人t-PAP区在大肠杆菌中表达及其活性研究

人t-PAP区在大肠杆菌中表达及其活性研究 Expression and Purification of t-PA P Region and Its Activity 赵月娥 ZHAO Yue-e 1 first-author 郜金荣 GAO Jin-rong 1 叶林柏 YE Lin-bai 1 徐进平 XU Jin-ping 1 刘源洁 LIU Yuan-jie 1 吴正辉 WU Zheng-hui 1 武汉大学病毒研究所,湖北,武汉,430072 武汉大学病毒研究所,湖北,武汉,430072 Institute of Virology,Wuhan University Institute of Virology,Wuhan University 用 PCR方法获得编码人组织型纤溶酶原激活物 （tissure- type plasminogen activator,t- PA） C端 P区肽段的 DNA,并经测序证实 ,长 810 bp,含有全部的 t- PA的丝氨酸蛋白酶编码序列 .将这段序列克隆到表达载体p QE30中转化大肠杆菌 JM10 9菌株 ,经 SDS- PAGE检测 :转化菌株中表达出分子量约 2 9× 10 3的蛋白 ,用纤维平板法测定激活纤溶活性 ,结果表明表达的 t- PA蛋白 C端 P区具有很强的激活纤溶蛋白酶原的活性 .证实 t- PA蛋白 C端 P区的酶结构区的功能是独立的 ,其生物活性不受其它区域影响 The DNA fragment encoding t-PA P region protein was obtained by PCR,using plasmid pTAhtpa（F） harborong the full length t-PA cDNA as template. This P region fragmemt was cloned into pQE30 and transformed to JM109 strain.P region proteins fused to 6 His peptides at its N terminus with molecular weight of 29×10 3 were expressed in JM109 induced by IPTG.The expressed protein were purified by Ni-NTA-agarose and were studied its activity by fibrin plate activity assay（FPAA）.The results show that the function of t-PA P region is indepent for other regions of t-PA. and it has significant activity to stimulate plasminogen. t-PAP区 表达 活性 t-PA P region expression activity Q78 湖北省科委重点项目 !（0 0 15 10 49） 2001-02-01 2021-04-01 2