CN1280983A - 白细胞介素－11的衍生物及其制备方法 - Google Patents
白细胞介素－11的衍生物及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种重组白细胞介素11及其制备方法,该IL－11的cDNA是在野生型IL－11的cDNA的N端缺失1—8个氨基酸或N端1—8个氨基酸被其它氨基酸取代。改造后的cDNA放在表达载体转化入大肠杆菌后能直接表达产生游离的IL－11,与野生型IL－11具有相同的生物学活性。经收菌—菌体破碎—固液分离—分子筛层析—离子交换层析等步骤,可制得高纯度、高比活的新型重组IL－11用于治疗血小板数低的病人。本发明的特点是:①直接表达游离IL－11,表达水平达20%以上;②纯化工艺简单;③极少的异源蛋白污染;④生产成本低廉。
Description
本发明属医药领域中的重组蛋白质药物。
白细胞介素11(Interleukin11，简称IL-11)是人体内分泌的一类重要细胞因子，它作用于骨髓细胞中原始的造血干细胞，促进巨核细胞成熟，增加血小板数目，促进血小板再生。重组白细胞介素11于1997年11月由美国FDA批准上市，成为治疗癌症病人经放、化疗后血小板数降低的唯一药物。
目前，用基因工程方法生产IL-11是唯一有效的途径。但重组野生型IL-11在大肠杆菌中表达时遇到很大的困难，即游离的完整的IL-11不能直接在大肠杆菌中表达。这是因为完整的IL-11cDNA5’端GC含量特别丰富，尤其是前8个氨基酸中有6个脯氨酸，严重地阻碍了完整的IL-11直接表达。为了表达IL-11，只能用隔合蛋白的表达方法。这样一来又造成了融合蛋白需要酶切的困难，产生了诸如切割用专一蛋白酶价格昂贵及异源蛋白污染等问题。
本发明的目的是用基因工程中最常用的大肠杆菌表达系统直接表达游离的IL-11。在保证IL-11的生物学活性不变的前提下，发明出具有表达量高、纯化工艺简单、生产成本低廉的新型重组白细胞介素11及其制备方法。
本发明的原理：我们试验中发现，在野生型IL-11cDNA5’端缺失一部分氨基酸，从而降低GC含量和脯氨酸数目，可以提高IL-11的表达。当IL-11N端缺的氨基酸数目从1个增加至8个时，游离IL-11的表达量从无到有并且逐步增加，而表达产物的生物学活性与野生型IL-11相比则基本不变。同样，若以苏氨酸和蛋氨酸取代IL-11前8个氨基酸中的脯氨酸或甘氨酸，表达效果与缺失氨基酸相同。这样，便可通过缺失N端几个氨基酸或用苏氨酸取代脯氨酸和甘氨酸来达到在大肠杆菌中直接表达游离的IL-11。
经上述改造转化的生产用菌种，在发酵表达结束后，经收菌--菌体破碎--固液分离--分子筛层析--离子交换层析等纯化步骤，即可制得纯度大于95％，比活性达到8×106IU/mg的具有相同于野生型IL-11生物活性的新型重组白细胞介素11。
最佳实施例：在构建新型重组IL-11的克隆过程中，其N端比野生型的IL-11缺失前5个氨基酸(Pro-Gly-Pro-Pro-Pro)，且第6-8个氨基酸换成Met-Thr-Thr，其余的氨基酸序列与野生型的IL-11相同。上述改造的IL-11cDNA放在表达载体PBV220中，转化入大肠杆菌DH5α，用温控诱导的方式可以直接表达产生游离的IL-11，表达水平达20％。游离表达的IL-11是以可溶状态存在于大肠杆菌的细胞浆中，且具天然活性，这一优势可以免去包涵体形式的蛋白所需的变性、复性及异构体分离等问题，从而大大简化了纯化工艺及降低了IL-11的生产成本。
表达和制备方法如下：在菌种平板上挑取一单菌落，接种于2mlLBA培养基中(LBA：每升含10g蛋白胨，5g酵母膏，5gNaCl，60mg氨苄青霉素，PH7.0)30℃培养过夜作为种子，按1％的接种量接种于50mlLBA中，30℃生长至0D600=0.5时，迅速升温至42℃诱导表达4小时，离心收获菌体。将菌体悬浮在50mMPB(Na2HPO4+NaH2PO4，PH8.0)缓冲液中，超声破碎菌体，20000g×20分钟离心，取上清。上清过分子筛SephacrylS-200凝胶层析纯化，上样量为柱体积的1-5％，20mMPB、PH8.0缓冲液洗脱，收集IL-11富集峰。再用阳离子交换柱CM-Sepharose进一步纯化，用0-1MNaCl、20mMPB、PH8.0线性梯度洗脱，在0.25MNaCl左右浓度处洗下IL-11活性峰。SDS-PAGE电泳分析表明，得到的IL-11纯度大于95％，比活性达8×106IU/mg。通过上述方法，可以得到高纯度、高比活性的新型重组IL-11。
本发明提供了一种新的表达和制备IL-11的方法，与世界上唯一生产IL-11的美国Genetics Institute生产工艺相比具有如下特点：①表达水平高，本发明的游离IL-11表达水平达20％，而G·I·公司的IL-11表达水平只相当于5％；②不需要切割用专一蛋白酶，而G·I·公司的工艺需用价格昂贵的enterokinase来切割融合蛋白；③极少的异源蛋白污染，而G·I·公司的工艺中存在着融合蛋白前导肽和加入eterokinase的异源蛋白污染问题；④生产工艺简单且成本低廉。上述优势使本发明具有明显的技术创新性和工艺先进性，使之更适合于工业化生产且经济实用。
Claims (5)
1．白细胞介素-11的衍生物，其特征在于该IL-11的cDNA在野生型的IL-11 cDNA的N端缺失1-8个氨基酸或N端1-8个氨基酸被其它氨基酸取代。
2．白细胞介素-11的衍生物的制备工艺，其特征在于经大肠杆菌发酵--温度诱导表达--收菌--菌体破碎--固液分离--分子筛层析--离子交换层析等步骤完成。
3．如权利要求2所述的固液分离，其特征在于用离心方法分离，工艺参数为15000g～25000g，离心时间是10～30分钟。
4．如权利要求2所述的分子筛层析，其特征在于采用S-100或S-200、S-300 Sephacryl凝胶柱层析，上样量为柱体积的1-5％，洗脱缓冲液是10～30mMPB(PB：Na2HPO4+NaH2PO4、PH6～9)。
5．如权利要求2所述的离子交换层析，其特征在于采用CM-Sepharose阳离子交换柱，洗脱条件是0-1MNaCl，PH7～10，线性梯度洗脱，最佳洗脱浓度是NaCl200～300mM。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007067602A1 (en) * | 2005-12-06 | 2007-06-14 | Wyeth | Interleukin-11 compositions and methods of use |
| CN100441596C (zh) * | 2003-01-13 | 2008-12-10 | 重庆富进生物医药有限公司 | 缺失型人白细胞介素11微小化变异体 |
| CN102161700A (zh) * | 2011-01-19 | 2011-08-24 | 温州医学院 | 一种新型人白细胞介素-11 |
| US10376557B2 (en) * | 2006-06-13 | 2019-08-13 | Helix Biomedix Inc. | Peptide fragments for inducing synthesis of extracellular matrix proteins |
-
1999
- 1999-07-16 CN CN 99112322 patent/CN1280983A/zh active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100441596C (zh) * | 2003-01-13 | 2008-12-10 | 重庆富进生物医药有限公司 | 缺失型人白细胞介素11微小化变异体 |
| WO2007067602A1 (en) * | 2005-12-06 | 2007-06-14 | Wyeth | Interleukin-11 compositions and methods of use |
| US10376557B2 (en) * | 2006-06-13 | 2019-08-13 | Helix Biomedix Inc. | Peptide fragments for inducing synthesis of extracellular matrix proteins |
| CN102161700A (zh) * | 2011-01-19 | 2011-08-24 | 温州医学院 | 一种新型人白细胞介素-11 |
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