Marine Applied Microbes and Aquatic Organism Disease Control Lab, Department of Aquatic Biomedical Sciences, School of Marine Biomedical Sciences, College of Ocean Sciences & Marine and Environmental Research Institute, Jeju National University, Jeju 690-756, South Korea.
Fish Shellfish Immunol. 2011 Oct;31(4):588-94. doi: 10.1016/j.fsi.2011.06.019. Epub 2011 Jul 6.
Lysozymes are the key molecules in innate immune system and possess high bactericidal properties. In the present study, a full-length c-type lysozyme cDNA has been cloned and characterized by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques from kelp grouper, Epinephelus bruneus (Eb-CTL). The cDNA consists of 753 base pairs (bp) with a 432 bp open reading frame (ORF) that encodes 144 amino acids (aa) residues and polyadenylation signal sequence AATAAA. The deduced aa sequence polypeptide had a predicted molecular weight of 16 kDa and theoretical isoelectric point of 7.3. The deduced aa sequence have a long lysozyme domain which contains all catalytic sites and other conserved residues required for lysozyme activity. Pair-wise alignments showed a higher identity (76.4%) with Psetta maxima lysozyme and low identity (38.9%) with lysozymes of insect Bombyx mori. Interestingly, phylogenetic analysis revealed that the kelp grouper lysozyme was more closely related to other fish and vertebrate lysozymes. Quantitative real-time reverse transcriptase-PCR analysis showed that Eb-CTL transcripts are constitutively expressed in hematopoietic organs such as heart, spleen, and kidney after stimulation with LPS or infection with Vibrio anguillarum, indicating the involvement of Eb-CTL in the innate immunity of kelp grouper. This study is a first step on the genetics and functions of the c-type lysozyme in kelp grouper, and their role in anti-bacterial activity with reference to immunological properties which might have biotechnological applications.
溶菌酶是先天免疫系统的关键分子，具有很强的杀菌特性。本研究通过表达序列标签（EST）和快速扩增 cDNA 末端（RACE）技术，从褐菖鲉（Epinephelus bruneus）克隆并鉴定了全长 c 型溶菌酶 cDNA（Eb-CTL）。cDNA 由 753 个碱基对（bp）组成，其中包含一个 432 bp 的开放阅读框（ORF），编码 144 个氨基酸（aa）残基和多聚腺苷酸化信号序列 AATAAA。推导的 aa 序列多肽预测分子量为 16 kDa，理论等电点为 7.3。推导的 aa 序列具有一个长的溶菌酶结构域，其中包含溶菌酶活性所需的所有催化位点和其他保守残基。两两比对显示，它与大菱鲆溶菌酶的同源性较高（76.4%），与昆虫家蚕溶菌酶的同源性较低（38.9%）。有趣的是，系统进化分析表明，褐菖鲉溶菌酶与其他鱼类和脊椎动物的溶菌酶更为密切相关。实时定量 RT-PCR 分析显示，Eb-CTL 转录本在受到 LPS 刺激或感染鳗弧菌后在造血器官（如心脏、脾脏和肾脏）中持续表达，表明 Eb-CTL 参与了褐菖鲉的先天免疫。本研究是褐菖鲉 c 型溶菌酶在遗传学和功能方面的第一步，其在抗菌活性方面的作用以及与免疫特性相关的生物技术应用可能具有重要意义。
