蚯蚓纤溶酶基因（Efp-Ⅰ）在大肠杆菌中的克隆、表达及活性分析

Cloning, Expression of Fibrinolytic Enzyme Gene Efp-Ⅰ from Eisenia fetida in Escherichia coli and Activity Analysis

摘要:

从GenBank查询到的蚯蚓纤溶酶（earthworm fibrinolytic enzyme，EFE）基因序列中，只有AY438624翻译的蛋白质序列与天然EfP-Ⅰ在N-端具有较高的相似性。根据该基因的5′与3′序列设计引物，通过RT-PCR从赤子爱胜蚓(Eisenia fetida)获得一个完整的基因（GenBank，DQ418454）。序列分析证明，由该基因编码蛋白质的N-末端与天然EfP-Ⅰ的N-末端的氨基酸顺序完全相同。ScanProsite prediction programs分析显示，该基因与AY438624相似性极高，二者均属于胰蛋白酶家族；不同的是，该基因编码蛋白质的序列中含有N-糖苷键的结构域，所以DQ418454是EfP-Ⅰ中的一个新基因。在此基础上，构建了该基因的原核表达载体pMAL-c2X-Efp-Ⅰ，并进行了转化、诱导和表达。Western blotting证明，表达产物同时具有MBP和EfP-Ⅰ的抗原特异性，是MBP和EfP-Ⅰ的融合蛋白（MBP-EfP-Ⅰ）。经亲和层析分离纯化的MBP-EfP-Ⅰ，在酪蛋白平板和纤维蛋白平板上表现出明显的纤溶酶活性。

Abstract:

Earthworm fibrinolytic enzyme (EFE) is a group of protease having fibrinolytic and plasminogen-activator activities isolated from earthworm. Molecular biology research showed that there were 21 EFE coding sequences, in which only one sequence, AY438624， whose translated protein had similar N-terminal amino-acid sequence toEfP-Ⅰpurified from Eisenia fetida. To obtain coding sequence ofEfP-Ⅰ, we designed specific primers according to 5′ and 3′ sequences of AY438624. A new DNA sequence was obtained by RT-PCR, sequence analysis showed that the protein translated from the coding sequence had identical N-terminal amino-acid sequence with EfvP-Ⅰpurified from Eisenia fetida and Lumbricus rubellus. Analysis by using ScanProsite prediction programs proved that the sequence had high similarity to AY438624 and belonged to trypsin family of serine protease. But there was difference between two sequences, that was there was a domain of characteristic amino acids of N-glycosylation site Asn-Xaa-Ser/Thr（N-x-S/T）in the new sequence（DQ418454）. Then the expressed vector pMAL-c2X-Efp-Ⅰwas constructed by cloning the gene into the plasmid pMAL-c2X，and was transformed to E.coli TB1. After induction and expression of the recombinant, the product MBP-EfP-Ⅰ was purified by MBP affinity chromatography. Western blotting analysis showed that the product reacted with both anti-MBP and anti-EfP-Ⅰ-1 serum. Casein plate test and fibrin plate test showed that the protein expressed had fibrinolytic activity.