Li Rong-Gui, Qian Dong-Meng, Guo Dao-Sen, Du Gui-Cai, Yan Zhi-Yong, Wang Bin
Department of Biology, Qingdao University, Qingdao 266071, China.
Acta Biochim Biophys Sin (Shanghai). 2006 Aug;38(8):543-8. doi: 10.1111/j.1745-7270.2006.00192.x.
The cDNA encoding a protease of Perinereis aibuhitensis Grube (PPA) was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMAL-PPA. pMAL-PPA was introduced into Escherichia coli BL21(DE3) and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-beta-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.
克隆了编码双齿围沙蚕蛋白酶（PPA）的cDNA。推导的氨基酸序列分析表明，该蛋白与大豆孢囊线虫丝氨酸蛋白酶C末端氨基酸169 - 246具有49%的同一性。Northern印迹分析表明，该cDNA可与从海蚯蚓分离的约260个碱基的mRNA杂交。通过聚合酶链反应扩增该cDNA，并克隆到pMAL-p2中构建表达载体pMAL-PPA。将pMAL-PPA导入大肠杆菌BL21(DE3)，通过异丙基-β-D-硫代半乳糖苷诱导实现与麦芽糖结合蛋白融合的PPA的过表达。融合蛋白通过直链淀粉树脂柱亲和层析和二乙氨基乙基 - 琼脂糖4B柱离子交换层析进行纯化。用纯化的蛋白免疫兔子并制备抗血清。Western印迹分析表明，该抗体可与从海蚯蚓中提取的约9 kDa的蛋白发生反应。重组PPA的活性分析表明它可能是一种纤溶酶原激活剂。
