱用10mMPB pH6.0缓冲液进行平衡，然后将样品用HCl调至pH5.8上样，上样流速为50ml/分，上样总体积为7000ml，总蛋白21g，将上样后的柱用平衡液洗涤，280nm波长处测定约至基线。
洗脱1：用10mMPB pH6.0 0.5050ml/分洗脱
洗脱2：用10mMPB,0.25M NaCl梯度洗脱，洗脱后收集活性峰，如图1所示，其体积总蛋白、总活性见总结表1和表2。
如图2所示为SDS-PAGE分析：
1为上样液(冻融样品)
2为穿流峰
3-7为活性峰随机取样分析
疏水层析材料：
层析柱型号 55×35 上海锦华厂
层析介质 Phenyl Sepharose FF pharmacia Co.
柱床体积 430ml
以15ml/分的流速，对10MmPB、1M NaCl pH6.2的床柱体积，进行平衡，然后在离子交换洗脱活性峰加入去热原NaCl，使其成为1M，保持pH值不变，过0.45μ膜去杂质。
上样流速为15ml/分，上样总体积为3250ml，总蛋白6.1g，上样后用平衡液将A280洗至基线。
洗脱1：用0.5M NaCl、10mMPB、pH6.2,15ml/分速度洗脱洗脱2：用10mMPB,pH6.2洗脱，洗脱后收集活性峰，如图3所示，图中
Lane 1.marker 2.9603 3.9603 4.9603 5.Marker
其体积总蛋白、总活性见总结表1和表2。
如图4所示，SDS-PAGE分析：
SDSPAGE分析标准蛋白为31KD,20.4/19.7KD,16.9KD,14.4KD,8.1KD,6.2KD和2.5KD,左右两边为标准蛋白，中间三个样品为洗脱活性峰混合后的取样。
表1出发菌株375g，经冻融、离子交换、疏水层析，其蛋白活性、比活性比较
| 总蛋白 | 总活性 | 总活性 | 比活性 | 纯化倍数 | 活性比率 | |
| 冻出液 | 7000 | 21g | 6.3×108Au | 3.1×104Au/ml | ||
| 离子交换 | 3250ml | 6.1g | 3.96×108Au | 6.5×104Au/ml | 2.1 | 63％ |
| 疏水去 | 850ml | 3.1g | 3.01×108Au | 9.7×104Au/ml | 3.1 | 48％ |
表2出发菌株375g，用匀浆法(压榨法)破菌与冻融法提取，其蛋白活性比较
| 总体积 | 总蛋白 | 总活性 | 比活性 | 活性比率 | |
| 压榨法 | 3500ml | 37g | 8.8×108Au | 2.3×104Au/ml | 100％ |
| 冻融法 | 7000ml | 21g | 6.3×108Au | 3×104Au/ml | 72％ |
本发明与各研究单位的葡激酶核苷酸序列及其对应氨基酸异同的比较：氨基酸次序 11 34 35 36 43 92 116资料来源上海植生所 AAA AGT AAA GGA CAT TAT TTA本发明
Lys Ser Lys Gly His Tyr Leu成都地奥 AAA GGT AAA GGA GAT TTT TTA专利申请号95111222
Lys Gly Lys Gly His Phe Leu上海医大 AAA GGT AAA AGA CGT TAT ATA专利申请号94112105
Lys Gly Lys Arg Arg Tyr Leu青岛双龙 AAA GGT AAG GGA CGT TAT TTA专利申请号97105988
Lys Gly Lys Gly Arg Tyr Leu医科院 AAA GGT AAA GGA CGT TAT ATA专利申请号98102132流研所 Lys Gly Lys Gly Arg Tyr Leu医科院 AAA GGT AAA GGA CGT TAT TTA专利申请号98102132基础所 Lys Gly Lys Gly Arg Tyr Leu日本 AAG GGT AAA GGA CAT TAT TTA Nucleic Acid Research 11
Lys Gly Lys Gly Ilis Tyr Leu (22)P.679(83)德国 AAG GGT AAA AGA CGT TAT TTA M.G.G.210,528(87)
Lys Gly Lys Arg Arg Tyr Leu比利时 AAG AGT AAA GGA CAT TAT TTA Fibrinolysis 6,226(92)
Lys Ser Lys Gly His Tyr Leu
Claims (10)
1．一种重组葡激酶的生产方法，包括利用已构建好的葡激酶工程菌株，在培养基中培养，达到一定OD值，离心收集菌体，将菌体处理后，离心收集上清液，再用两步纯化得到产品，其特征在于葡激酶纯化和除热原杂质，采用：
(1)将葡激酶基因的高表达工程菌菌体经冰冻-解冻-冰冻反复循环至少两次；
(2)在经过循环后的菌体中加入冻融液；
(3)将冻融液离心提取上清液；
(4)上清液经两步柱层析纯化；
(5)上样经洗脱、收集、超滤脱盐、浓缩、除菌过滤得到葡激酶原液；加入保护剂，冻干后得到重组葡激酶产品。
2．如权利要求1所述的溶血栓药物重组葡激酶，其特征在于菌体冰冻-解冻-冰冻反复循环2-10次。
3．如权利要求1所述的溶血栓药物重组葡激酶，其特征在于冰冻温度为-100℃～-15℃。
4．如权利要求1所述的溶血栓药物重组葡激酶，其特征在于冻融液为磷酸盐缓冲液(PBS)。
5．如权利要求1或4所述的溶血栓药物重组葡激酶，其特征在于冻融液离心提取上清液时，高速离心机的转速为4000-5000rpm。
6．如权利要求1所述的溶血栓药物重组葡激酶，其特征在于上清液在两步纯化过程中采用SP.sepharose FF和Phenyl Sepharose FF柱层析。
7．如权利要求1所述的溶血栓药物重组葡激酶，其特征在于重组葡激酶具有高表达的工程菌株EcoliH15(中国微生物保藏中心保藏号CGMCC0432)。
8．如权利要求1或7所述的溶血栓药物重组葡激酶，其特征在于重组葡激酶具有如下核苷酸全序列及其编码的氨基酸序列：TCA AGT TCA TTC GAC AAA GGA AAA TAT AAA AAA GGC GAT GAC GCG AGTSer Ser Ser Phe Asp Lys Gly Lys Tyr Lys Lys Gly Asp Asp Ala SerTAT TTT GAA CCA ACA GGC CCG TAT TTG ATG GTA AAT GTG ACT GGA GTT GATTyr Phe Glu Pro Thr Gly Pro Tyr Leu Met Val Asn Val Thr Gly Val AspAGT AAA GGA AAT GAA TTG CTA TCC CCT CAT TAT GTC GAG TTT CCT ATT AAA CCTSer Lys Gly Asn Glu Leu Leu Ser Pro His Tyr Val Glu Phe Pro Ile Lys ProGGG ACT ACA CTT ACA AAA GAA AAA ATT GAA TAC TAT GTC GAA TGG GCA TTA GATGly Thr Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val Glu Trp Ala Leu AspGCG ACA GCA TAT AAA GAG TTT AGA GTA GTT GAA TTA GAT CCA AGC GCA AAG ATCAla Thr Ala Tyr Lys Glu Phe Arg Val Val Glu Leu Asp Pro Ser Ala Lys IleGAA GTC ACT TAT TAT GAT AAG AAT AAG AAA AAA GAA GAA ACG AAG TCT TTC CCTGlu Val Thr Tyr Tyr Asp Lys Asn Lys Lys Lys Glu Glu Thr Lys Ser Phe ProATA ACA GAA AAA GGT TTT GTT GTC CCA GAT TTA TCA GAG CAT ATT AAA AAC CCTIle Thr Glu Lys Gly Phe Val Val Pro Asp Leu Ser Glu His Ile Lys Asn ProGGA TTC AAC TTA ATT ACA AAG GTT GTT ATA GAA AAG AAA TAAGly Phe Asn Leu Ile Thr Lys Val Val Ile Glu Lys Lys End
9．如权利要求1或7或8所述的溶血栓药物重组葡激酶，其特征在于将葡激酶活性高的菌株中分离的噬菌体α的DNA为模板，采用引物Ⅰ和引物Ⅱ经PCR扩增，得到含有完整葡激酶基因的DNA小片段，该基因DNA片段与pJLA502载体质粒，分别以BamHⅠ和NcoⅠ酶切；再将酶切后的上述质粒和葡激酶基因DNA小片段，用T4 DNA连接酶连接获得含有葡激酶基因的pJLA502质粒DNA，再转化入HB101大肠杆菌细胞内，构建成葡激酶高表达的工程菌株EcoliH15(中国微生物保藏中心保藏号CGMCC0432)。
10．根据权利要求9所述的溶血栓药物重组葡激酶，其特征在于所述的引物Ⅰ，其核苷酸序列为：
5′ATCCATGGCCATGCTCAAAAGAAGT3′；引物Ⅱ，其核苷酸序列为：
5′CTTATAGAAAAGAAATAAGGATCCCC 3′。
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| CN104031901A (zh) * | 2014-05-21 | 2014-09-10 | 丽珠集团丽珠制药厂 | 一种纯化尿激酶的方法 |
| CN107353049A (zh) * | 2017-08-10 | 2017-11-17 | 黑龙江九穗谷农业科技发展有限公司 | 一种多效生物菌酶的制备方法 |
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| CN100383240C (zh) * | 2005-07-21 | 2008-04-23 | 山东大学 | 一种快速制备转糖基β-半乳糖苷酶的方法 |
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| CN104031901A (zh) * | 2014-05-21 | 2014-09-10 | 丽珠集团丽珠制药厂 | 一种纯化尿激酶的方法 |
| CN104031901B (zh) * | 2014-05-21 | 2016-08-24 | 丽珠集团丽珠制药厂 | 一种纯化尿激酶的方法 |
| CN107353049A (zh) * | 2017-08-10 | 2017-11-17 | 黑龙江九穗谷农业科技发展有限公司 | 一种多效生物菌酶的制备方法 |
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Owner name: TONGHUA YUJIN PHARMACEUTICAL CO., LTD. Free format text: FORMER OWNER: CHENGDU JINPENG BIOLOGY TECHNOLOGY CO., LTD.; SHANGHAI INST. OF PLANT PHYSIOLOGY, CHINESE ACADEMY OF SCIENCES Effective date: 20060908 |
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| C41 | Transfer of patent application or patent right or utility model | ||
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Effective date of registration: 20060908 Address after: 134001 No. 258 water spring road, Jilin, Tonghua Patentee after: Tonghua jade gold pharmaceutical Limited by Share Ltd. Address before: 610041 5-1-10, 6 West tree road, Gaoxin District, Sichuan, Chengdu Co-patentee before: SHANGHAI INST OF PLANT PHYSIOL Patentee before: JINPENG BIO TECH Co.,Ltd. SHANGH |
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| C56 | Change in the name or address of the patentee |
Owner name: TONGHUA ZHONGXIN YUJIN PHARMACEUTICAL CO., LTD. Free format text: FORMER NAME: JILIN YUJIN PHARMACEUTICAL CO., LTD. Owner name: JILIN YUJIN PHARMACEUTICAL CO., LTD. Free format text: FORMER NAME: TONGHUA YUJIN PHARMACEUTICAL CO., LTD. |
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| CP01 | Change in the name or title of a patent holder |
Address after: 134001 No. 258 water spring road, Jilin, Tonghua Patentee after: Tonghua Xinyu Pharmaceutical Co.,Ltd. Address before: 134001 No. 258 water spring road, Jilin, Tonghua Patentee before: Jilin jade gold pharmaceutical Limited by Share Ltd. Address after: 134001 No. 258 water spring road, Jilin, Tonghua Patentee after: Jilin jade gold pharmaceutical Limited by Share Ltd. Address before: 134001 No. 258 water spring road, Jilin, Tonghua Patentee before: Tonghua jade gold pharmaceutical Limited by Share Ltd. |
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| CP01 | Change in the name or title of a patent holder |
Address after: 134001 No. 258 water spring road, Jilin, Tonghua Patentee after: Tonghua Kangyuan Yujin Pharmaceutical Co.,Ltd. Address before: 134001 No. 258 water spring road, Jilin, Tonghua Patentee before: Tonghua Xinyu Pharmaceutical Co.,Ltd. |
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| CP01 | Change in the name or title of a patent holder | ||
| CX01 | Expiry of patent term |
Granted publication date: 20040310 |
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| CX01 | Expiry of patent term |