文章摘要
斜带石斑鱼甘露糖受体基因的克隆表达及其功能研究
Molecular cloning and functional study of mannose receptor gene in Epinephelus coioides
投稿时间:2020-08-25  修订日期:2020-12-04
DOI:
中文关键词: 斜带石斑鱼  甘露糖受体  赤点石斑鱼神经坏死病毒  凋亡
英文关键词: Epinephelus coioides  mannose receptor  red-spotted grouper nervous necrosis virus  apoptosis
基金项目:广东省海洋与渔业厅项目,(GDME-2018C006),单聚体壳寡糖的免疫调控及其推广应用,主持,在研,100万,2018/7-2020/7
作者单位邮编
张梦兰 华南师范大学生命科学学院 510631
秦真东 仲恺农业工程学院动物科技学院 
卢志杰 仲恺农业工程学院动物科技学院 
赵丽娟 仲恺农业工程学院动物科技学院 
潘淦 华南师范大学生命科学学院 
林蠡 仲恺农业工程学院动物科技学院 510225
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中文摘要:
      甘露糖受体(Mannose receptor,MR)是一种参与病原体识别和抗原递呈的内吞性受体,在免疫应答中发挥着重要的作用。为了研究斜带石斑鱼甘露糖受体(Epinephelus coioides mannose receptor, EcMR)在抗赤点石斑鱼神经坏死病毒(Red-spotted grouper nervous necrosis virus,RGNNV)感染中的免疫功能,本研究成功克隆与表达了EcMR基因。研究结果显示,EcMR cDNA全长4877 bp,共编码1446 个氨基酸。EcMR的蛋白结构域包括1个信号肽(Signal peptide),1 个富含蓖麻类β型三叶草结构域(RICIN),1个Ⅱ型纤维连接蛋白结构域(FN Ⅱ),8个串联的C-型凝集素样结构域(CLECTs)以及1个跨膜结构域(Transmembrane region)。实时定量PCR(qRT-PCR)和细胞免疫荧光(IF)分析结果显示,EcMR在斜带石斑鱼的8个组织中均有表达,其相对表达量顺序为鳃>头肾>脑>脾脏>肝脏>外周血>心脏>肌肉。在研究MR是否参与RGNNV入侵过程中时发现,RGNNV可以在GF-1(Grouper fin cell)细胞中快速增殖,同时显著激活EcMR基因的表达。为进一步探究RGNNV对GF-1细胞的影响,本研究通过双染色法检测了RGNNV感染GF-1细胞后的细胞凋亡情况,其结果表明,RGNNV感染可以促进GF-1的细胞凋亡,并且细胞凋亡率随着RGNNV感染时间延长而增加。同时,qRT-PCR和酶活检测结果显示,RGNNV的感染可以显著促进凋亡相关基因的转录水平以及Caspase-3、Caspase-8、Caspase-9的酶活水平。综上所述,本研究成功克隆了EcMR基因,并且揭示了其在RGNNV入侵过程中一定的相关性。本研究结果将为石斑鱼病毒性疾病的防控提供一定的基础理论参考。
英文摘要:
      Mannose receptor (MR) which plays an important role in immune response is an endocytosis receptor involved in pathogen recognition and antigen presentation. In order to study the immune function of EcMR gene against red grouper nerve necrosis virus (Red-spotted grouper nervous necrosis virus, RGNNV), EcMR gene was cloned and expressed. The results shown that the EcMR cDNA full length sequence was 4877 bp, and its open reading frame encoded 1446 amino acids. The protein domain of EcMR was composed of a signal peptide, an extracellular ricin-like β-type clover domain (RICIN), a fibronectin type Ⅱ domain (FN Ⅱ), eight tandem C-type lectin-like domains (CLECTs) and a transmembrane domain. The results of real-time quantitative PCR (qRT-PCR) shown that EcMR gene was widely existed in 8 examined tissues in the following order of gill > head kidney > brain > spleen > liver > peripheral blood > heart > muscle. The results showed that RGNNV could proliferate rapidly in GF-1 cells and significantly activate the expression of EcMR gene at 6 h and 24 h after infection. In order to further explore the effect of RGNNV on GF-1 cells, the apoptosis of GF-1 cells infected by RGNNV was detected by the Alexa Fluor? 488 annexin V/Dead Cell Apoptosis Kit. The results showed that RGNNV infection could promote the apoptosis of GF-1 cells, and the apoptosis rate increased with the prolongation of infection time. At the same time, the results of qRT-PCR and enzyme activity assay showed that RGNNV infection could significantly promote the transcription level of apoptosis-related genes and the enzyme activity levels of Caspase-3, Caspase-8 and Caspase-9. To sum up, this study successfully cloned the EcMR gene and revealed its correlation in the process of RGNNV invasion. The results of this study will provide some basic theoretical reference for the prevention and control of grouper viral diseases.
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