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1. 发明申请

US20090087421A1 Novel Recombinant Staphylokinase Derivatives and the Preparations and Applications thereof 审中-公开
标题翻译：新型重组葡激酶衍生物及其制备及应用
公开(公告)号：US20090087421A1
公开(公告)日：2009-04-02
申请号：US12185601
申请日：2008-08-04
申请人： Houyan Song , Gang Song
发明人： Houyan Song , Gang Song
IPC分类号： A61K38/45 , C12P21/04 , C12N15/11 , C12N15/00 , C12N1/21
CPC分类号： C07K14/31
摘要： The present invention relates to the biotechnology field, more particularly, to novel recombinant staphylokinase (RGD/KGD-Sak) derivatives and the preparation the thereof. The derivatives, have a low polymerizing ability, low immunogenicity and a bifunctionality of thrombolytics and anticoagulant. Based on the line, structural analysis of the monomer and dimer of recombinant staphylokinases and their biochemical properties, we designed two novel bifunctional staphylokinase molecular structures. Mutant genes were constructed by PCR site-directed mutagenesis which were then recombined with a prokaryotic vector and used to transform E. coli. Engineered strains with a high expression level were selected by screening and propagated by fermentation, followed by disruption of the cells, centrifugation to collect inclusion bodies, renaturation, and purification of RGD/KGD-SAK through a two-step method. After lyophilized, the polymerizing ability and immunogenicity of the products decreased significantly. The derivatives can not only activate fibrinogen to lyse thrombus, but also significantly inhibit the platelet aggregation induced by ADP, suggesting that they have the bifunctionality of thrombolytics and anticoagulant.
摘要翻译：本发明涉及生物技术领域，更具体地涉及新型重组葡激酶（RGD / KGD-Sak）衍生物及其制备。衍生物具有低聚合能力，低免疫原性和溶栓剂和抗凝血剂的双功能性。基于线条，重组葡萄球菌激酶单体和二聚体的结构分析及其生物化学性质，设计了两种新型双功能葡萄球菌激酶分子结构。突变基因通过PCR定点诱变构建，然后与原核载体重组，并用于转化大肠杆菌。选择具有高表达水平的工程菌株，通过发酵筛选和繁殖，随后破碎细胞，通过两步法离心收集RGD / KGD-SAK的包涵体，复性和纯化。冻干后，产物的聚合能力和免疫原性显着降低。该衍生物不仅可以激活纤维蛋白原裂解血栓，还可以显着抑制ADP诱导的血小板聚集，这表明它们具有溶血栓剂和抗凝血剂的双功能性。

2. 发明授权

US07407789B2 Recombinant staphylokinase derivatives and the preparation and applications thereof 失效
标题翻译：重组葡激酶衍生物及其制备及应用
公开(公告)号：US07407789B2
公开(公告)日：2008-08-05
申请号：US10182160
申请日：2001-01-23
申请人： Houyan Song , Gang Song
发明人： Houyan Song , Gang Song
IPC分类号： C12N9/52 , C12N9/00 , C12N9/24 , C12N9/48 , C12N9/50 , C12N15/00 , A61K38/48 , A61K39/085 , A61K39/02 , A61K38/00 , C07K1/00
CPC分类号： C07K14/31
摘要： The present invention relates to the biotechnology field, more particularly, to novel recombinant staphylokinase (RGD/KGD-Sak) derivatives and the preparation the thereof. The derivatives, have a low polymerizing ability, low immunogenicity and a bifunctionality of thrombolytics and anticoagulant. Based on the trimensional structural analysis of the monomer and dimer of recombinant staphylokinases and their biochemical properties, we designed two novel bifunctional staphylokinase molecular structures. Mutant genes were constructed by PCR site-directed mutagenesis, which were then recombined with a prokaryotic vector and used to transform E. coli. Engineered strains with a high expression level were selected by screening and propagated by fermentation, followed by disruption of the cells, centrifugation to collect inclusion bodies, renaturation, and purification of RGD/KGD-SAK through a two-step method. After lyophilized, the polymerizing ability and immunogenicity of the products decreased significantly. The derivatives can not only activate fibrinogen to lyse thrombus, but also significantly inhibit the platelet aggregation induced by ADP, suggesting that they have the bifunctionality of thrombolytics and anticoagulant.
摘要翻译：本发明涉及生物技术领域，更具体地涉及新型重组葡激酶（RGD / KGD-Sak）衍生物及其制备。衍生物具有低聚合能力，低免疫原性和溶栓剂和抗凝血剂的双功能性。基于重组葡激酶单体和二聚体的三维结构分析及其生化特性，设计了两种新型双功能葡萄球菌激酶分子结构。突变基因通过PCR定点诱变构建，然后与原核载体重组，并用于转化大肠杆菌。选择具有高表达水平的工程菌株，通过发酵筛选和繁殖，随后破碎细胞，通过两步法离心收集RGD / KGD-SAK的包涵体，复性和纯化。冻干后，产物的聚合能力和免疫原性显着降低。该衍生物不仅可以激活纤维蛋白原裂解血栓，还可以显着抑制ADP诱导的血小板聚集，这表明它们具有溶血栓剂和抗凝血剂的双功能性。

3. 发明申请

US20050202000A1 Novel remombinant staphylokinase derivant and its preparation method and application thereof 失效
标题翻译：新型重组葡激酶衍生物及其制备方法及应用
公开(公告)号：US20050202000A1
公开(公告)日：2005-09-15
申请号：US10182160
申请日：2001-01-23
申请人： Houyan Song , Gang Song
发明人： Houyan Song , Gang Song
IPC分类号： C07K14/31 , C12N15/54 , A61K38/48 , C07H21/04 , C12N9/64 , C12N15/09 , C12P21/06
CPC分类号： C07K14/31
摘要： The present invention relates to the biotechnology field, more particularly, to novel recombinant staphylokinase (RGD/KGD-Sak) derivatives and the preparation the thereof. The derivatives, have a low polymerizing ability, low immunogenicity and a bifunctionality of thrombolytics and anticoagulant. Based on the line, structural analysis of the monomer and dimer of recombinant staphylokinases and their biochemical properties, we designed two novel bifunctional staphylokinase molecular structures. Mutant genes were constructed by PCR site-directed mutagenesis which were then recombined with a prokaryotic vector and used to transform E. coli. Engineered strains with a high expression level were selected by screening and propagated by fermentation, followed by disruption of the cells, centrifugation to collect inclusion bodies, renaturation, and purification of RGD/KGD-SAK through a two-step method. After lyophilized, the polymerizing ability and immunogenicity of the products decreased significantly. The derivatives can not only activate fibrinogen to lyse thrombus, but also significantly inhibit the platelet aggregation induced by ADP, suggesting that they have the bifunctionality of thrombolytics and anticoagulant.
摘要翻译：本发明涉及生物技术领域，更具体地涉及新型重组葡激酶（RGD / KGD-Sak）衍生物及其制备。衍生物具有低聚合能力，低免疫原性和溶栓剂和抗凝血剂的双功能性。基于线条，重组葡萄球菌激酶单体和二聚体的结构分析及其生物化学性质，设计了两种新型双功能葡萄球菌激酶分子结构。突变基因通过PCR定点诱变构建，然后与原核载体重组，并用于转化大肠杆菌。选择具有高表达水平的工程菌株，通过发酵筛选和繁殖，随后破碎细胞，通过两步法离心收集RGD / KGD-SAK的包涵体，复性和纯化。冻干后，产物的聚合能力和免疫原性显着降低。该衍生物不仅可以激活纤维蛋白原裂解血栓，还可以显着抑制ADP诱导的血小板聚集，这表明它们具有溶血栓剂和抗凝血剂的双功能性。

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