重组人尿激酶原氨基末端片段的制备及活性测定  

Expression and Characterization of Recombinant Human Amino-terminal Fragment of Prourokinase

作　　者：汪晶[1] 陈新园[1] 孙自勇[1] 姚宏伟[1] 陈均勇[1] 刘建宁[1] 

机构地区：[1]南京大学分子医学研究所,南京210093

出　　处：《南京大学学报（自然科学版）》2004年第1期66-74,共9页Journal of Nanjing University（Natural Science）

基　　金：教育部重大科技研究项目(00-03);杰出青年基金(30025011);教育部优秀青年教师资助计划

摘　　要：　尿激酶原的N 末端135个氨基酸片段包含了表皮生长因子结构域和环柄结构域,参与了尿激酶原和其受体的结合以及尿激酶原诱导的化学趋化活性.利用RT PCR,从人脐带静脉内皮细胞中克隆ATF基因.将ATF基因插入pET 29a(+)中构建表达质粒pET 29a(+)/ATF,并转化至大肠杆菌BL21(DE3)中,经IPTG诱导表达及纯化后,从每升培养液中获得15mgrhATF,其纯度超过95%.活性测定结果表明rhATF能够阻断尿激酶原与尿激酶受体的结合,并能抑制尿激酶对纤溶酶原以及纤溶酶对尿激酶原的激活.ATF is the amino terminal Ser1 Lys 135 fragment of pro urokinase (proUK) containing an epidermal growth factor like (EGF) domain and a kringle domain. It is critically involved in some important functions of proUK. The EGF domain participates in receptor binding and promoting cell adhesion and migration. The kringle domain is associated with the chemotactic action, anti angiogenic and anti tumor activities. ATF can bind to the urokinase type plasminogen activator receptor (uPAR) with high affinity (K_d=4×10^(-10) mol/L) but it is enzymatically inactive, and specifically prevent binding of pro uPA synthesized by tumor cells to the receptors, which inhibits the proliferation and invasion of tumor cells. Fabbrini MS et al. has constructed a chimera consisting of the human ATF fused to a cytotoxin saporin isoform (SAP 3) to target and destroy tumor cells. Therefore, ATF is a potential regent against cancer. Recent studies suggest that ATF also inhibits the replication, assembly and budding of HIV 1, providing a novel therapeutic strategy for AIDS.  In this report, the cDNA fragment encoding ATF was cloned from the endothelial cells of human umbilical vein (HUVEC) by reverse transcriptase polymerase chain reaction (RT PCR). The ATF gene was inserted between the NdeI XhoI sites of pET 29a(+) vector to construct recombinant expression plasmid pET 29a(+)/ATF. The host cell strain E. coli BL21(DE3) transformed with pET 29a(+)/ATF was induced with IPTG to overexpress recombinant human ATF (rhATF) as insoluble inclusion body. The amount of rhATF expressed accounts for 20% of total bacterial protein. After puritication of inclusion body, renaturation, CM cation exchange chromatography and Superdex G 75 gel filtration, 15 mg rhATF was obtained from one liter of culture medium with homogeneity greater than 95%. The results of zymograph assay demonstrated that the purified rhATF could block the binding of proUK to the uPAR on U973 cell surface. Kinetic analysis showed that both Glu plasminogen activation by urokinase and

关 键 词：ATF 尿激酶受体 纯化 抑制 制备 活性测定 多肽 

分 类 号：Q5[生物学—生物化学] Q78