牙鲆FEL1基因的表达及功能分析

Expression and Functional Analysis of FEL 1 Gene in Paralichthys olivaceus

凝集素(Lectin)是一类能与糖进行非共价可逆结合的蛋白质,具有凝集细胞和聚糖的作用。在牙鲆的集约化养殖过程中,会面临许多病原微生物的入侵,造成大量的经济损失,研究表明鱼卵凝集素(Fish egg lectin)是凝集素超家族中的一员,在硬骨鱼的抗菌免疫反应中发挥作用。在本研究中,克隆验证了牙鲆FEL1基因(Fish egg lectin 1,PoFEL 1),其中开放阅读框(Open reading frame,ORF)为675 bp,编码224个氨基酸,蛋白分子量为24.8 kDa。PoFEL 1组织表达分析显示,PoFEL 1在卵巢、肠以及肝脏中的表达量比较高;胚胎发育过程中PoFEL 1表达分析显示,在胚胎发育的早期PoFEL 1的表达量比较高,随着胚胎的逐渐成熟,其表达量逐渐降低,呈母源性表达。体外细胞刺激实验表明LPS、PGN、Poly(I∶C)和E.tarda刺激牙鲆卵巢细胞系后,PoFEL 1的表达上调。利用牙鲆卵巢细胞研究了PoFEL 1蛋白的亚细胞定位,证实其分布在细胞质中。用LPS刺激过表达PoFEL 1的牙鲆卵巢细胞系,发现细胞因子p38、IL-6和TNF-α的表达显著上调。通过双荧光素酶检测系统发现,过表达PoFEL 1能显著上调NF-κB报告基因的表达,从而引起机体的免疫应答,说明PoFEL 1对NF-κB通路可能具有调节作用。以上研究结果说明PoFEL 1在牙鲆先天性免疫过程中发挥作用。

Lectin is a type of protein that can be non-covalently reversibly bound to sugar,and has the function of agglutinating cells and glycans.In the intensive breeding process of the Japanese flounder,many pathogenic microorganisms are invaded,causing a large amount of economic loss.Studies have shown that Fish Egg Lectin is a member of the lectin superfamily and is antibacterial in teleost fish playing a role in the immune response.In this study,we cloned the FEL 1 gene(Fish Egg Lectin 1,PoFEL 1),which has a complete open reading frame(ORF)of 675 bp,and encoding 224 amino acids.The protein molecular weight(MV)according to the predicted protein sequence is 24.8 kDa.The expression analysis of PoFEL 1 showed that the expression of PoFEL 1 was higher in ovary,intestine and liver.During embryonic development of the Paralichthys olivaceus,qRT-PCR analysis revealed that the expression of PoFEL 1 gradually decreased with the gradual maturation of the Paralichthys olivaceus embryos as maternal expression.In vitro immune-stimulation experiments showed that the expression of PoFEL 1 was up-regulated after stimulation ovarian cell lines with LPS,PGN,Poly(I∶C)and E.tarda.The subcellular localization of PoFEL 1 protein was studied using ovary cells and confirmed to be distributed in the cytoplasm.When the cells overexpressed with PoFEL 1 were stimulated with LPS,the expression levels of the cytokines p38,IL-6 and TNF-αwere found to be significantly up-regulated.Using a dual luciferase assay system,we found that overexpression of PoFEL 1 can significantly up-regulate the expression of NF-κB reporter gene,thereby causing the body’s immune response,indicating that PoFEL 1 may regulate the NF-κB signaling pathway.In summary,PoFEL 1 plays a role in the innate immunity of the Paralichthys olivaceus.