CN103897054A - 一种牛肺抑肽酶异性制备层析介质的合成和应用 - Google Patents
一种牛肺抑肽酶异性制备层析介质的合成和应用 Download PDFInfo
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Abstract
本发明涉及一种牛肺抑肽酶特异性制备层析介质，由人工配体和层析介质连接而成。其配体为2,4,6-三甲基-3,5-二氨基苯磺酸，层析介质是sepharose与sephadex，亲和配体和层析介质通过间隔臂1,3-二氨基-2-羟基丙烷与间隔臂三聚氯氰进行连接。建立了一种牛肺抑肽酶特异性制备层析介质的合成方法，以及利用上述的层析介质高效纯化牛肺抑肽酶的方法，利用该亲和介质可以大规模从原料中快速分离纯化分子量为~6kDa的牛肺抑肽酶。整个过程只需要经过一步亲和层析，纯化得到的人尿胰蛋白酶抑制剂纯品，其收率大于85%，比活大于15000IU/mg.pr。整个纯化步骤消耗时间短，活性回收率及比活较高，适于大规模推广应用。
Description
技术领域
本发明涉及生物工程、医疗、诊断用酶生产技术领域，具体是指一种牛肺抑肽酶异性制备层析介质的合成和应用。
背景技术
抑肽酶( aprotinin) 是非特异性丝氨酸蛋白酶抑制剂, 由58 个氨基酸残基组成的单链碱性蛋白, 链内有3 个二硫键相互交联, 等电点为10. 5。长期以来用于急性胰腺炎的治疗, 90 年代以来, 继英国、加拿大之后, 1993 年12 月美国食品药品监督管理局(FDA) 批准临床用于胸外科手术中, 用于保护血小板, 减少出血和渗血, 并应用于有凝血障碍、可能出现大出血的病人的治疗。抑肽酶通过抑制激肽释放酶以及纤溶酶而达到止血的目的, 能显著减少手术中及手术后病人的出血, 临床研究表明其副作用小、安全性好。
其在临床医疗中有广泛的应用，适用症包括急性胰腺炎（包括外伤性、术后及内窥镜逆行性胰胆管造影术后的急性胰腺炎）、慢性复发性胰腺炎的急性恶化期，急性循环衰竭（出血性休克、细菌性休克、外伤性休克、烧伤性休克）；也广泛用于胸外科手术、消化系统手术、肿瘤手术、器官移植、器官切除手术及CPB手术；还用于治疗与预防肿瘤化疗产生的肾功能障碍。
抑肽酶的临床用量较大, 国内按中国药典2013版规定，分子量要求为6 kDa，目前抑肽酶制剂为牛肺、胰等器官的提取物, 工艺复杂, 成本高, 价格较贵, 产品纯度也不高。基因工程抑肽酶的产品尚未上市, 在大肠杆菌中较难以活性形式表达, 体外复性极易发生二硫键的错配, 复性得率低。已报道的分泌表达体系产量普遍偏低。
为了大规模生产牛肺抑肽酶，必须减少分离纯化的步骤。因此，需要提供快速高效的牛肺抑肽酶分离方法。
发明内容
本发明的目的是提供一种牛肺抑肽酶异性制备层析介质的合成和应用，利用该介质可以大规模快速分离纯化牛肺抑肽酶，可在仅用一步层析的情况下，得到牛肺抑肽酶纯品，整个纯化步骤消耗时间短，活性回收率及比活较高，适于大规模推广应用。
为了实现上述目的，在本发明的第一方面，提供了一种牛肺抑肽酶异性制备层析介质合成方法，其特点是，所述牛肺抑肽酶异性制备层析介质由人工配体和层析介质连接而成。所述人工配体是2,4,6-三甲基-3,5-二氨基苯磺酸，所述层析介质是sepharose与Sephadex。牛肺抑肽酶异性制备层析介质由人工配体和层析介质通过1,3-二氨基-2-羟基丙烷与三聚氯氰作为间隔臂进行连接，其分子结构式为：
在本发明的第二方面，提供了一种利用上述的牛肺抑肽酶异性制备层析介质大规模纯化牛肺抑肽酶的方法，其特点是，将含有牛肺抑肽酶的原料，加入溶解液溶解、过滤、浓缩的溶液流经所述牛肺抑肽酶异性制备层析介质进行吸附，然后采用pH值6.8清洗液冲洗亲和介质，最后用PH值9.0洗脱缓冲液洗脱介质上吸附的牛肺抑肽酶，从而纯化牛肺抑肽酶，获得的牛肺抑肽酶分子量为~6kDa，比活大于15000 IU/mg.pr。
本发明的整个过程只需要经过一步亲和层析，纯化得到的人尿胰蛋白酶抑制剂纯品，其收率大于85%，比活大于15000 IU/mg.pr。整个纯化步骤消耗时间短，活性回收率及比活较高，适于大规模推广应用。
附图说明
图1为本发明纯化牛肺抑肽酶的电泳图。
具体实施方式
实施例1：基于sepharose的牛肺抑肽酶异性制备层析介质合成及应用
（1）介质合成：
取sepharose 4B 2000 g，用10倍体积的纯化水洗涤，抽干成湿饼状；加2000 ml 活化缓冲液（0.5 M NaOH, 10% 二甲亚砜，6%环氧氯丙烷），40 ℃搅拌2.5 h，然后倒入漏斗中，用10倍体积纯化水清洗，抽干成湿饼状。活化的sephadex G75介质加2000 ml 含~5% 1,3-二氨基-2-羟基丙烷的水溶液, 60 ℃搅拌 20 h，用10倍体积纯化水清洗，抽干成湿饼状。加2000 L 0.1M/L三聚氯嗪的水-丙酮溶液，使用0.1M氢氧化钠控制体系pH为7.5，4℃冰浴搅拌1h，用10倍体积纯化水清洗，抽干成湿饼状。加2000 ml 0.15mol 2,4,6-三甲基-3,5-二氨基苯磺酸水溶液，40 ℃搅拌12 h，PH维持9-10，用纯化水清洗，抽干成湿饼状抽干成湿饼状即可。
（2）纯化牛肺抑肽酶：
取牛肺抑肽酶异性制备层析介质质2000ml，装柱，用10L 0.02M磷酸盐缓冲液PH7.0平衡，取115.2百万单位抑肽酶粗品，经溶解、过滤、浓缩至体积3.5L，过以上经过平衡的层析介质吸附，然后用10L 0.02M磷酸盐缓冲液PH6.8洗涤干净，再用0.05MGLY-NaOH PH9.0甘氨酸溶液洗脱，收集洗脱流出蛋白高峰段，检测效价为100.4 百万单位，收率为87.2%，电泳纯度为95%以上，见附图1，比活为15538 IU/mg.pr。
实施例2：基于sephadex的牛肺抑肽酶异性制备层析介质合成及应用
(1) 介质合成：
取sephadex G75 2000 g，用10倍体积的纯化水洗涤，抽干成湿饼状；加2000 ml 活化缓冲液（0.5 M NaOH, 10% 二甲亚砜，6%环氧氯丙烷），40 ℃搅拌2.5 h，然后倒入漏斗中，用10倍体积纯化水清洗，抽干成湿饼状。活化的sephadex G75介质加2000 ml 含~5% 1,3-二氨基-2-羟基丙烷的水溶液, 60 ℃搅拌 20 h，用10倍体积纯化水清洗，抽干成湿饼状。加2000 L 0.1M/L三聚氯嗪的水-丙酮溶液，使用0.1M氢氧化钠控制体系pH为7.5，4℃冰浴搅拌1h，用10倍体积纯化水清洗，抽干成湿饼状。加2000 ml 0.15mol 2,4,6-三甲基-3,5-二氨基苯磺酸水溶液，40 ℃搅拌12 h，PH维持9-10，用纯化水清洗，抽干成湿饼状抽干成湿饼状即可。
(2) 纯化牛肺抑肽酶：
取牛肺抑肽酶异性制备层析介质质2000ml，装柱，用10L 0.02M磷酸盐缓冲液PH7.0平衡，取115.2百万单位抑肽酶粗品，经溶解、过滤、浓缩至体积3.5L，过以上经过平衡的层析介质吸附，然后用10L 0.02M磷酸盐缓冲液PH6.8洗涤干净，再用0.05MGLY-NaOH PH9.0甘氨酸溶液洗脱，收集洗脱流出蛋白高峰段，检测效价为97.6 百万单位，收率为84.7%，电泳纯度为95%以上，比活为16032 IU/mg.pr。
Claims (5)
1.一种牛肺抑肽酶特异性制备层析介质，其特征在于，所述牛肺抑肽酶特异性制备层析介质由人工配体和层析介质连接而成。
2.根据权利要求1所述的牛肺抑肽酶特异性制备层析介质，其特征在于，所述人工配体是2,4,6-三甲基-3,5-二氨基苯磺酸，所述层析介质是sepharose与sephadex。
3.根据权利要求1所述的牛肺抑肽酶特异性制备层析介质，其特征在于，所述牛肺抑肽酶特异性制备层析介质由人工配体和层析介质通过1,3-二氨基-2-羟基丙烷与三聚氯氰作为间隔臂进行连接，其分子结构式为：
4.一种根据权利要求1所述的牛肺抑肽酶特异性制备层析介质的合成方法，其特征在于，将所述人工配体和所述层析介质进行连接得到。
5.一种利用根据权利要求1所述的牛肺抑肽酶特异性制备层析介质大规模纯化牛肺抑肽酶的方法，其特征在于，将含有牛肺抑肽酶的原料进行溶解、超滤浓缩后的溶液流经所述牛肺抑肽酶特异性制备层析介质进行吸附，然后采用pH值6.8清洗液冲洗介质，最后用pH值9.0洗脱缓冲液洗脱介质上吸附的抑肽酶，从而纯化牛肺抑肽酶纯品，分子量为~6 kDa，其收率大于85%，比活大于15000 IU/mg.pr。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1160720A (zh) * | 1996-11-18 | 1997-10-01 | 安徽省生物研究所 | 直接从黄牛肺中制取高纯度抑肽酶的方法 |
| CN1587394A (zh) * | 2004-07-29 | 2005-03-02 | 南昌市万华生化制品有限公司 | 一种从牛肺中直接提取纯化抑肽酶的方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1160720A (zh) * | 1996-11-18 | 1997-10-01 | 安徽省生物研究所 | 直接从黄牛肺中制取高纯度抑肽酶的方法 |
| CN1587394A (zh) * | 2004-07-29 | 2005-03-02 | 南昌市万华生化制品有限公司 | 一种从牛肺中直接提取纯化抑肽酶的方法 |
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| IN SEOK YANG ET AL: "Crystal structures of aprotinin and its complex with sucrose octasulfate reveal multiple modes of interactions with implications for heparin binding", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
| XIN YU ET AL: "Affinity purification of urinary trypsin inhibitor from human urine", 《J SEP SCI》 * |
| 丁鸿等: "猪肺抑肽酶分离纯化及部分性质分析", 《广东农业科学》 * |
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