皱纹盘鲍Hdh-MMP-1基因cDNA的克隆及原核表达

利用同源克隆方法和cDNA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)cDNA全长序列(GenBank登录号:KR537291)。结果表明，Hdh-MMP-1 cDNA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pET28a-catMMP-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。

cDNA Cloning and Prokaryotic Expression of Matrix Metalloproteinase-1 from Haliotis discus hannai

Matrix metalloproteinase-1 gene (Hdh-MMP-1)from the muscle of abalone(Haliotis discus hannai) was cloned by RT-PCR and RACE technology(GenBank accession number KR537291).The full-length of cDNA 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 pI of 5.99.The Hdh-MMP-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 pET28a-catMMP-1 containing the catalytic domain of MMP-1 was constructed.The recombinant MMP-1 was successfully expressed in Escherichia coli BL21cells.