重组刺桐胰蛋白酶抑制剂的制备及活性鉴定(英文)  

Recombinant Preparation and Characterization of Erythrina Trypsin Inhibitor

作　　者：马志峰[1] 孙自勇[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）》2005年第5期462-469,共8页Journal of Nanjing University（Natural Science）

摘　　要：刺桐胰蛋白酶抑制剂(ETI)是一种Kunitz型胰蛋白酶抑制剂,能够抑制胰蛋白酶及组织型纤溶酶原激活剂的活性,因此ETI可以作为配体与固定相偶联用于亲和层析.利用化学合成及聚合酶链式反应(PCR)的方法获得ETI的编码基因,然后用NdeI/SacI酶切并插入载体pET25b ( +)中构建重组表达质粒pET25b ( +)/ETI .重组表达质粒转化大肠杆菌BL21(DE3)后,用IPTG诱导使重组ETI(rETI)过量表达形成包涵体.包涵体经过洗涤、复性及CM离子交换柱纯化,每升培养液可获得14 mgrETI ,其纯度超过92 %.活性测定表明,rETI对凝血酶及尿激酶的活性没有抑制作用,但能显著抑制胰蛋白酶、瑞替普酶及纤溶酶的活性,抑制常数Ki分别为5 .14×10-9,9 .4×10-8和2 .08×10-8mol/L.Erythrina trypsin inhibitor （ETI） is a Kunitz-type trypsin inhibitor, which inhibits both trypsin and tissue-type plasminogcn activator, Immobilized ETI is an effective ligand for the affinity purification of these enzymes. In the study, the DNA encoding ETI gene was chemically synthesized and amplified with polymerase chain reaction （PCR）. The amplified ETI gene was digested with Ndel/Sacl and inserted into the pET25b （ ＋ ） vector to construct the rETI plasmid. The recombinant expression plasmid was transformed into E. coli strain BL21 （DE3）, and induced with IPTG to overexpress recombinant ETI as insoluble inclusion belly. After inclusion body purification, renaturation and CM cation-exchange chromatography, 14 mg rETI were obtained from one liter of culture medium （3.5 g pellet mass） with homogeneity greater than 92%. Kinetic analysis showed that rETI had no inhibitory effect on thrombin and urokinase, but significantly inhibit the activity of trypsin, reteplase and plasmin with a Ki of 5.14×10^-9, 9.4×10^-8 and 2.08×10^-8 mol/L respectively.

关 键 词：刺桐胰蛋白酶抑制剂(ETI) 胰蛋白酶 瑞替普酶 纤溶酶 抑制 

分 类 号：Q78[生物学—分子生物学]