鸡DDX 41基因克隆表达、细胞定位及其在禽腺病毒4型感染调控天然免疫中的作用  

Cloning,expression,cellular localization of chicken DDX41 gene and its role in regulation of natural immunity by avian adenovirus type 4 infection

作　　者：刘琪 曹影丽 魏宁波 杨侃侃 梁月巧 宋祥军 邵颖 涂健 祁克宗 LIU Qi;CAO Yingli;WEI Ningbo;YANG Kankan;LIANG Yueqiao;SONG Xiangjun;SHAO Ying;TU Jian;QI Kezong(Anhui Key Laboratory of Veterinary Pathobiology and Disease Control,Anhui Province Animal Food Quality and Biosafety Engineering Laboratory,Hefei 230036,China)

机构地区：[1]兽医病理生物学与疫病防控安徽省重点实验室,安徽省动物性食品质量与生物安全工程实验室,安徽合肥230036

出　　处：《浙江农业学报》2023年第5期1028-1036,共9页Acta Agriculturae Zhejiangensis

基　　金：国家自然科学基金(31972642)。

摘　　要：为获得鸡DDX41(chDDX41)的原核表达蛋白,探讨chDDX41的细胞定位及其在禽腺病毒4型(FAdV-4)感染引起的天然免疫中的作用。根据GenBank中chDDX41基因序列,设计特异性引物,以LMH细胞cDNA为模板扩增,将扩增序列连接至pET-32a、pCAGGS-HA、pCMV-3×Flag和pmCherry-C1空载体,构建重组质粒。将pET-32a-chDDX 41转化至大肠埃希菌BL21(DE3)感受态细胞中,经IPTG诱导表达。利用激光共聚焦显微镜观察chDDX41在鸡肝癌细胞(LMH)和鸡巨噬细胞(HD11)中的亚细胞定位;利用实时荧光定量PCR(qRT-PCR)检测在LMH细胞中过表达chDDX 41对IFN-β等细胞因子表达量的影响。结果表明,克隆得到1854 bp的chDDX 41基因,共编码617个氨基酸。SDS-PAGE结果显示,重组蛋白pET-32a-chDDX41主要以可溶性蛋白的方式在上清液中表达,经Western Blot鉴定,在90 ku左右有特异性条带,与预期结果一致;chDDX41在LMH和HD11细胞中主要定位在细胞核;FAdV-4感染过表达chDDX 41基因后的LMH细胞,发现IFN-β、IL-6、IL-1β和IL-8等细胞因子在mRNA水平上的表达量上调,表明chDDX41参与了FAdV-4引起的天然免疫应答。研究结果可为进一步研究DDX 41基因功能及其在天然免疫中的分子机制提供参考。To obtain the prokaryotic expression protein of chicken DDX41(chDDX41)and to investigate the subcellular localization of chDDX41 and its in natural immunity induced by avian adenovirus type 4(FAdV-4)infection,based on the sequence of chDDX 41 gene in GenBank,specific primers were designed to amplify and ligation of amplified sequences to pET-32a,pCAGGS-HA,pCMV-3×Flag and pmCherry-C1 empty vectors using LMH cell cDNA as template to construct recombinant plasmids.The pET-32a-chDDX 41 was transformed into Escherichia coli BL21(DE3)receptor cell with IPTG-induced expression.The subcellular localization of chDDX41 in chicken liver cancer cells(LMH)and chicken macrophage cell line(HD11)cells was observed by laser confocal microscopy,and the effect of overexpression of chDDX 41 on cytokine expression in FAdV-4-infected LMH cells was examined by real-time fluorescence quantitative PCR(qRT-PCR).The result showed that chDDX 41 gene was of 1854 bp in size,encoding 617 amino acids.SDS-PAGE showed that the recombinant protein pET-32a-chDDX41 was mainly expressed as soluble protein in the supernatant,and the specific band appeared at around 90 ku was identified by Western Blot,which was consistent with the expected results.chDDX41 was mainly localized in the nucleus in LMH and HD11 cells;FAdV-4 infection of LMH cells after overexpression of the chDDX 41 gene revealed an upregulation of the expression of cytokines such as IFN-β,IL-6,IL-1βand IL-8 at the mRNA level,indicating that chDDX41 was involved in the natural immune response induced by FAdV-4.The result provided a basis for further study of the gene function of DDX41 and its specific molecular mechanism in natural immunity.

关 键 词：chDDX41 原核表达 细胞定位 禽腺病毒4型 天然免疫 

分 类 号：S855.3[农业科学—临床兽医学]