花鲈ToLL样受体家族部分基因的克隆及其在细菌刺激下的表达特征研究

发布时间：2018-01-30 05:19

  本文关键词： 花鲈 Toll样受体 哈维弧菌 无乳链球菌 细胞转染表达分析　出处：《上海海洋大学》2017年硕士论文　论文类型：学位论文

【摘要】：花鲈(Lateolabrax japonicus)是中国南方重要的经济鱼类,近年来随着养殖模式的扩大和水环境的恶化,导致水环境中的病菌极易爆发流行,这极大限制了花鲈养殖业的发展。非特异免疫是鱼类抵御外界病菌刺激的第一道防线,其免疫激活主要通过特异性识别一系列病原相关分子模式(Pathogen-associated molecular patterns,PAMPs)的模式识别受体(pattern recognition receptor,PRR)来实现。其中Toll样受体(Toll-like receptors,TLR)是一类至关重要的PRR,属于Ⅰ型跨膜受体,能够抵御病原体感染。TLR结构具有高度的保守性,主要包含胞外的富含亮氨酸重复区域(LRR)、跨膜区和胞内的TIR受体区(Toll/interleukin-1 receptor,TIR)。TLR主要通过LRR接受胞外刺激信号,通过TIR与衔接蛋白的结合继续信号转导,激活核因子NF-κB免疫途径,产生炎症因子消灭病菌。因此,本实验筛选克隆得到花鲈LjTLR1、LjTLR3、LjTLR5三个TLR基因,进行细菌刺激,以期研究三个TLR基因对病原菌的抵御作用。本文根据从转录组筛选得到的LjTLR1、LjTLR3、LjTLR5基因片段,利用RACE PCR技术获得三个TLR基因cDNA序列全长,利用不同软件进行序列生物信息学分析,进一步利用实时荧光定量PCR技术分析TLR基因的组织表达分布。鉴于TLR蛋白对PAMP的抵抗作用,利用哈维弧菌(Vibrio harveyi)和无乳链球菌(Streptococcus agalactiae)注射刺激花鲈,研究三个TLR基因在面对不同病菌刺激后的表达差异。利用原位杂交技术进一步确定病菌对TLR基因的表达影响。利用细胞转染技术将三个TLR基因的真核重组质粒转入人胚肾293T(HEK-293T)细胞中,以期初步探究三个TLR蛋白的细胞定位。具体研究结果如下:(1)LjTLR1、LjTLR3、LjTLR5基因cDNA全长分别为2755、3265和3907bp,开放阅读框分别包含2484、2769和2676bp。LjTLR1蛋白理论分子量大小和等电点分别是93.55 kDa和6.56,LjTLR3的是103.85 kDa和8.73,LjTLR5的是101.15 kDa和6.10。三个基因都具有典型的TLR蛋白结构域:LRR、LRRCT、跨膜区和TIR结构域,只是LRR的数量有所不同。多重序列比对结果显示,LjTLR1、LjTLR3、LjTLR5氨基酸序列与其他鱼类的相似性更高。系统进化树也显示,三个TLR基因在进化关系上与鱼类更为接近,而与哺乳动物有一定的遗传距离。(2)组织分布表达结果显示,LjTLR1、LjTLR3、LjTLR5在检测组织中均有表达,并且在免疫组织中具有高表达水平。LjTLR1在头肾、肠和肝脏中表达较高,在肌肉中表达最低;LjTLR3在脾脏、头肾和肝脏中表达较高,在心脏中表达最低;LjTLR5在头肾、肌肉和肠中表达较高,在肝脏中表达最低。(3)经哈维弧菌和无乳链球菌刺激后,LjTLR1、LjTLR3、LjTLR5在免疫组织中均有显著表达上调,在肌肉中表达上调比例较平稳,而且哈维弧菌的刺激程度要高于无乳链球菌。经哈维弧菌刺激后,头肾中三者表达均显著上调,均在6h开始显著的表达上调,LjTLR1、LjTLR3、LjTLR5的最高表达量分别出现在48h、48 h和24 h;脾脏中三者表达均显著上调,均在6 h开始表达上调,LjTLR1、LjTLR3、LjTLR5基因的最高表达量分别出现在48 h、48 h和24 h。肝脏中三者表达均显著上调,均在6 h开始表达上调,LjTLR1、LjTLR3、LjTLR5基因的最高表达量分别出现在72 h、24 h和24 h。经无乳链球菌刺激后,头肾中三者表达均显著上调,均在6 h开始表达上调,LjTLR1、LjTLR3、LjTLR5基因的最高表达量分别出现在72 h、96 h和24 h;脾脏中三者表达均显著上调,均在6 h开始表达上调,LjTLR1、LjTLR3、LjTLR5基因的最高表达量分别出现在48 h、48 h和24h;肝脏中三者表达均显著上调,均在6 h开始表达上调,LjTLR1、LjTLR3、LjTLR5基因的最高表达量分别出现在24 h、6 h和6 h。在肌肉中三者均在免疫后期才开始出现较显著的表达上调。(4)原位杂交结果显示,在脾脏和头肾中,PBS处理后仅监测到少量阳性信号,而经不同菌刺激后LjTLR1、LjTLR3、LjTLR5的阳性信号明显多于PBS对照组。在经不同处理的同一组织中,哈维弧菌的LjTLR1、LjTLR3、LjTLR5阳性信号要明显强于无乳链球菌。这也就进一步证明了LjTLR1、LjTLR3、LjTLR5在面对细菌刺激时的表达特征,均出现了显著的表达上调。(5)本文成功构建了真核重组表达质粒,LjTLR1、LjTLR3、LjTLR5三个TLR重组蛋白在HEK-293T细胞中成功表达。三个TLR重组蛋白定位均在细胞质和膜附近,说明三者可能都属于典型的TLR蛋白。
[Abstract]:Japanese sea bass (Lateolabrax japonicus) is an important economic fish Chinese south, in recent years with the expansion of farming mode and deterioration of water environment, resulting in water environment bacteria easily outbreak, which greatly limits the development of aquaculture. Japonicus nonspecific immunity is the first line of defense against external stimulation of the fish pathogen, its main immune activation through a series of specific recognition of pathogen associated molecular patterns (Pathogen-associated molecular, patterns, PAMPs) pattern recognition receptors (pattern recognition, receptor, PRR) to achieve. The Toll like receptor (Toll-like receptors TLR) is a kind of important PRR, belongs to the type I transmembrane receptor, can resist pathogen infection is highly.TLR the conservative, mainly contains the extracellular leucine rich repeat region (LRR), TIR receptor region, transmembrane region and intracellular (Toll /interleukin-1 receptor, TIR.TLR) Mainly through the LRR to receive extracellular stimuli, through a combination of TIR and adaptor protein to signal transduction and activation of nuclear factor kappa NF- B immune pathway, inflammatory cytokines kill bacteria. Therefore, the screening of cloned LjTLR1 LjTLR3, LjTLR5 japonicus, three TLR genes, bacterial stimulation, in order to study the resistant effect of three the TLR gene of pathogenic bacteria. According to the selected from the transcriptome of LjTLR1, LjTLR3, LjTLR5 gene, full-length three TLR cDNA gene sequence using RACE PCR technology, the sequence bioinformatics analysis using different software, further by using real-time quantitative PCR analysis of TLR gene expression in resistance distribution. The TLR protein of PAMP, by Harvey (Vibrio harveyi) and Vibrio agalactiae (Streptococcus agalactiae) injection stimulation on three TLR based latelabrajaponicus, because in the face of different bacteria thorn Differential expression stimulated by in situ hybridization. The expression of TLR gene in bacteria further determine the effect of using cell transfection technique. The eukaryotic recombinant plasmid of three TLR gene into human embryonic kidney 293T (HEK-293T) cells, cellular localization in order to preliminary explore three TLR protein. The specific results are as follows: (1 LjTLR1, LjTLR3), the full-length LjTLR5 gene of cDNA were 27553265 and 3907bp, respectively. The open reading frame contains 24842769 2676bp.LjTLR1 protein and theoretical molecular weight and isoelectric point were 93.55 kDa and 6.56 LjTLR3, 103.85 kDa and 8.73 LjTLR5 are 101.15 kDa and 6.10. three genes with TLR protein domain typical: LRR, LRRCT, transmembrane region and TIR domain, but the number of LRR different. Multiple sequence alignment showed that LjTLR1, LjTLR3, LjTLR5 amino acid sequence similarity with other species. The phylogenetic tree is also higher Show that the three TLR genes in the evolutionary relationship with fish closer, and a certain genetic distance and mammals. (2) the expression of tissue distribution showed that LjTLR1, LjTLR3, LjTLR5 in the detection of tissues, and has a high expression level of.LjTLR1 in head kidney in immune tissues, high expression of intestinal and in the liver, muscle was the lowest; LjTLR3 high expression in the spleen, head kidney and liver, in the heart was the lowest; LjTLR5 high expression in head kidney, muscle and intestine and in the liver was the lowest. (3) by Harvey Vibrio and Streptococcus agalactiae after stimulation, LjTLR1, LjTLR3, LjTLR5 were in immune tissues were significantly up-regulated in the muscle expression ratio is relatively stable, and the degree of stimulation of Vibrio Harvey is higher than that of Streptococcus agalactiae. By Vibrio Harvey stimulation, three head kidney expression were significantly increased in 6h, significantly the expression of 璋，

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