三倍体湘云鲫2号IFNa3基因的克隆及功能初探  

Molecular cloning and preliminary functional study of IFNa3 in No.2 of Xiangyun crucian carp

作　　者：吴慧 侯利芬 黄嘉仪 冯浩[1] WU Hui;HOU Lifen;HUANG Jiayi;FENG Hao(State Key Laboratory of Developmental Biology of Freshwater Fish,College of Life Science,Hunan Normal University,Changsha 410081,China)

机构地区：[1]湖南师范大学,省部共建淡水鱼类发育生物学国家重点实验室,湖南长沙410081

出　　处：《水产学报》2021年第9期1478-1490,共13页Journal of Fisheries of China

基　　金：国家自然科学基金(31920103016,32002415);湖南省自然科学基金(2019RS1038);湖南省现代农业(水产)产业项目;湖南省学位与研究生教育改革项目(2019JGYB095);湖南省研究生优秀教学团队项目。

摘　　要：三倍体湘云鲫2号具有不育、生长快、抗病抗逆性强等优良特性。为探究其抗病优势分子机制,实验克隆及鉴定了三倍体湘云鲫2号干扰素a3(3nIFNa3)。3nIFNa3的CDS由552个核苷酸组成,编码184个氨基酸。经预测,3nIFNa3 N端23位氨基酸为信号肽;3nIFNa3成熟肽中存在2个半胱氨酸残基并参与形成二硫键,表明其隶属于Ⅰ型一组干扰素。实时荧光定量PCR(qPCR)结果显示,宿主细胞经poly I∶C刺激后8 h、草鱼呼肠孤病毒(GCRV)或鲤春病毒血症病毒(SVCV)感染后48 h,3nIFNa3的转录水平达到最高。免疫印迹和免疫荧光结果显示,3nIFNa3为分泌型蛋白,分子量约为21.8 ku,其在出胞前主要分布于细胞质中。进一步研究发现,在宿主细胞中过表达3nIFNa3或含3nIFNa3的上清培养基孵育宿主细胞均能诱导内源ISG基因转录水平的显著提高。其中,3nSTAT1及3nVIPERIN在含3nIFNa3的上清培养基孵育后2 h转录水平达到最高,3nPKR则在4 h表达水平最高。此外,病毒滴度测定及结晶紫染色实验表明,EPC细胞经3nIFNa3孵育或过表达3nIFNa3后,其抗GCRV和SVCV的能力均显著增强。研究表明,3nIFNa3为可分泌的细胞因子,在宿主抗病毒天然免疫反应中发挥作用。No.2 of Xiangyun crucian carp possesses the advantages of sterility, rapid growth and improved disease and stress resistance. In order to explore the molecular mechanism of its disease resistance, triploid hybrid interferon a3(3 nIFNa3) has been cloned and characterized. The CDS of 3 nIFNa3 comprised 552 nucleotides, encoding 184 amino acids. The first 23 amino acids of N-terminal were signal peptides. There are 2 cysteine residues involved in the formation of disulfide bonds in mature peptides of 3 nIFNa3, which indicates that 3 nIFNa3 belongs to group I type I interferon. qPCR results showed that the mRNA levels of 3 nIFNa3 in the host cells reached the peak at 8 h after poly(I: C) stimulation and reached the highest at 48 h after GCRV or SVCV infection. Both Western blot and immunofluorescence results showed that 3 nIFNa3 was a secretory protein and its molecular weight was around 21.8 ku, which was mainly distributed in the cytoplasm before secretion. Further studies showed that the transcription of endogenous ISG genes were significantly up-regulated after the host cells overexpressed 3 nIFNa3 or were incubated with the 3 nIFNa3-containing conditioned media. The transcription levels of3 nSTAT1, 3 nVIPERIN or 3 nPKR reached the highest at 2, 2 or 4 h after the host cells were incubated with the3 nIFNa3-containing conditioned media. Besides, the results of the classic plaque assay and the crystal violet staining showed that EPC cells presented significantly enhanced antiviral ability against GCRV and SVCV after incubation with 3 nIFNa3 or overexpression of 3 nIFNa3. The above results show that 3 nIFNa3 is a secretory cytokine and plays an important role in the host antiviral innate immune response.

关 键 词：三倍体湘云鲫2号 Ⅰ型干扰素(IFN-Ⅰ) 鲤春病毒血症病毒(SVCV) 草鱼呼肠孤病毒(GCRV) 天然免疫 

分 类 号：Q786[生物学—分子生物学] S917.4[农业科学—水产科学]