仿刺参基质金属蛋白酶基因MMP-16的克隆及表达

基质金属蛋白酶(MMPs)是一种能够降解细胞外基质的蛋白水解酶类。为研究MMPs在仿刺参免疫防御中的作用,本实验采用RACE技术克隆了仿刺参基质金属蛋白酶16基因(Aj-MMP-16)的cDNA全长序列,并对其序列特征和功能进行了初步分析;采用实时荧光定量PCR(qRT-PCR)方法,分别分析了Aj-MMP-16基因在仿刺参不同组织、不同"化皮"体壁组织以及病原菌刺激后体腔细胞中的表达情况。结果显示,Aj-MMP-16基因的cDNA全长为2 976 bp,包括一个342 bp的5′非编码区,一个963 bp的3′非编码区;开放阅读框(ORF)为1 671 bp,编码557个氨基酸,预测蛋白分子量为63.11 ku,等电点为4.79。Aj-MMP-16具有典型的MMPs家族蛋白结构:N-端前肽区、铰链区、催化区、C-端类血红素结合区和跨膜区。Aj-MMP-16与其他物种的MMPs具有一定的相似性,与紫色球海胆的MMP-16相似性最高。Aj-MMP-16基因mRNA在仿刺参各组织中均有表达,表达量由高到低为呼吸树、肠、体腔细胞、管足、肌肉、体壁;在"化皮病"不同阶段,AjMMP-16基因mRNA在"化皮"体壁组织中的表达量显著高于正常体壁组织;灿烂弧菌和蜡样芽孢杆菌刺激后,体腔细胞中Aj-MMP-16基因mRNA表达量显著升高。Aj-MMP-16基因可能在仿刺参内脏再生、炎症发生以及免疫应答中起着重要的作用。

Cloning and expression analysis of MMP-16 gene from sea cucumber (Apostichopus japonicus)

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade extracellular matrix. In order to study the function of MMPs in the immune defense of Apostichopus japonicus, the full-length cDNA sequence of the matrix metalloproteinase 16 gene, named Aj-MMP-16, was cloned using RACE method. The sequence characteristics and function of Aj-MMP-16 were preliminarily analyzed. The results showed that the full-length cDNA of this gene was 2 976 bp, including a 5'' non-coding region of 342 bp, a 3'' non-coding region of 963 bp, and an open reading frame (ORF) of 1 671 bp encoding 557 amino acids. The predicted molecular weight of Aj-MMP-16 protein was 63.11 ku and isoelectric point was 4.79. Functional domain analysis revealed the typical MMPs family protein structure of Aj-MMP-16 including N-terminal propeptide region, hinge region, catalytic region, hemopexin-like domain and transmembrane region. Multiple sequence alignment and phylogenetic analysis showed that Aj-MMP-16 shared a certain degree of conservatism with MMPs of other species and had the highest identity with MMP-16 of Strongylocentrotus purpuratus. Quantitative real time PCR showed that the Aj-MMP-16 mRNA was expressed in all tissues of A. japonicus, and the expression levels were from high to low in the order of respiratory tree, intestine, coelomocytes, tube feet, muscle and body wall. At different stages of skin ulceration syndrome progression, the expression of Aj-MMP-16 mRNA in ulcerate body wall was significantly higher than that of the normal body wall. After the pathogenic bacteria challenge, Aj-MMP-16 mRNA expression increased significantly in coelomocytes. The results suggested that Aj-MMP-16 may play important roles in visceral regeneration, inflammation and immune response in sea cucumber.