斜带石斑鱼CCR6a基因的克隆、表达模式及抗病毒能力研究

Cloning,expression pattern and antiviral function of CCR6a gene in Epinephelus coioides

趋化因子受体是具有7个疏水性跨膜结构的G蛋白偶联受体,是一类低分子蛋白质,可以募集并激活白细胞作用于感染部位。相关研究表明,趋化因子及其受体在病原体感染、创伤修复、肿瘤形成及其转移等方面起重要作用。本研究从斜带石斑鱼(Epinephelus coioides)组织中克隆出了CCR6a(CC chemokine receptor 6a,CCR6a)基因ORF序列,并对其序列和进化关系进行了分析,研究该基因组织分布、亚细胞定位和抗病毒功能情况。研究结果表明:斜带石斑鱼CCR6a基因ORF序列全长为1131 bp,编码376个氨基酸;进化树分析结果表明斜带石斑鱼CCR6a与鞍带石斑鱼CCR6a聚为一支;荧光定量PCR(RT-qPCR)检测CCR6a基因组织分布发现CCR6a在所有检测组织中均有表达,在免疫组织肾脏和鳃的表达量最高,在皮肤中的表达量次之;亚细胞定位显示该基因为膜分布,在石斑鱼细胞中过表达CCR6a可显著抑制赤点石斑鱼神经坏死病毒(Red-spotted grouper nervous necrosis virus,RGNN)的复制。研究结果将有助于理解鱼类趋化因子在病毒感染中的作用。

Chemokine receptor is a kind of transmembrane receptor(GPCR)which includes seven hydrophobic transmembrane structures.Studies have shown that chemokine and their receptors play an important role in inflammatory reaction,pathogen infection,wound repair,tumor formation and metastasis and so on.But,the role of chemokine receptors in teleost fish,especially in fish antiviral remains unclear.In this study,the ORF sequence of CC chemokine receptor 6a(CCR6a)gene was cloned from tissue of orange-spotted grouper,and its sequence and evolutionary relationship were analyzed.At the same time,tissue distribution,subcellular localization and antiviral function of this gene were studied.The results showed that the full length of ORF sequence of CCR6a gene in oblique grouper was 1131 bp,encoding 376 amino acids.Evolutionary tree analysis showed that orange-spotted grouper CCR6a was the closest relative between giant grouper.Quantitative real-time PCR(RT-qPCR)detection of CCR6a tissue distribution found that CCR6a was high expression in immune tissue like kidney,gill,and skin.Subcellular localization showed that CCR6a was distributed in membrane.Overexpression of CCR6a in GS cells could significantly inhibit the replication of red-spotted grouper nervous necrosis virus(RGNN).These results would help to understand the role of fish chemokines in viral infections.