鲤天然免疫识别受体TLR1和TLR21的基因克隆及免疫功能的初步研究

发布时间：2018-02-03 03:35

本文关键词： 鲤鱼 TLRs受体基因克隆 poly(I:C) 嗜水气单胞菌　出处：《山东师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：Toll样受体(TLRs)是重要的模式识别分子,是连接适应性免疫和天然免疫的桥梁。TLRs受体可以分为六大亚家族,TLR1家族、TLR3家族、TLR4家族、TLR5家族、TLR7家族、TLR11家族。鱼类是目前已知的TLRs种类最多的物种,目前已经在鱼类中发现21种TLRs。对鱼类TLRs的研究可以为天然免疫的进化研究提供基础数据。本研究运用PCR技术和RACE技术首次从鲤中克隆得到TLR1和TLR21基因的全长。TLR1的cDNA全长为2626 bp,开放阅读框(ORF)为2166 bp,编码721个氨基酸残基,TLR21的cDNA全长为3604 bp,ORF包含2892 bp,编码963个氨基酸残基,利用SMART软件对两者的结构域进行分析,发现其具有典型的TLRs受体结构,包含了一个胞内的TIR结构域、一个跨膜区、一个胞外亮氨酸重复序列结构域。使用BioEdit软件对不同物种的氨基酸序列进行比对,发现胞外具有天冬酰胺位点,推测与马蹄状结构形成有关,胞内TIR结构域保守性较高。利用I-TASSER软件在线模拟TLR1和TLR21的三维空间结构,证实了两者典型的TLRs受体家族特征。使用MEGA6软件对不同鱼类及部分哺乳动物构建进化树,发现鲤鱼的TLR1属于TLR1家族成员,与草鱼的亲缘关系最近,而TLR21属于TLR11家族成员,与斑马鱼和大西洋鲑鱼的亲缘关系最近。利用实时荧光定量PCR技术检测鲤TLR1和TLR21在肝脏、脾脏、头肾、前肠、后肠、鳃、性腺、皮肤、肌肉、口腔上皮和脑等11种组织中表达分布情况。结果显示,TLR1在脑、脾脏、头肾和性腺中表达较高,在后肠和口腔上皮细胞中表达较低,TLR21在脾脏和头肾中表达较高,在皮肤和肝脏中表达较低。对鲤进行嗜水气单胞菌注射刺激后,TLR1 mRNA的表达量在前肠、后肠和肝脏中有明显升高的趋势,其中在后肠和肝脏中升高为对照组的7倍,TLR21 mRNA的表达量在肝脏、前肠和后肠中有升高的趋势。对鲤进行腹腔注射poly(I:C),诱导了其肝脏、脾脏、头肾、前肠和后肠中TLR1和TLR21基因的表达,肝脏和前肠中的TLR1 mRNA的表达量有升高趋势,分别升高为对照组的3倍和2倍,而TLR21 m RNA的表达量在肝脏、脾脏、前肠和后肠中有明显上调趋势,肝脏中升高的倍数最高,可为对照组的20倍。本研究为丰富鱼类天然免疫提供了基础资料,同时也为鲤疾病发病机制的研究和防治提供了理论基础。
[Abstract]:TLRs receptor is an important pattern recognition molecule. It is a bridge between adaptive immunity and innate immunity. TLRs receptor can be divided into six subfamilies: TLR1 family. TLR3 family, TLR4 family, TLR5 family, TLR7 family, TLR11 family. Fish are the most known species of TLRs. At present, 21 species of TLRs have been found in fish. The study on fish TLRs can provide the basic data for the evolutionary study of innate immunity. This study used PCR and RACE technology for the first time from carp. The total length of cDNA of TLR1 and TLR21 gene. TLR1 is 2626. Bp. Open reading frame (ORF) was 2166bp, and the cDNA encoding 721 amino acid residues TLR21 was 3604 BP ORF containing 2892bp. Encoding 963 amino acid residues, SMART software was used to analyze their domains. It was found that they had a typical TLRs receptor structure, including an intracellular TIR domain. A transmembrane domain and an extracellular leucine repeat domain were used to compare the amino acid sequences of different species using BioEdit software. It is speculated that the formation of horseshoe structure is related to the formation of horseshoe structure, and the conserved domain of intracellular TIR is high. I TASSER software is used to simulate the 3D spatial structure of TLR1 and TLR21 on line. The phylogenetic tree of different fish and some mammals were constructed by using MEGA6 software. It was found that the TLR1 of carp belongs to the TLR1 family. TLR21 is a member of the TLR11 family. Real-time fluorescence quantitative PCR was used to detect TLR1 and TLR21 in liver, spleen, head and kidney, foregut, hindgut, Gill, gonad and skin. The results showed that the expression of TLR1 was higher in brain, spleen, head and kidney, and gonad, but lower in hindgut and oral epithelial cells. The expression of TLR21 was higher in spleen and head kidney than in skin and liver. The expression of TLR1 mRNA was found in foregut after injection of Aeromonas hydrophila. The expression of TLR21 mRNA in the hindgut and liver was 7 times higher than that in the control group. The expression of TLR1 and TLR21 genes in liver, spleen, head and kidney, foregut and hindgut of common carp were induced by intraperitoneal injection of polyI: Con. The expression of TLR1 mRNA in liver and foregut was 3 times and 2 times higher than that in control group, while the expression of TLR21 mRNA was found in liver and spleen. There was a significant upward trend in the foregut and hindgut, and the highest increase in liver was 20 times higher than that in the control group. This study provided basic information for enriching the innate immunity of fish. It also provides a theoretical basis for the study and prevention of common carp disease.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S943

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