CN1077390A - 一种新型溶栓药物及其制备 - Google Patents
一种新型溶栓药物及其制备 Download PDFInfo
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Abstract
本发明涉及一种新型溶血栓药物及其制备方
法。从蛇毒得到了一种新的纤维蛋白(原)溶解酶,并
将此酶与抗人纤维蛋白单克隆抗体连接,制成蛇毒溶
栓导向药物。
Description
本发明涉及一种新型溶血栓药物及其制备方法。
尖吻蝮蛇毒中提取的纤维蛋白(原)溶解酶可降解血栓的主要成分纤维蛋白。目前报道的该酶仅作用于纤维蛋白原的α(A)链,而对β(B)链无作用(l.Ouyang C,Huang T-F.Purification and characterization of the fibrinolytic principle of Agkistrodon acutus venom.Biochim Biophys Acta 1976;439:146-53.2.Ouyang C,Huang T-F.The properties of the purified fibrinolytic principle from Agkistrodon acutus snake venom.Toxicon 1977;15:161-7)。
尿激酶也是一个溶血栓剂,有人将尿激酶与抗人纤维蛋白单克隆抗体连接,制成单抗导向溶血栓剂,其纤溶效率比单纯尿激酶增加100倍(Bode C,et al.Antibody-directed urokinase:a specific fibrinolytic agent,Science 1985;229:765)。但是尿激酶价格昂贵。
本发明的目的是从蛇毒中得到一种新的纤维蛋白(原)溶解酶,并将此酶与抗人纤维蛋白单克隆抗体共价连接,制成单抗导向蛇毒溶血栓剂。
本发明从尖吻蝮蛇毒中得到一个对纤维蛋白原的α(A)和β(B)链均有降解作用的酶,分子量为25000~28000,等电点为8.00~9.00。并从含有抗人纤维蛋白β链N-端七肽单克隆抗体的小鼠腹水中亲和层析提纯该单克隆抗体。将上述纤维蛋白(原)溶解酶与双功能交连剂N-琥珀酰亚胺基3-(2-吡啶二硫基)丙酸酯(简称SPDP,下同)连接,再与经巯基修饰的单克隆抗体连接,最后得到纤维蛋白(原)溶解酶与抗人纤维蛋白β-链N-端七肽单克隆抗体的连接物。
本发明的主要优点是,所得到的溶栓药物其体外溶解血浆凝块活力较单纯纤维蛋白(原)溶解酶高,特异性好。与国外实验室现有的尿激酶抗体导向溶血栓剂相比,本发明所得到的溶血栓剂成本低,原料易得,更具临床应用价值。
实施例:
1 纤维蛋白(原)溶解酶的鉴定:
分子量测定:采用十二烷基硫酸钠(简称SDS,下同)-聚丙烯酰胺凝胶电泳方法。凝胶浓度12.5%,照常规方法电泳,标准蛋白采用Pharmacia公司电泳分子量测定试剂盒。测得纤维蛋白(原)溶解酶的分子量为26800。
等电点测定:采用等电聚焦电泳法。照常规方法进行等电聚焦电泳。电泳完毕,以1厘米为间隔,测量电泳胶从阳极到阴极各点的pH值,绘制pH梯度曲线,从曲线求出纤维蛋白(原)溶解酶的等电点。测得纤维蛋白(原)溶解酶的等电点为8.65。
对纤维蛋白的溶解方式:将纤维蛋白(原)溶解酶与纤维蛋白凝块在37℃反应,取反应液作SDS-聚丙烯酰胺凝胶电泳,胶浓度为12.5%。反应15分钟后,纤维蛋白的α亚基全部降解,反应60分钟后,β亚基大部分降解。
对纤维蛋白原的溶解方式:将纤维蛋白(原)溶解酶与纤原在37℃反应,取反应液作SDS-聚丙烯酰胺凝胶电泳,胶浓度为7.5%。反应15分钟后,纤维蛋白原的α亚基全部降解。60分钟后,β亚基大部分降解。
2 抗人纤维蛋白β链N-端七肽单克隆抗体与纤维蛋白(原)溶解酶连接物的制备:
抗纤维蛋白β链N-端七肽单克隆抗体的纯化:取小鼠腹水5毫升,用0.45μ膜过滤。将滤液加于用0.1M pH8.0的磷酸缓冲液平衡好的蛋白-A亲和层析柱上,用相同缓冲液洗脱至280nm的吸光度<0.01,改用0.1M,pH3.5的柠檬酸钠洗脱(洗脱组分用2M Tris-Hcl pH6.0,含0.15M Nacl的溶液中和)。将收集组分对0.005M pH8.0的磷酸缓冲液透析24hr,测抗体含量,冰干备用。
抗纤维蛋白β链N-端七肽单克隆抗体与纤维蛋白(原)溶解酶的连接:
纤维蛋白(原)溶解酶-SPDP连接物的制备:取纤维蛋白(原)溶解酶8毫克,溶于1毫升pH7.4的磷酸钠溶液中,加入20倍的SPDP无水乙醇溶液,室温搅拌25分钟,反应液对pH7.4的磷酸钠溶液(含0.2M精氨酸,0.01%Tween 80)透析12hr。
单克隆抗体-巯基化试剂(iminothiolane)连接物的制备:取抗体4.43毫克,溶于0.85毫升pH7.4的磷酸钠溶液中,对相同溶液透析6hr,加入100倍的iminothiolane(溶与25mM pH9.3的硼酸钠溶液中),室温搅拌25分钟,过Sephadex G-25脱盐柱。(预先用pH6.6的磷酸缓冲液平衡)。
单克隆抗体-纤维蛋白(原)溶解酶的连接、纯化:将上述方法得到的纤维蛋白(原)溶解酶-SPDP连接物溶液与抗体-iminothiolane溶液混和,室温搅拌过夜,加10倍量的马来酰亚氨溶液终止反应,反应液过Sephacryl S-200凝胶过滤柱,用pH6.6的磷酸缓冲液洗脱,收集洗脱峰。
所用的单克隆抗体来源于中国医学科学院放射医学研究所提供的含抗人纤维蛋白β链N-端七肽单克隆抗体的小鼠腹水。
3 抗纤维蛋白β链N-端七肽单克隆抗体与纤维蛋白(原)溶解酶连接物的鉴定:
采用放射性同位素125I标记的纤维蛋白(原)溶解酶,进行各步反应,将反应液过Sephacryl S-200凝胶过滤柱(予先用标准分子量蛋白标定),测定各组分分子量和放射性活性,确定具有放射性活性,分子量约300KD的组分为单克隆抗体与纤维蛋白(原)溶解酶的连接物。
4 体外溶解血浆凝块活力测定:
放射性标记血浆凝块的制备:取1毫升枸橼酸钠抗凝人血浆,与痕量125I标记的纤维蛋白原、50微升CaCl2(0.5M)混和,加25微升凝血酶(100NIH U/ml),将混和物吸进内径4mm胶管内,在37℃保温1小时。按1厘米长度切割,推进平皿,用0.15M NaCl洗60分钟,测量凝块的放射性计数。
取单抗-纤维蛋白(原)溶解酶溶液1毫升,加入2毫升血浆,从加入放射性标记的血凝块起,1分钟后,取反应液50微升测定放射性计数,作为本底,分别于0.5、1、1.5、2hr取50微升反应液测量放射性记数。
取同样活力单位的纤维蛋白(原)溶解酶溶液1毫升,加入2毫升血浆,加入放射性标记的血凝块,照上述方法测量反应液的放射性记数,分别计算血块溶解率。单抗-纤维蛋白(原)溶解酶溶解血浆凝块活力较单纯纤维蛋白(原)溶解酶高2.5~4.9倍。
Claims (5)
1、一种新型溶血栓药物,其特征为含有从蛇毒中提取的酶和单克隆抗体。
2、根据权利要求1所述的酶是尖吻蝮蛇毒酶。
3、根据权利要求1所述的酶,其分子量为25000~28000,等电点为8.00~9.00。
4、根据权利要求1所述的酶既能溶解纤维蛋白原的α(A)链,又能溶解β(B)链。
5、根据权利要求1所述的溶血栓药物的制备是将蛇毒溶栓酶与单克隆抗体连接而得。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1060806C (zh) * | 1997-11-12 | 2001-01-17 | 中国科学院上海生物化学研究所 | 尖吻蝮蛇蛇毒类凝血酶的基因序列 |
CN1102173C (zh) * | 1999-03-26 | 2003-02-26 | 福建医科大学 | 蕲蛇酶及其生产工艺 |
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1993
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1060806C (zh) * | 1997-11-12 | 2001-01-17 | 中国科学院上海生物化学研究所 | 尖吻蝮蛇蛇毒类凝血酶的基因序列 |
CN1102173C (zh) * | 1999-03-26 | 2003-02-26 | 福建医科大学 | 蕲蛇酶及其生产工艺 |
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