【摘要】 利用同源克隆方法和c DNA末端快速扩增(RACE)技术,从皱纹盘鲍(Haliotis discus hannai)肌肉组织中克隆得到基质金属蛋白酶-1基因(Hdh-MMP-1)c DNA全长序列(Gen Bank登录号:KR537291)。结果表明,Hdh-MMP-1 c DNA全长2136 bp,其中ORF长度为1551 bp,编码区含有516个氨基酸残基,预测其分子质量为58.94 ku,理论等电点为5.99。Hdh-MMP-1具有MMPs家族典型的N-端前肽区、催化区、铰链区和C-端类血红素结合区。利用SOPMA和SWISS-MODEL软件对该基因编码蛋白质高级结构进行了预测分析。氨基酸序列相似性结果显示,Hdh-MMP-1不仅与多种生物MMP-1基因具有序列相似性,且与某些软体动物和虫类的MMP-14及MMP-19也具有序列相似性。多序列比对结果显示,Hdh-MMP-1与红螺鲍、杂色鲍、美洲牡蛎的MMP-1相似性分别为95.29%、82.35%、38.85%。随后,构建了表达载体p ET28a-cat MMP-1,利用大肠杆菌原核表达系统,在大肠杆菌BL21(DE3)中成功对该蛋白质催化区进行异源表达。更多还原

【Abstract】 Matrix metalloproteinase-1 gene( Hdh-MMP-1) from the muscle of abalone( Haliotis discus hannai) was cloned by RT-PCR and RACE technology( Gen Bank accession number KR537291). The fulllength of c DNA Hdh-MMP-1 was 2136 bp,including an open reading frame( ORF) of 1551 bp coding 516 amino acid residues with an estimated molecular weight of 58. 94 ku and a theoretical p I of 5. 99. The HdhMMP-1 contains N-terminal prodomain,catalytic domain,hinge region and C-terminal hemopexin domain,which was typical in matrix metalloproteinases. Higher-order structure of Hdh-MMP-1 was predicted using SOPMA and SWISS-MODEL. The results of multiple sequence alignment analysis showed that there was about 38. 9% ~ 95. 3% identities in amino acid sequence with some other organisms. Prokaryotic expression plasmid p ET28a-cat MMP-1 containing the catalytic domain of MMP-1 was constructed. The recombinant MMP-1 was successfully expressed in Escherichia coli BL21 cells.更多还原