Antioxidant enzymes from the crab Scylla paramamosain: Gene cloning and gene/protein expression profiles against LPS challenge
Antioxidant enzymes from the crab Scylla paramamosain: Gene cloning and gene/protein expression profiles against LPS challenge
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DOI：
10.1016/j.fsi.2010.02.008
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发表时间：
2010-05-01
影响因子：
4.7
通讯作者：
Wang, Ke-Jian
中科院分区：
文献类型：
--
作者：
Liu, Hai-Peng;Chen, Fang-Yi;Wang, Ke-Jian
近年来的研究表明，抗氧化酶在机体代谢过程或病原菌入侵引起的抗氧化反应中起着重要作用。过氧化氢酶是这些关键酶中的一种，从无脊椎动物到脊椎动物都有其特有的功能，并且高度保守。本研究从拟穴青蟹血细胞抑制消减杂交文库中克隆了过氧化氢酶的全长cDNA序列。Sp-CAT基因cDNA全长2551 bp，开放阅读框1551 bp，编码517个氨基酸残基。在Sp-CAT的氨基酸序列中预测了保守的催化活性残基His-71、Asn-144和Tyr-354，推测Sp-CAT蛋白的分子量为59 kDa，等电点为6.4。多序列比对分析表明，Sp-CAT基因与其它物种的氨基酸序列同源性较高（75.4%）。Sp-CAT mRNA转录本在正常S.拟穴龙实时荧光定量PCR检测结果显示，LPS刺激后3、6 h血细胞和6 h肝胰腺中Sp-CAT基因的表达量显著增加。此外，还测定了LPS攻击后不同组织和血清中CAT和SOD的活性。血细胞、血清和肝胰腺中CAT活性分别在LPS刺激后3、6、24和48 h显著升高。此外，SOD活性显着诱导后3和6 h的血细胞裂解物，3和12 h的血清，12和48 h的肝胰腺LPS刺激，表明组织和时间依赖性的抗氧化反应在螃蟹。这些数据表明，在LPS攻击的螃蟹发生了强烈的抗氧化反应，这可能是在保护宿主免受微生物感染。(C)2010爱思唯尔有限公司保留所有权利。
Recent studies revealed that antioxidant enzymes play important roles in antioxidant responses caused by metabolic process or pathogen invasion. Catalase is one of these key enzymes which has been characterized and highly conserved from invertebrates to vertebrates. In the present study, a full-length cDNA sequence of catalase was cloned from the hemocyte suppression subtractive hybridization library of the crab Scylla paramamosain. The Sp-catalase (Sp-CAT) cDNA sequence contained 2551 bp with an open reading frame of 1551 bp encoding 517 amino acid residues. The conserved catalytic active residues His-71, Asn-144 and Tyr-354 were predicted in the amino acid sequence of Sp-CAT. The deduced Sp-CAT protein had a calculated molecular mass of 59 kDa with an estimated isoelectric point of 6.4. Multiple alignment analysis revealed that the deduced amino acid sequence of Sp-CAT shared high identity (75.4%) with those of other species. The Sp-CAT mRNA transcripts were demonstrated in multiple tissues of normal S. paramamosain. After LPS challenge, the expression level of Sp-CAT gene was increased significantly in hemocyte at 3 and 6 h, and in hepatopancreas at 6 h, respectively, determined by quantitative real-time PCR. Furthermore, the activities of CAT and SOD were also measured in different tissues and serum after LPS challenge. The CAT activity was significantly increased at 3, 6, 24 and 48 h in hemocyte lysate, at 3 h in serum, and at 24 and 48 h in hepatopancreas after LPS challenge. In addition, the SOD activity was significantly induced at 3 and 6 h in hemocyte lysate, 3 and 12 h in serum, 12 and 48 h in hepatopancreas post LPS stimulation, indicating a tissue and time-dependent antioxidant response in the crab. Taken together, these data demonstrated that a strong antioxidant response occurred in the LPS-challenged crab, which might be involved in the protection of host against microbial infections. (C) 2010 Elsevier Ltd. All rights reserved.