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徐华,徐昕.抗凝溶栓重组葡激酶-水蛭素融合蛋白的构建与表达[J].内科急危重症杂志,2015,21(21):
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| DOI： |
| CN KeyWords: 抗凝 溶栓 重组葡激酶-水蛭素融合蛋白 酵母表达体系 |
| EN KeyWords:Anticoagulation thrombolysis Sak - hirudin fusion protein yeast expression system |
| Fund Project:湖北省教育厅课题：抗凝溶栓Hir-RGD-hPLMsp融合蛋白的构建、表达和活性研究，编号：B2013105 |
| Author:xuhua |
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| CN Abstract: |
| 目的：通过间接实验探讨抗凝溶栓重组葡激酶-水蛭素融合蛋白的构建效果与抗凝溶栓活性表达情况。方法：在间接实验中，选择pBV-重组葡激酶-水蛭素融合蛋白质质粒进行酵母大量表达，然后进行两级纯化分析；去纯化的单位进行抗凝溶栓的检测。结果：工程菌酵母发酵培养诱导1 h 后就可以检测到重组葡激酶-水蛭素融合蛋白表达，表达量随发酵时间的增加而递增。两级纯化的重复性非常好，纯度经RP-HPLC检定在98%以上。重组葡激酶-水蛭素融合蛋白的溶栓活性明显高于水蛭素与葡激酶的单纯溶栓活性(P<0.05)。重组葡激酶-水蛭素融合蛋白具有更强的纤溶酶原激活活性，明显高于水蛭素与葡激酶的单纯纤溶酶原激活活性(P<0.05)。结论：通过间接实验建立的重组葡激酶-水蛭素融合蛋白生产工艺具有很好的诱导与纯化效果，而重组葡激酶-水蛭素融合蛋白能发挥比较好的抗凝与溶栓作用。 |
| EN Abstract: |
| Objective: To investigate the construction and expression of anticoagulation and thrombolysis staphylokinase-hirudin fusion protein by the indirect experiments. Methods: In the indirect experiments, The pBV- taphylokinase-hirudin fusion protein plasmid were given abundantly yeast expressing, then were given the two purification analysis; the purification units were given the anticoagulant and thrombolytic detection. Results: When the yeast fermentation engineering bacteria were induced 1 h, it can be detected staphylokinase-hirudin fusion protein expression, the expression were related to the increasing fermentation time. Two-stage purification repeatability was very good, The accredited by RP-HPLC purity were more than 98%. The staphylokinase-hirudin fusion protein thrombolytic activity were significantly higher than hirudin and Staphylokinase simple thrombolytic activity (P <0.05). And the staphylokinase-hirudin fusion protein had stronger plasminogen activator activity that were significantly higher than hirudin and Sak's simple plasminogen activator activity (P <0.05). Conclusion: The staphylokinase-hirudin fusion protein production process has better induction and purification effects by the indirect experiments, and the staphylokinase-hirudin fusion protein can play better anticoagulation and thrombolysis. |
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