皱纹盘鲍Hdh-MMP-1基因cDNA的克隆及原核表达  

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

作　　者：段雪昆[1] 杜翠红[1,2] 胡健健[1] 蔡秋凤[1,2] 刘光明[1,2] 曹敏杰[1,2] DUAN Xue- kun DU Cui- hong HU Jian- jian CAI Qiu- feng LIU Guang-ming CAO Min- jie(College of Food and Biological Engineering, Jimei University, Xiamen 361021,China National and Local Joint Engineering Research Center of Deep Processing Technology for Aquatic Products, Xiamen 361021, China)

机构地区：[1]集美大学食品与生物工程学院,福建厦门361021 [2]水产品深加工技术国家地方聚合工程研究中心,福建厦门361021

出　　处：《集美大学学报（自然科学版）》2016年第5期321-329,共9页Journal of Jimei University：Natural Science

基　　金：国家自然科学基金资助项目(31471640);国家海洋局海洋公益性行业科研专项(201305015)

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。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 fu l l- length of cDNA Hdh -MMP-1 was 2136 b p, 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 a-bout 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 BL21 cells.

关 键 词：皱纹盘鲍 基质金属蛋白酶 c DNA克隆 生物信息学 原核表达 

分 类 号：Q785[生物学—分子生物学] S917.4[农业科学—水产科学]