IL-17N的基因克隆、表达及促炎作用
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S941

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中央级基本科研业务费项目(2018HY-ZD02,2016HY-JC03,2020JBFJ02).


Cloning, expression and pro-inflammatory effect of interleukin-17N in Cyprinus carpio
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    摘要:

    白细胞介素17(Interleukin-17,IL-17)在炎症和宿主防御中起重要作用。为了解鲤(Cyprinus carpioIL-17N基因的功能,本研究使用同源搜索和基因克隆的方法在鲤基因组挖掘鉴定到两个IL-17N基因(CcIL-17NaCcIL-17Nb),均由TOP3B基因内含子2的互补序列编码。共线性比较显示,除了东方红鳍鲀(Takifugu rubripes)外,在其他已研究的鱼类中,与该基因相邻的均为SDF2LPPM1F基因。两个CcIL-17Ns都有3个外显子,编码136个氨基酸,两者相似性高达为97.1%,CcIL-17Na、CcIL-17Nb和其他硬骨鱼类的IL-17N相似性分别为65.3%~97.1%、64.7%~96.3%,和鲤IL-17家族其他成员的相似性分别为32.9%~51.4%、31.4%~50.7%。鱼类IL-17家族系统树显示7个成员形成6支,其中IL-17A/F1和IL-17A/F3组成一支,其余6个成员单独成支,鲤IL-17N先与鲤科鱼类以94%置信值聚在一起,然后与虹鳟(Oncorhynchus mykiss)、大西洋鲑(Salmo salar)、青鳉(Oryzias latipes)、东方红鳍鲀(Takifugu rubripes)和尼罗罗非鱼(Oreochromis niloticus)等以85%的置信值聚在一起,再与雀鳝(Lepisosteus oculatus)和腔棘鱼(Coelacanth)的IL-17N以95%置信值聚为一支。实时定量PCR结果显示CcIL-17Ns在受精后0.5 h和12 h的表达量极显著高于受精后25 h、35 h、60 h、120 h及仔鱼阶段的表达量(P<0.01);CcIL-17Ns在鲤夏花和成鱼脑中的表达量均最高,显著(P<0.05)或极显著(P<0.01)高于其他组织。嗜水气单胞菌(Aeromonas hydrophila)感染导致CcIL-17Ns在各组织的表达均上调,感染6 h,脑中的表达量显著增加(P<0.05),1 d,CcIL-17Ns在其他组织中均显著增加(P<0.05),感染3 d和7 d,表达量均下降至与对照组无显著差异(P>0.05)。构建原核重组表达载体pMAL-c2X-17N,在大肠杆菌(Escherichia coli)Transetta(DE3)中进行原核表达,使用镍柱纯化获得可溶的IL-17N重组蛋白(MBP-17N)。使用0.1 ng/mL、1 ng/mL、10 ng/mL和100 ng/mL的MBP-17N孵育鲤肾组织8 h,结果显示,不同浓度的MBP-17N均使IL-1βNF-κB显著上调(P<0.05);1 ng/mL和10 ng/mL的MBP-17N极显著上调IFN-γP<0.01);0.1 ng/mL、1 ng/mL的MBP-17N显著上调IL-6,1 ng/mL、10 ng/mL、100 ng/mL的MBP-17N显著上调CCL20P<0.05);1 ng/mL的MBP-17N使TRAF6的表达显著高于对照组(P<0.05)。本研究通过分析鲤IL-17Ns的系统发育、测定该基因的时空表达特征,发现嗜水气单胞菌感染后各组织CcIL-17Ns表达上调、原核表达蛋白能引起炎症因子的表达,从而证实了鲤IL-17N参与炎症反应。

    Abstract:

    Interleukin-17 (IL-17) plays an important role in inflammation and host defense in mammals. To investigate the biological function of CcIL-17N, two CcIL-17N genes were identified from the common carp (Cyprinus carpio) whole-genome using BLAST and gene cloning performed by genomics, named as CcIL-17Na and CcIL-17Nb, respectively. They were encoded by the complementary sequence of intron 2 of the TOP3B gene. Synteny analysis indicated that SDF2L and PPM1F were present on both sides of IL-17N, except for Takifugu rubripes. These two IL-17N isoforms encoded 136 amino acids, including 3 exons, and exhibited 97.1% similarity in sequence. The similarity between CcIL-17Na and CcIL-17Nb of common carp with other teleosts were estimated to be 65.3%-97.1%, 64.7%-96.3%, and of 32.9%-51.4%, 31.4%-50.7% in comparison with other IL-17 family members in common carp. The phylogenetic tree of teleost IL-17 family members showed that 7 members constitute 6 credible branches, of which IL-17A/F1 and IL-17A/F3 constitute a branch, and the remaining six members form a branch separately. IL-17N of common carp was clustered with zebrafish and grass carp with a bootstrap of 95%, with medaka, T. rubripes, tilapia, etc. clustered with a bootstrap of 84%, with Lepisosteus osseus IL-17N with a bootstrap of 70%, and finally coelacanth IL-17N with a bootstrap of 97%. CcIL-17Ns expression level at 0.5 and 12 h after fertilization was significantly higher than that at 25, 35, 60, 120 h, and larval fish stage (P<0.01). CcIL-17Ns expression in the brain of summerlings and adult fish was the highest, which was significantly higher than other tissues (P<0.05 or P<0.01). CcIL-17Ns expression in all tissues was up-regulated under Aeromonas hydrophilis infection. CcIL-17Ns expression in the brain was significantly up-regulated for 6 h after infection (P<0.05). At 1 d, CcIL-17Ns expression in other tissues was significantly up-regulated, and the expression was decreased for 3 d and 7 d after infection, with no significant difference from the control group (P>0.05). Recombinant prokaryotic expression plasmid pMAL-c2X-17N was constructed and transformed into Escherichia coli Transetta (DE3) for prokaryotic expression. Soluble recombinant common carp IL-17N protein (rccIL-17N, MBP-17N) was obtained by Ni-NTA. Under the induction of different concentrations of MBP-17N protein (0.1, 1, 10, and 100 ng/mL), kidney tissue was incubated for 8 h. qPCR indicated that IL-1β and NF-κB was significantly up-regulated at 0.1, 1, 10, and 100 ng/mL of MBP-17N. IFN-γ was significantly up-regulated under 1 and 10 ng/mL MBP-17N, whereas IL-6 was significantly up-regulated at 0.1 and 1 ng/mL. CCL20 was significantly up-regulated at 1, 10, and 100 ng/mL. Furthermore, TRAF6 gene significantly upregulated at 1 ng/ml than that of the control group. In conclusion, this study revealed that ccIL-17N was highly conserved in evolution. Meanwhile, ccIL-17N is involved in inflammatory response based on its role in A. hydrophila infection and rccIL-17N function.

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张磊,沈泽恩,李红霞,徐逾鑫,李迎宾,俞菊华.鲤IL-17N的基因克隆、表达及促炎作用[J].中国水产科学,2020,27(12):1402-1414
ZHANG Lei, SHEN Ze'en, LI Hongxia, XU Yuxin, LI Yingbin, YU Juhua. Cloning, expression and pro-inflammatory effect of interleukin-17N in Cyprinus carpio[J]. Journal of Fishery Sciences of China,2020,27(12):1402-1414

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  • 收稿日期:2020-04-06
  • 最后修改日期:2020-05-11
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  • 在线发布日期: 2020-12-17
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