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首页> 外文期刊>Molluscan Research >The activity parameters, cDNA cloning and mRNA expression of lysozyme in Cyclina sinensis (Bivalvia, Veneridae) responding to the pathogenic bacterium Vibrio anguillarum
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The activity parameters, cDNA cloning and mRNA expression of lysozyme in Cyclina sinensis (Bivalvia, Veneridae) responding to the pathogenic bacterium Vibrio anguillarum

机译：Cyclina sinensis（Bivalvia，Veneridae）对病原菌鳗弧菌的反应活性参数，cDNA克隆和溶菌酶的mRNA表达

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摘要

Lysozyme is an important component of the innate immune response against pathogen infection. The functional forms have been well-studied in the c-type lysozymes of vertebrates but less so in i-type lysozymes prevalent in most invertebrate animals. Cyclina sinensis is a commercially important marine venerid bivalve that is abundant and widely distributed around the maritime coasts of Asia. We obtained an i-type lysozyme (i-lyz) gene in C. sinensis by large scale EST sequencing of a SMART-cDNA library. In C. sinensis, the i-lyz gene encodes 181 amino acids. The lysozyme activities and the mRNA levels of the i-lyz gene in haemocytes were upregulated during the infection by a bacterium, Vibrio anguillarum. The lysozyme activity in haemocytes was increased after 12h infection by V. anguillarum, and reached the highest level at 24h post-infection, which was significantly higher than that of the control group (P<0.01). The expression level of the i-lyz gene in haemocytes was increased after 3h infection of Vibrio, and reached the highest level at 6h post-infection. A recombinant i-lyz was obtained through prokaryotic expression which, when isolated and purified, had a molecular weight of approximately 19kDa. Western blotting was used to validate the expression of the fusion protein. Furthermore, we demonstrate that the recombinant i-lyz protein has obvious bacteriostatic activity upon Escherichia coli whereby the K value of the curve was significantly lower than control and blank groups. Our study shows that the C. sinensis i-type lysozyme plays an important role in the immune defence against V . anguillarum and provides a theoretical reference for clarifying the mechanism of immune response against pathogen invasion in this bivalve.
机译：溶菌酶是针对病原体感染的先天免疫应答的重要组成部分。功能形式已在脊椎动物的c型溶菌酶中得到了很好的研究，但在大多数无脊椎动物中普遍存在的i型溶菌酶中却很少研究。 Cyclina sinensis是一种商业上重要的海洋marine类双壳类动物，在亚洲海洋沿岸丰富且广泛分布。通过SMART-cDNA文库的大规模EST测序，我们在中华绒螯蟹中获得了i型溶菌酶（i-lyz）基因。在中华绒螯蟹中，i-lyz基因编码181个氨基酸。在被细菌弧菌感染期间，血细胞中的溶菌酶活性和i-lyz基因的mRNA水平被上调。鳗弧菌感染12h后，血细胞中的溶菌酶活性增加，并在感染后24h达到最高水平，显着高于对照组（P <0.01）。感染弧菌3小时后，血细胞中i-lyz基因的表达水平升高，并在感染后6h达到最高水平。通过原核表达获得重组i-lyz，当分离和纯化时，其分子量约为19kDa。使用蛋白质印迹法验证融合蛋白的表达。此外，我们证明了重组i-lyz蛋白对大肠杆菌具有明显的抑菌活性，因此曲线的K值显着低于对照组和空白组。我们的研究表明，中华绒螯蟹i型溶菌酶在针对V的免疫防御中起着重要作用。并为阐明该双壳类动物对抗病原体入侵的免疫反应机制提供了理论参考。

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