Liang Fu-Rui, Hong Yue-Hui, Ye Cong-Cong, Deng Hailin, Yuan Jian-Ping, Hao Yun-Fang, Wang Jiang-Hai
Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-Sen University, Guangzhou 510006, People's Republic of China.
Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-Sen University, Guangzhou 510006, People's Republic of China; South China Sea Bioresource Exploitation and Utilization Collaborative Innovation Center, School of Marine Sciences, Sun Yat-Sen University, Guangzhou 510006, People's Republic of China.
Fish Shellfish Immunol. 2017 Aug;67:280-292. doi: 10.1016/j.fsi.2017.06.024. Epub 2017 Jun 7.
Cathepsin L (CatL) has been widely known for its involvement in the innate immunity. However, it still remains poorly understand how CatL modulates the immune system of teleosts. Moreover, the CatL of Nile tilapia (NtCatL) has not been cloned or characterized. In this study, the gene encoding NtCatL was cloned, and was characterized by bioinformatics analysis, heterologous expression and protease activity assay. The coding sequence of NtCatL is 1017 bp in length and encodes 338 amino acid residues with a predicted molecular weight of 38.487 kDa and a theoretical isoelectric point of 5.79. NtCatL possesses the features of a typical cathepsin L, including one signal peptide, one propeptide region, and one papain family cysteine protease domain containing four active site residues (Gln, Cys, His, and Asn). The prediction of protein-protein interaction shows that NtCatL may interact with some functional proteins for realizing an immune function. Real-time quantitative PCR revealed the widespread transcriptional expression of NtCatL in six tissues of healthy Nile tilapia, and the NtCatL mRNA is significantly up-regulated after Streptococcus agalactiae challenge. These results suggest that NtCatL is likely to be involved in the immune reaction of Nile tilapia. Recombinant proteins from the mature domain (residues 117-337) of NtCatL were obtained by heterologous expression using pET28a and Rosetta (DE3) competent cells. A protein product with the high purity was obtained by using TALON Superflow purification rather than adopting HisTrap HP columns. The protease activity of the recombinant protein was verified by using a substrate hydrolyzing assay. This work has cloned and characterized the CatL from Nile tilapia for the first time, and contributes to elucidating the immunological functions of CatL.
组织蛋白酶L（CatL）因其参与先天免疫而广为人知。然而，关于CatL如何调节硬骨鱼免疫系统仍知之甚少。此外，尼罗罗非鱼的CatL（NtCatL）尚未被克隆或鉴定。在本研究中，克隆了编码NtCatL的基因，并通过生物信息学分析、异源表达和蛋白酶活性测定对其进行了鉴定。NtCatL的编码序列长度为1017 bp，编码338个氨基酸残基，预测分子量为38.487 kDa，理论等电点为5.79。NtCatL具有典型组织蛋白酶L的特征，包括一个信号肽、一个前肽区和一个含有四个活性位点残基（Gln、Cys、His和Asn）的木瓜蛋白酶家族半胱氨酸蛋白酶结构域。蛋白质-蛋白质相互作用预测表明，NtCatL可能与一些功能蛋白相互作用以实现免疫功能。实时定量PCR显示，NtCatL在健康尼罗罗非鱼的六个组织中广泛转录表达，无乳链球菌攻击后NtCatL mRNA显著上调。这些结果表明，NtCatL可能参与尼罗罗非鱼的免疫反应。使用pET28a和Rosetta (DE3)感受态细胞通过异源表达获得了来自NtCatL成熟结构域（第117 - 337位氨基酸残基）的重组蛋白。通过使用TALON Superflow纯化而非HisTrap HP柱获得了高纯度的蛋白质产物。通过底物水解测定验证了重组蛋白的蛋白酶活性。这项工作首次克隆并鉴定了尼罗罗非鱼的CatL，有助于阐明CatL的免疫功能。
