大弹涂鱼(Boleophthalmus pectinirostris)Gal-8L基因序列及其细菌凝集活性的鉴定

GAL-8L OF MUDSKIPPER BOLEOPHTHALMUS PECTINIROSTRIS:CHARACTERIZATION OF THE SEQUENCE AND BACTERIAL AGGLUTINATION ACTIVITY

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,Bp Gal-8L)c DNA序列。首先确定其序列特征,其次采用实时定量PCR(real-time quantitative PCR,q PCR)的方法鉴定Bp Gal-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Bp Gal-8L-N和Bp Gal-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,Bp Gal-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Bp Gal-8L属于鱼类Gal-8的一种亚型。q PCR结果显示,Bp Gal-8L基因m RNA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Bp Gal-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,Bp Gal-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。

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-SL-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 13-galactoside binding motifs of the galectin family. Meanwhile, BpGal-8L belongs to Gal-SL 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.