牙鲆半乳糖凝集素6基因的克隆、表达及功能研究

Cloning,Expression and Function of Galectin-6 from Paralichthys olivaceus

半乳糖凝集素6(Galectin-6)是β-半乳糖苷结合凝集素家族的成员之一。本研究首次分离并鉴定了牙鲆(Paralichthys olivaceus)Galectin-6(PoGalectin-6),分析了其分子特征和表达模式,并对其免疫相关功能进行了研究。PoGalectin-6基因的开放阅读框长为1089 bp,共编码362个氨基酸,其中包含2个糖识别结构域(CRDs)。同源序列比对和系统进化树分析显示,PoGalectin-6与大菱鲆(Scophthalmus maximus)Galectin-4的相似性为80.9%。组织分布结果显示,PoGalectin-6基因主要在肠组织中特异性表达。迟缓爱德华氏菌(Edwardsiella tarda)感染后,PoGalectin-6基因在肠组织中的表达显著升高,感染后12 h表达量最高,随后逐渐降低并恢复至正常水平。细菌结合实验证实,PoGalectin-6重组蛋白(rPoGalectin-6)能够结合枯草芽孢杆菌(Bacillus subtilis)、蜡样芽孢杆菌(Bacillus cereus)、杀鲑气单胞菌(Aeromonas salmonicida)、迟缓爱德华氏菌和创伤弧菌(Vibrio vulnificus),但并不结合短小芽孢杆菌(Bacillus pumilus)。此外,rPoGalectin-6以钙离子依赖的方式对短小芽孢杆菌、枯草芽孢杆菌、杀鲑气单胞菌和迟缓爱德华氏菌表现出明显的凝集作用。研究表明,PoGalectin-6基因参与了由迟缓爱德华氏菌感染引起的免疫应答,这一发现为探索Galectin-6在硬骨鱼类中的免疫功能奠定了基础。

Galectin-6 is a member of theβ-galactoside-binding lectin family,which has been widely studied in mammals(e.g.,Lgals6).Galectin-6 has been cloned from several teleost fishes,such as Maylandia zebra,Esox lucius,and Poecilia formosa,but there are few studies on its function.In this study,we analyzed the molecular characteristics of Galectin-6 extracted from Paralichthys olivaceus(PoGalectin-6)and studied its immune-related functions.The full length of the open reading frame(ORF)of PoGalectin-6 cDNA is 1089 bp,encoding 362 amino acids containing two carbohydrate recognition domains(CRDs).Multiple sequence alignment and phylogenetic tree analysis showed that PoGalectin-6 is highly similar to Scophthalmus maximus Galectin-4(approximately 80.9%).Tissue distribution experiments demonstrated that PoGalectin-6 is specifically expressed in intestinal tissues.The expression level of PoGalectin-6 in the intestine significantly increased after Edwardsiella tarda stimulation,and the highest expression level was observed at 12 h after infection.Recombinant PoGalectin-6(rPoGalectin-6)exhibited binding ability to gram-positive(Bacillus subtilis,and B.cereus)and gram-negative(Aeromonas salmonicida,E.tarda,and Vibrio vulnificus)bacteria.rPoGalectin-6 showed Ca2+-dependent agglutination activity against B.pumilus,B.subtilis,A.salmonicida,and E.tarda.This study suggests that PoGalectin-6 may play an important role in the immune response against E.tarda infection,laying the foundation for exploring the immune functions of Galectin-6 in teleosts.