| 李飞 李忠培 占志 王冬雪 朱美财.抗PAI 抑制作用的纤溶酶原激活剂突变体的活性研究[J].,2014,14(16):3029-3032 |
| 抗PAI 抑制作用的纤溶酶原激活剂突变体的活性研究 |
| Expression and Activity of Tissue-type Plasminogen Activator MutantReteplase with Deletion of PAI-1 Binding Sites |
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| DOI： |
| 中文关键词: 组织型纤溶酶原激活剂 突变体 蛋白表达 纯化 酶动力学 |
| 英文关键词: t-PA Mutant Protein expression Purification Enzyme kinetics |
| 基金项目:国家“重大新药创制”科技重大专项（2009 ZX09103-603） |
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| 中文摘要: |
| 目的：重组表达抗PAI 抑制作用的t-PA 突变体，经诱导表达、复性、纯化后进行生物学活性和酶动力学分析。方法：构建
pBV220-tpa 重组表达质粒，经DNA测序确认后，转化至大肠杆菌DH5a，温控诱导表达，凝胶过滤法对包涵体蛋白进行初步纯化，
复性后，过刺桐胰蛋白酶亲和层析柱纯化，酶动力学分析其活性。结果：测序证实,t-PA突变体的DNA序列正确,表达蛋白占总菌
体蛋白的30%,经纯化后纯度达90％以上，比活性为7.0× 108IU/mg，t-PA 突变体与PAI-1 反应后，其活性未受到抑制。t-PA 突变
体酶的米氏常数Km为0.5298，最大水解速度Vmax为0.0595。结论：经生物学活性测定, 表达蛋白能够明显抵抗PAI 的抑制作用，
并具有良好的生物活性，该突变体有可能成为用量更少、疗效更佳的新型溶栓药物。 |
| 英文摘要: |
| Objective: To construct a prokaryotic expression vector for tissue-type plasminogen activator mutantreteplase with
deletion of a PAI-1 binding site, and valuate its biological activity by enzyme kinetics analysis.Methods:The recombinant plasmid
pBV220-t-PA was transformed into E.coli for expression after DNA sequencing. The recombinant mutant t-PA inclusion body was
purified with gel filtration, and then was purified with erythrina trypsin affinity chromatography after refolding.Results:The mutant t-PA
encoding sequence was confirmed by DNA sequencing. The expression product was about 30% of whole lysis protein. After purified
with gel filtration, the protein purity was more than 95%. The specific activity of mutant t-PA with deletion of PAI-1 binding site was
4.2× 105 IU/mg. This protein was not inhibited by PAI-1. The Kmwas 0.3298 umol·L-1 and Vmax of mutant t-PA was 0.0476 umol·min-1·
g-1.Conclusion:Recombinant mutant t-PA with deletion of PAI-1 binding site prepared from E.coli could resist the inhibitions of PAI-1,
and harbor a better biological activity. |
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