Department of Bio-science, Institute of Life Science, Nanchang University, 999 Xuefu Road, Nanchang 330031, Jiangxi Province, China.
Fish Shellfish Immunol. 2013 May;34(5):1033-41. doi: 10.1016/j.fsi.2012.12.009. Epub 2013 Jan 17.
Lysozymes are important proteins to bivalve in the innate immune responses against bacterial infections, and provide nutrition as digestion enzymes. A new LYZ1 from the freshwater mussel Cristaria plicata was cloned by rapid amplification of cDNA ends (RACE) and nested PCR method. The full-length cDNA sequence of CpLYZ1 was 763 bp. The cDNA contained a 5'-terminal untranslated region (UTR) of 21 bp, a 3'- terminal UTR of 259 bp with a 29 bp poly(A) tail, a tailing signal (AATAAA) and the open reading frame of 483 bp. The CpLYZ1 cDNA encoded a polypeptide of 160 amino acids with a predicted molecular mass of 17.8 kDa, and a theoretical isoelectric point of 6.07. The comparison of the deduced amino acid sequences with LYZs from other species showed that the enzyme belonged to i-type lysozyme. The mRNA transcript of CpLYZ1 could be detected in all the examined tissues with the highest expression level in hepatopancreas. The expression levels of CpLYZ1 in hemocytes, hepatopancreas and gill significantly increased after Aeromonas hydrophila challenge. The expression level of CpLYZ1 in hemocytes sharply decreased from 6 h to 24 h and significantly increased at 48 h, and was the highest level in hepatopancreas at 24 h, and was the maximum level in gill at 48 h. Furthermore, the recombinant CpLYZ1 was induced to be expressed as an inclusion body form by IPTG at 37 °C for 4 h, and then was purified by using the Ni(2+) affinity chromatography. The relative enzyme activity of the recombinant CpLYZ1 was influenced on pH and temperature. The optimal pH and temperature was 5.5 and 50 °C, respectively. Against Escherichia coli, A. hydrophila, Staphyloccocus aureus, Bacillus subtilis, Streptococcus sp. and Staphylococcus epidermidis, the recombinant CpLYZ1 had bacteriolytic activity.
溶菌酶是贝类先天免疫反应中抵抗细菌感染的重要蛋白，同时也是消化酶为贝类提供营养。本研究通过快速扩增 cDNA 末端（RACE）和巢式 PCR 方法从淡水贻贝褶纹冠蚌中克隆到一个新的 LYZ1 基因（CpLYZ1）。CpLYZ1 的全长 cDNA 序列为 763bp，包含一个 21bp 的 5′非翻译区（UTR），一个 259bp 的 3′UTR 区，其中包含一个 29bp 的 poly(A)尾、一个尾信号（AATAAA）和一个 483bp 的开放阅读框。该基因编码的蛋白由 160 个氨基酸组成，理论分子量为 17.8kDa，理论等电点为 6.07。与其他物种的 LYZs 氨基酸序列比较分析显示，该酶属于 i 型溶菌酶。实时荧光定量 PCR 结果显示，CpLYZ1 在褶纹冠蚌各组织中均有表达，在肝胰腺中表达量最高。在受到嗜水气单胞菌刺激后，血细胞、肝胰腺和鳃中 CpLYZ1 的表达水平显著上调。在血细胞中，CpLYZ1 的表达水平在 6h 到 24h 时急剧下降，在 48h 时显著上调，在肝胰腺中于 24h 时达到最高水平，在鳃中于 48h 时达到最高水平。此外，CpLYZ1 重组蛋白在 IPTG 诱导下于 37°C 表达 4h 后形成包涵体，再经 Ni(2+)亲和层析柱纯化。重组 CpLYZ1 的相对酶活性受 pH 和温度影响，最适 pH 和温度分别为 5.5 和 50°C。该酶对大肠杆菌、嗜水气单胞菌、金黄色葡萄球菌、枯草芽孢杆菌、链球菌和表皮葡萄球菌均具有溶菌活性。
