鲤IL-17B基因序列特征、表达模式、原核重组蛋白的获得及其促炎作用  

Sequence analysis,expression patterns,acquisition of prokaryotic recombinant protein and pro-inflammatory effect of IL-17B in Cyprinus carpio

作　　者：王钰婧 徐逾鑫 冯文荣 李建林[1,2] 李红霞[1,2] 苏胜彦[1,2] 宋长友[1,2] 唐永凯 俞菊华[1,2] WANG Yujing;XU Yuxin;FENG Wenrong;LI Jianlin;LI Hongxia;SU Shengyan;SONG Changyou;TANG Yongkai;YU Juhua(Wuxi Fisheries College,Nanjing Agricultural University,Wuxi 214128,China;Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,Wuxi 214081,China)

机构地区：[1]南京农业大学无锡渔业学院,江苏无锡214128 [2]中国水产科学研究院淡水渔业研究中心,江苏无锡214081

出　　处：《水产学报》2023年第8期33-45,共13页Journal of Fisheries of China

基　　金：中国水产科学研究院基本科研业务费(2018HY-ZD02,2020TD37);国家现代农业产业技术体系专项(CARS-45)。

摘　　要：为了解鲤白细胞介素17B基因(IL-17B)的功能,实验使用同源搜索和基因克隆技术在鲤基因组中挖掘到2个IL-17Bs基因(CcIL-17B1和CcIL-17B2),均有3个外显子和2个内含子,编码198个氨基酸,内含IL-17家族特有的由4个半胱氨酸形成的2个二硫键,蛋白序列一致性高达91.92%。共线性分析显示,在硬骨鱼类染色体加倍过程中,IL-17B及其附近基因出现了丢失,大部分硬骨鱼类有1个IL-17B、斑马鱼2个IL-17Bs均丢失,而鲤特有的染色体加倍致使存在2个CcIL-17Bs。实时荧光定量PCR(qPCR)结果显示,CcIL-17Bs在鲤受精卵发育早期(0~12 h)和成鱼性腺中高表达。使用大肠杆菌表达系统,获得了可溶的重组蛋白NusA-17B。肛灌不同浓度的NusA-17B,结果显示,高浓度(500μg/kg)组的1和3 d的鲤肠道组织肠绒毛缺损,出现大量的杯状细胞和炎性细胞,定量分析显示,炎症因子基因IL-1β、IFN-γ、IL-6、趋化因子CCL20和NF-κB的表达均显著上调;7 d时肠道组织结构和炎症相关基因的表达均得到恢复,与对照组无显著差异。低浓度(5μg/kg)和中浓度(50μg/kg)肛灌实验,除了中浓度1和3 d鲤肠道TRAF6的表达显著上调,其余被检测基因的表达均与对照组无显著差异。使用不同浓度(0.1、1.0、10.0和100.0 ng/mL)NusA-17B孵育鲤肾组织8 h,结果显示,IL-1β在0.1、1.0和100.0 ng/mL的NusA-17B刺激下显著上调,IFN-γ和NF-κB分别只在10.0和0.1 ng/mL的NusA-17B刺激下显著上调,IL-6及趋化因子CCL20能够在10.0和100.0 ng/mL的NusA-17B刺激下显著表达,TRAF6则在0.1、1.0和10.0 ng/mL的NusA-17B刺激下显著表达。体内和体外实验结果表明CcIL-17B参与了炎症反应。Interleukin-17(IL-17)is an important inflammatory factor,which is able to promote the production of various inflammation-related factors,such as IL-1β,IFN-γ,IL-6,CCL20 and so forth.It plays an important role in autoimmune disease,host defense,inflammation and so on.In order to study the pro-inflammatory function of IL-17B in the Cyprinus carpio,the IL-17B gene was cloned,the NusA-17B protein was recombinantly expressed by Escherichia coli system and the pro-inflammatory effect of the IL-17B was then investigated by in vivo and in vitro experiments.In this study,by homology search and gene cloning method,we found two IL-17Bs in the genome of the C.carpio,CcIL-17B1 and CcIL-17B2 respectively.Both of the two genes have open reading frames(ORF)of 597 bp,which are composed of three exons and two introns.The length of their exons are the same,24,320 and 253 bp respectively.While the introns are different,the CcIL-17B1 intron are 84,420 bp respectively and the CcIL-17B2 intron are 81,851 bp respectively.The two CcIL-17Bs encode 198 amino acids,containing 2 disulfide bonds formed by 4 cysteines,which were unique to the IL-17 family.Sequence blast analysis showed that the consistency of the two CcIL-17Bs proteins reached 91.92%.The collinearity analysis results are as follows.During the doubling of the chromosomes of teleost,IL-17B and its nearby genes were lost.Most teleost have one IL-17B and Danio rerio have both IL-17Bs that were lost.The common carp-specific chromosomes doubled resulting in the presence of two CcIL-17Bs.Real time quantitative PCR(qPCR)was used to measure the expression of CcIL-17B1 and CcIL-17B2 in fertilized eggs,larvae and different tissues of adult C.carpio.The results showed that the expression levels of both CcIL-17B1 and CcIL-17B2 were significantly higher at 0-12 hours post fertilization than that at 25-90 hours post fertilization(P<0.05).While the expression levels have no significant difference at 25-90 hours after fertilization and 1-14 days after hatching(P>0.05).In adult fish,the expres

关 键 词：鲤 IL-17B 基因表达 重组蛋白 炎症反应 

分 类 号：Q785[生物学—分子生物学] S917.4[农业科学—水产科学]