重组tPA及其突变体纤溶活性的比较

重组tPA及其突变体纤溶活性的比较 The Comparison of the Thrombolytics Activity between Recombinant tPA and Its Mutated Form 李宝宗 LI Bao-zong 1 2 first-author 郑竑 ZHENG Hong 1 2 叶林柏 YE Lin-bai 1 2 郜金荣 GAO Jin-rong 1 2 佘应龙 SHE Ying-long 1 2 贺石汉 WU Zheng-hui 1 2 吴正辉 1 2 武汉大学生命科学学院 武汉大学生命科学学院 College of Life Sciences, Wuhan University College of Life Sciences, Wuhan University 武汉大学生命科学学院,湖北,武汉,430072 武汉大学生命科学学院,湖北,武汉,430072 将重组基因rtPA及其突变体rtPAm分别克隆到原核表达载体pET his上,通过平板筛选,双酶切和PCR鉴定获得阳性克隆子,重组质粒转化大肠杆菌BL21（DE3）.经IPTG诱导,表达出的两种tPA蛋白都形成不可溶的包涵体.SDS PAGE结果显示两种tPA蛋白分子量为4.0×104左右,表达产量较高.包涵体经尿素变性,透析复性后柱层析纯化,纤溶平板法测定激活纤溶蛋白酶的活性,结果显示rtPAm比活性比rtPA高70%左右. The recombinant tPA gene and its mutated form were subcloned into pET-his prokaryotic expression vector and screened with Amp-LB plate .The recombinants plasmids were confirmed by double digestion and PCR and then transformed into E.coli BL21（DE3） . The proteins of both kinds of tPA were expressed by IPTG induction led to formation insoluble inclusion aggregates. SDS-PAGE showed fat bands of molecular mass of （4.0×10 4 ） referred to the both expressed proteins. The inclusion bodies were denatured, renatured and purified by Ni-NTA-agarose and the activities of the purified proteins were measured by fibrin plate activity assay. Our results showed that the activity of the mutated tPA was 70% higher than the wild type. 组织型纤溶酶原激活物 克隆 表达 活性比较 tissue-type plasminogen activator（tPA） cloning expression the comparison of activity TQ464 国家科技攻关项目（2001BA749C）;湖北省重点项目（2001AA304B01） 2004-06-01 2021-04-01 6