Molecular cloning and expression of a C-type lectin-like protein from orange-spotted grouper Epinephelus coioides.
Molecular cloning and expression of a C-type lectin-like protein from orange-spotted grouper Epinephelus coioides.
复制标题
DOI：
10.1111/jfb.12296
复制
发表时间：
2014-02
影响因子：
2
通讯作者：
H. Ji;J. Wei;S. Wei;Y. Yan;Y. Huang;X. Huang;S. Zhou;Y. Zhou;Q. Qin
中科院分区：
文献类型：
--
作者：
H. Ji;J. Wei;S. Wei;Y. Yan;Y. Huang;X. Huang;S. Zhou;Y. Zhou;Q. Qin
克隆了斜带石斑鱼C型凝集素样蛋白（Ec-CTLP）。该基因全长905 bp，开放阅读框522 bp，编码174个氨基酸残基。Ec-CTLP的推定氨基酸序列包含N末端19个残基的信号肽、Cys 43至Arg 169的CLECT结构域和保守的不完全WND（Trp-Asn-Asp）基序。与其它鱼类的同源性为32 ~ 42%。Ec-CTLP在大肠杆菌中的表达有不同程度的上调。在16和4° C下攻击后的Coioides脾（生殖系干细胞）细胞。细胞内定位显示Ec-CTLP仅分布在细胞质中。重组Ec-CTLP（rEc-CTLP）在大肠杆菌BL 21（DE 3）中表达并纯化，制备小鼠抗Ec-CTLP血清。rEc-CTLP融合蛋白不具有血凝活性，但提高了冷冻细菌的存活率。细菌（包括革兰氏阴性E.大肠杆菌和革兰氏阳性金黄色葡萄球菌）与rEc-CTLP浓度呈正相关。这些发现为进一步了解C型凝集素在海洋鱼类天然免疫中的作用提供了线索。
A C-type lectin-like protein (Ec-CTLP) was cloned from the grouper Epinephelus coioides. The full-length cDNA of Ec-CTLP was composed of 905 bp with a 522 bp open reading frame that encodes a 174-residue protein. The putative amino acid sequence of Ec-CTLP contains a signal peptide of 19 residues at the N-terminus and a CLECT domain from Cys43 to Arg169 and a conserved imperfect WND (Trp-Asn-Asp) motif. The homologous identity of deduced amino acid sequences is from 32 to 42% with other fishes. The expression of Ec-CTLP was differently upregulated in E. coioides spleen (germline stem) cells after being challenged at 16 and 4° C. Intracellular localization revealed that Ec-CTLP was distributed only in the cytoplasm. Recombinant Ec-CTLP (rEc-CTLP) was expressed in Escherichia coli BL21 (DE3) and purified for mouse Mus musculus anti-Ec-CTLP serum preparation. The rEc-CTLP fusion protein does not possess haemagglutinating activity, but improves survival from frozen bacteria. The survival of bacteria (including gram-negative E. coli and gram-positive Staphylococcus aureus) was positively correlated with the concentration of the rEc-CTLP. These findings can provide clues to help understand the probable C-type lectin in marine fish innate immunity.