作 者：
张明辉;章骞;林锦锦;张凌晶;刘光明;陈玉磊;曹敏杰
单 位：
集美大学海洋食品与生物工程学院;水产品深加工技术国家地方联合工程研究中心
关键词：
皱纹盘鲍;副溶血性弧菌;金属蛋白酶组织抑制剂;基质金属蛋白酶1;相互作用;先天性免疫
摘 要：
由弧菌感染引起的疾病是影响鲍存活率的主要因素,实验以皱纹盘鲍为研究对象,探讨金属蛋白酶组织抑制剂(tissue inhibitor of metalloproteinase, TIMP)在皱纹盘鲍抗弧菌免疫中的功能及其与基质金属蛋白酶1 (matrix metalloproteinase 1, MMP-1)的相互作用关系。实验通过PCR技术克隆得到了TIMP cDNA序列,全长为2 291 bp。利用NetNGlyc 1.0Server和NetOGlyc 4.0 Server软件对TIMP的氨基酸序列进行分析,结果显示,TIMP有3个潜在的N-糖基化位点,分别位于第47、77和152位的Asn,以及一个潜在的O-糖基化位点,即位于第108位的Thr。多序列比对结果显示,皱纹盘鲍TIMP氨基酸序列与杂色鲍、太平洋牡蛎、泥蚶中TIMP的序列相似性分别为76.0%、18.9%和19.3%。利用荧光定量PCR技术分析了副溶血性弧菌感染过程中皱纹盘鲍不同组织中TIMP的表达情况,发现在弧菌感染早期,TIMP在血淋巴细胞和鳃组织中的表达量均显著上调。利用RNA干扰技术分别敲低皱纹盘鲍TIMP和MMP-1的表达水平,研究TIMP与MMP-1在皱纹盘鲍抗弧菌免疫中的相互作用关系。结果显示,鲍体内MMP-1表达沉默后,TIMP表达量显著上调;而TIMP表达受到抑制时,MMP-1表达量显著下降,说明TIMP表达受MMP-1负调控,而MMP-1表达受TIMP正调控。本研究将有助于揭示TIMP及其与MMP-1相互作用在皱纹盘鲍先天免疫反应中的功能,也可为鲍养殖的病菌防治策略提供新的思路。
译 名：
Role of tissue inhibitor of metalloproteinase in the anti-Vibrio immunity of Haliotis discus hannai
作 者：
ZHANG Minghui;ZHANG Qian;LIN Jinjin;ZHANG Lingjing;LIU Guangming;CHEN Yulei;CAO Minjie;College of Ocean Food and Biological Engineering, Jimei University;National & Local Joint Engineering Research Center of Deep Processing Technology for Aquatic Products;
关键词：
Haliotis discus hannai;;Vibrio parahaemolyticus;;tissue inhibitor of metalloproteinase;;matrix metalloproteinase 1;;interaction;;innate immunity
摘 要：
As an important economic shellfish in coastal area of China, abalone is susceptible to bacterial infection,especially Vibiro parahaemolyticus. Similar to other invertebrates, Haliotis discus hannai resists pathogen infection by innate immune response. Tissue inhibitors of metalloproteinases(TIMPs) are endogenous protein regulators of the matrix metalloproteinases(MMPs) family. As MMP-1 was engaged in the innate immunity of abalone,the involvement of TIMP in the regulation of MMP-1 expression and activity as well as the immune responses merits further investigation. In this study, the role of TIMP in the innate immune responses of H. discus hannai against V. parahaemolyticus infection and the interaction between TIMP and MMP-1 were investigated. The full length cDNA sequence of TIMP was obtained, and the expressions of TIMP in different tissues of H. discus hannai were analyzed post Vibrio infection. The cloned TIMP cDNA sequence was 2 291 bp in length. NetNGlyc 1.0 Server and Netoglyc 4.0 Server were used to analyze the glycosylation sites of TIMP. The results showed that TIMP had three potential N-glycosylation sites, namely Asn at positions 47, 77 and 152, and a potential O-glycosylation site, namely Thr at position 108. Multiple alignment of TIMP amino acid sequences from different molluscs showed that the similarities of TIMP in H. discus hannai with those of H. diversicolor, Crassostrea gigas and Tegillarca granosa were 76%, 18.9% and 19.3%, respectively. In the early stage of V. parahaemolyticus infection,the expression of TIMP in hemocytes and gill tissues were significantly up-regulated. To study the interaction between TIMP and MMP-1 in the anti-Vibrio immunity of abalone, RNA interference technology was used to knock down the expression levels of TIMP and MMP-1. After the silencing of MMP-1 gene in abalone, TIMP expression was significantly up-regulated. When TIMP expression was inhibited, MMP-1 expression decreased significantly. These results indicated that MMP-1 expression was positively regulated by TIMP, while TIMP expression was negatively regulated by MMP-1 in abalone. In conclusion, the present study will help to reveal the interaction between TIMP and MMP-1 in abalone innate immunity against V. parahaemolyticus infection. It also provided new insights into the prevention and control of pathogens in abalone culture.
