| 摘要: |
| 半乳糖凝集素-8基因（galectin-8，Gal-8）属于凝集素家族一员。已知Gal-8在哺乳动物的先天免疫中发挥重要作用，然而在鱼类中却鲜有报道。本研究通过转录组测序获得大弹涂鱼（Boleophthalmus pectinirostris）半乳糖凝集素-8样基因（galectin-8 like，BpGal-8L） cDNA序列。首先确定其序列特征，其次采用实时定量PCR （real-time quantitative PCR，qPCR）的方法鉴定BpGal-8L组织表达分布及其与迟缓爱德华氏菌（Edwardsiella tarda）感染相关性；最后分别通过原核表达获得N端和C端CRD区重组蛋白BpGal-8L-N和BpGal-8L-C，并采用玻片法观察这两个CRD区的细菌凝集活性和糖类结合特性。多重序列比对显示，BpGal-8L蛋白含有两个糖识别结构域（carbohydrate recognition domain，CRD），但只有C端的CRD区存在Gal家族典型的β-半乳糖苷糖类结合基序。系统进化树表明BpGal-8L属于鱼类Gal-8的一种亚型。qPCR结果显示，BpGal-8L基因mRNA在各组织中普遍表达，并且在迟缓爱德华氏菌感染后大弹涂鱼肝和脾中表达量呈上调趋势。细菌凝集实验结果显示，只有BpGal-8L-C重组蛋白对检测的5种革兰氏阴性菌及2种革兰氏阳性菌有凝集作用，并与乳糖和脂多糖特异性结合。综上所述，BpGal-8L与大弹涂鱼抗菌免疫密切相关，且其N端和C端CRD区发挥不同作用。 |
| 关键词: 半乳糖凝集素-8样基因 细菌凝集 糖结合特异性 大弹涂鱼 |
| DOI：10.11693/hyhz20171000278 |
| 分类号:Q789;Q956 |
| 基金项目:国家自然科学基金项目，31402323号；浙江省自然科学基金项目，LY14C190007号；宁波市科技创新团队项目，2015C110018号；宁波市科技富民项目，2017C10037号；“水产”浙江省重中之重开放基金，xkzsc1417号。 |
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| Gal-8L OF MUDSKIPPER BOLEOPHTHALMUS PECTINIROSTRIS: CHARACTERIZATION OF THE SEQUENCE AND BACTERIAL AGGLUTINATION ACTIVITY |
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LIANG Ya-Fang, SHI Yu-Hong, MIAO Liang, CHEN Jiong
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Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China
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| Abstract: |
| The galectin-8 (Gal-8) belongs to the family of lectins. It is known that Gal-8 plays an important role in mammalian innate immunity. However, Gal-8 is rarely reported in fish. In this study, the cDNA sequence of galectin-8 like gene from mudskipper Boleophthalmus pectinirostris (BpGal-8L) was obtained by transcriptome sequencing. Firstly, the sequence was characterized. Secondly, tissues expression profile and the correlation between the expressions of BpGal-8L mRNA expressions and Edwardsiella tarda infection were identified by qPCR. Lastly, the BpGal-8L-N and BpGal-8L-C recombinant protein were obtained by prokaryotic expression, and then the bacterial agglutination activity and carbohydrate-binding properties were observed by slide method. By comparing the sequences, we found that the N-terminal and C-terminal of the BpGal-8L protein each contained a carbohydrate recognition domain (CRD), but only the C-terminal CRD had two typical β-galactoside binding motifs of the galectin family. Meanwhile, BpGal-8L belongs to Gal-8L cluster of the fish Gal-8 subset on the phylogenetic tree; BpGal-8L mRNAs were widely expressed in the tested tissues; and the expressions of BpGal-8L mRNA in liver and spleen were significantly up-regulated after E. tarda infection. Bacterial agglutination assay showed that BpGal-8L-C recombinant protein could agglutinate five Gram-negative and two Gram-positive bacteria and bind specifically to lactose and lipopolysaccharides. Therefore, BpGal8L is involved in antimicrobial immunity of mudskipper, and its N-terminal and C-terminal CRD may play different roles, which needs to be deciphered in the future. |
| Key words: galectin-8 like bacterial agglutination carbohydrate-binding specificity Boleophthalmus pectinirostris |
