Wang Guanghua, Jiang Zengjie, Yang Ning, Zhu Dongfa, Zhang Min
School of Marine Science, Ningbo University, Ningbo, 315211, China; Marine Science and Engineering College, Qingdao Agricultural University, Qingdao, 266109, China.
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, China; Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, China.
Fish Shellfish Immunol. 2017 May;64:251-259. doi: 10.1016/j.fsi.2017.03.029. Epub 2017 Mar 18.
Calreticulin (CRT) is a multifunctional calcium-binding chaperone shared among vertebrates and invertebrates. In this study, a novel CRT (CfCRT) was identified in the Zhikong scallop Chlamys farreri by rapid amplification of cDNA ends. The full-length cDNA was composed of 1345 bp, which included a 1158 bp open reading frame, a 25 bp 5'-untranslated region (UTR) and a 162 bp 3'-UTR. The predicted molecular mass of CfCRT was 44.8 kDa. CfCRT contained three highly conserved domains (N-, P- and C-domains) essential to the function of CRT. BLAST analysis revealed significant sequence similarity (73%-92%) with CRT proteins from other mollusks. The mRNA transcripts of CfCRT were present in all the tested tissues of Zhikong scallops, with the higher expression level in the hemocytes and mantle. After stimulation by Vibrio anguillarum, the mRNA transcript of CfCRT in hemocytes was significantly upregulated. Recombinant plasmid pBCRT was successfully expressed in Escherichia coli BL21 (DE3). The recombinant (r)CfCRT protein could bind to the surface of several bacteria including the Gram-negative bacteria V. anguillarum, E. coli, and the Gram-positive bacterium Staphylococcus aureus. Moreover, rCfCRT was able to suppress their growth significantly. These results indicate that CfCRT might act as an immune effector in Zhikong scallop innate immunity.
钙网蛋白（CRT）是脊椎动物和无脊椎动物共有的一种多功能钙结合伴侣蛋白。在本研究中，通过cDNA末端快速扩增技术，在栉孔扇贝中鉴定出一种新型钙网蛋白（CfCRT）。其全长cDNA由1345个碱基对组成，包括一个1158个碱基对的开放阅读框、一个25个碱基对的5'-非翻译区（UTR）和一个162个碱基对的3'-UTR。预测的CfCRT分子量为44.8 kDa。CfCRT包含对CRT功能至关重要的三个高度保守结构域（N-、P-和C-结构域）。BLAST分析显示，它与其他软体动物的CRT蛋白具有显著的序列相似性（73%-92%）。CfCRT的mRNA转录本存在于栉孔扇贝所有检测的组织中，在血细胞和外套膜中表达水平较高。经鳗弧菌刺激后，血细胞中CfCRT的mRNA转录本显著上调。重组质粒pBCRT在大肠杆菌BL21（DE3）中成功表达。重组（r）CfCRT蛋白能够结合包括革兰氏阴性菌鳗弧菌、大肠杆菌以及革兰氏阳性菌金黄色葡萄球菌在内的几种细菌的表面。此外，rCfCRT能够显著抑制它们的生长。这些结果表明，CfCRT可能在栉孔扇贝先天免疫中作为一种免疫效应分子发挥作用。
