抗PAI抑制作用的纤溶酶原激活剂突变体的活性研究

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。

Expression and Activity of Tissue-type Plasminogen Activator MutantReteplase with Deletion of PAI-1 Binding Sites

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 4.2×10^5IU/mg. This protein was not inhibited by PAI-1. The Km was 0.3298 μmol .L^-1 and Vmax of mutant t-PA was 0.0476 μmol.min^-1.g^-1. Conclusion： Recombinant mutant t-PA with deletion of PAl-1 binding site prepared from E.coli could resist the inhibitions of PAI-1, and harbor a better biological activity.