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Molecular characterization and functional analysis of IKKα in orange-spotted grouper (Epinephelus coioides).
Fish & Shellfish Immunology ( IF 4.7 ) Pub Date : 2020-03-17 , DOI: 10.1016/j.fsi.2020.03.029
Liangge He 1 , Yulin Zhao 1 , Lin Tang 1 , Xue Yu 1 , Zhifeng Ye 1 , Haoran Lin 2 , Yong Zhang 3 , Shuisheng Li 1 , Danqi Lu 1
Affiliation  

Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) plays crucial roles in regulating activation of nuclear factor kappa-B (NF-κB) in response to pathogens infections. Here, we cloned and identified IKKα gene of orange-spotted grouper (Epinephelus coioides), named as EcIKKα. The gene transcript contained a 2262 bp open reading frame, which encoded 753 amino acids. The typically conserved IKKα structure, including serine kinase domain (KD), leucine chain (LZ) structure, helix-loop-helix (HLH) motif and IKKβ-NEMO-binding domain, was identified in EcIKKα. Phylogenetic analysis suggested that EcIKKα had the closest relationship with large yellow croaker (Larimichthy crocea) IKKα. Ecikkα was ubiquitously expressed in all tissues tested and the highest expression level was in ovary. After lipopolysaccharide (LPS), flagellin, polyinosinic-polycytidylic acid (poly I:C), polyadenylic-polyuridylic acid (poly A:U), and Vibrio parahaemolyticus stimulation, the expression of Ecikkα increased in grouper spleen (GS) cells. In the luciferase assay, NF-κB-luc activity was significantly up-regulated when human embryonic kidney 293T (HEK 293T) cells were transfected with EcIKKα plasmid. Moreover, overexpression of EcIKKα significantly increased LPS- and flagellin-induced proinflammatory cytokines (interleukin-6 (il-6) and tumor necrosis factor-α (tnf-α)) expression, but did not significantly affect poly I:C- and poly A:U-induced cytokines (il-6 and tnf-α) expression. Overall, these results suggested that EcIKKα functions like that of mammals to activate NF-κB, and it could be involved in host defense against invading pathogens.

中文翻译:

橙斑石斑鱼(Epinephelus coioides)中IKKα的分子表征和功能分析。

核因子κB激酶亚基α(IKKα)的抑制剂在响应病原体感染而调节核因子κB(NF-κB)的激活中起关键作用。在这里,我们克隆并鉴定了橙色斑点石斑鱼(Epinephelus coioides)的IKKα基因,命名为EcIKKα。基因转录物包含2262bp的开放阅读框,其编码753个氨基酸。在EcIKKα中鉴定了典型的保守IKKα结构,包括丝氨酸激酶结构域(KD),亮氨酸链(LZ)结构,螺旋-环-螺旋(HLH)基序和IKKβ-NEMO结合结构域。系统发育分析表明,EcIKKα与大黄鱼(IKA)的关系最密切。Ecikkα在所有测试组织中普遍表达,最高表达水平在卵巢中。继脂多糖(LPS),鞭毛蛋白之后,聚肌苷酸-聚胞苷酸(poly I:C),聚腺苷酸-聚尿苷酸(poly A:U)和副溶血性弧菌刺激后,石斑鱼脾脏(GS)细胞中Ecikkα的表达增加。在荧光素酶测定中,当用EcIKKα质粒转染人胚肾293T(HEK 293T)细胞时,NF-κB-luc活性显着上调。此外,EcIKKα的过表达显着增加了LPS和鞭毛蛋白诱导的促炎细胞因子(白介素6(il-6)和肿瘤坏死因子-α(tnf-α))的表达,但并未显着影响poly I:C-和poly A:U诱导的细胞因子(il-6和tnf-α)表达。总体而言,这些结果表明,EcIKKα的功能类似于哺乳动物,以激活NF-κB,它可能参与宿主对入侵病原体的防御。聚腺苷酸-聚尿苷酸(poly A:U)和溶血弧菌刺激,石斑鱼脾(GS)细胞中Ecikkα的表达增加。在荧光素酶测定中,当用EcIKKα质粒转染人胚胎肾293T(HEK 293T)细胞时,NF-κB-luc活性显着上调。此外,EcIKKα的过表达显着增加了LPS和鞭毛蛋白诱导的促炎细胞因子(白介素6(il-6)和肿瘤坏死因子-α(tnf-α))的表达,但并未显着影响poly I:C-和poly A:U诱导的细胞因子(il-6和tnf-α)表达。总体而言,这些结果表明,EcIKKα的功能类似于哺乳动物,以激活NF-κB,它可能参与宿主对入侵病原体的防御。聚腺苷酸-聚尿苷酸(poly A:U)和溶血弧菌刺激,石斑鱼脾(GS)细胞中Ecikkα的表达增加。在荧光素酶测定中,当用EcIKKα质粒转染人胚胎肾293T(HEK 293T)细胞时,NF-κB-luc活性显着上调。此外,EcIKKα的过表达显着增加了LPS和鞭毛蛋白诱导的促炎细胞因子(白介素6(il-6)和肿瘤坏死因子-α(tnf-α))的表达,但并未显着影响poly I:C-和poly A:U诱导的细胞因子(il-6和tnf-α)表达。总体而言,这些结果表明,EcIKKα的功能类似于哺乳动物,以激活NF-κB,它可能参与宿主对入侵病原体的防御。Ecikkα在石斑鱼脾(GS)细胞中的表达增加。在荧光素酶测定中,当用EcIKKα质粒转染人胚胎肾293T(HEK 293T)细胞时,NF-κB-luc活性显着上调。此外,EcIKKα的过表达显着增加了LPS和鞭毛蛋白诱导的促炎细胞因子(白介素6(il-6)和肿瘤坏死因子-α(tnf-α))的表达,但并未显着影响poly I:C-和poly A:U诱导的细胞因子(il-6和tnf-α)表达。总体而言,这些结果表明,EcIKKα的功能类似于哺乳动物,以激活NF-κB,它可能参与宿主对入侵病原体的防御。Ecikkα在石斑鱼脾(GS)细胞中的表达增加。在荧光素酶测定中,当用EcIKKα质粒转染人胚胎肾293T(HEK 293T)细胞时,NF-κB-luc活性显着上调。此外,EcIKKα的过表达显着增加了LPS和鞭毛蛋白诱导的促炎细胞因子(白介素6(il-6)和肿瘤坏死因子-α(tnf-α))的表达,但并未显着影响poly I:C-和poly A:U诱导的细胞因子(il-6和tnf-α)表达。总体而言,这些结果表明,EcIKKα的功能类似于哺乳动物,以激活NF-κB,它可能参与宿主对入侵病原体的防御。用EcIKKα质粒转染人胚肾293T(HEK 293T)细胞后,NF-κB-luc活性显着上调。此外,EcIKKα的过表达显着增加了LPS和鞭毛蛋白诱导的促炎细胞因子(白介素6(il-6)和肿瘤坏死因子-α(tnf-α))的表达,但并未显着影响poly I:C-和poly A:U诱导的细胞因子(il-6和tnf-α)表达。总体而言,这些结果表明,EcIKKα的功能类似于哺乳动物,以激活NF-κB,并且它可能参与宿主对入侵病原体的防御。用EcIKKα质粒转染人胚肾293T(HEK 293T)细胞后,NF-κB-luc活性显着上调。此外,EcIKKα的过表达显着增加了LPS和鞭毛蛋白诱导的促炎细胞因子(白介素6(il-6)和肿瘤坏死因子-α(tnf-α))的表达,但并未显着影响poly I:C-和poly A:U诱导的细胞因子(il-6和tnf-α)表达。总体而言,这些结果表明,EcIKKα的功能类似于哺乳动物,以激活NF-κB,并且它可能参与宿主对入侵病原体的防御。U诱导的细胞因子(IL-6和tnf-α)表达。总体而言,这些结果表明,EcIKKα的功能类似于哺乳动物,以激活NF-κB,它可能参与宿主对入侵病原体的防御。U诱导的细胞因子(IL-6和tnf-α)表达。总体而言,这些结果表明,EcIKKα的功能类似于哺乳动物,以激活NF-κB,它可能参与宿主对入侵病原体的防御。
更新日期:2020-03-19
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