本文關(guān)鍵詞： 花鱸 Toll樣受體 哈維弧菌 無乳鏈球菌 細(xì)胞轉(zhuǎn)染表達(dá)分析　出處：《上海海洋大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：花鱸(Lateolabrax japonicus)是中國南方重要的經(jīng)濟(jì)魚類,近年來隨著養(yǎng)殖模式的擴(kuò)大和水環(huán)境的惡化,導(dǎo)致水環(huán)境中的病菌極易爆發(fā)流行,這極大限制了花鱸養(yǎng)殖業(yè)的發(fā)展。非特異免疫是魚類抵御外界病菌刺激的第一道防線,其免疫激活主要通過特異性識(shí)別一系列病原相關(guān)分子模式(Pathogen-associated molecular patterns,PAMPs)的模式識(shí)別受體(pattern recognition receptor,PRR)來實(shí)現(xiàn)。其中Toll樣受體(Toll-like receptors,TLR)是一類至關(guān)重要的PRR,屬于Ⅰ型跨膜受體,能夠抵御病原體感染。TLR結(jié)構(gòu)具有高度的保守性,主要包含胞外的富含亮氨酸重復(fù)區(qū)域(LRR)、跨膜區(qū)和胞內(nèi)的TIR受體區(qū)(Toll/interleukin-1 receptor,TIR)。TLR主要通過LRR接受胞外刺激信號(hào),通過TIR與銜接蛋白的結(jié)合繼續(xù)信號(hào)轉(zhuǎn)導(dǎo),激活核因子NF-κB免疫途徑,產(chǎn)生炎癥因子消滅病菌。因此,本實(shí)驗(yàn)篩選克隆得到花鱸LjTLR1、LjTLR3、LjTLR5三個(gè)TLR基因,進(jìn)行細(xì)菌刺激,以期研究三個(gè)TLR基因?qū)Σ≡牡钟饔�。本文根�?jù)從轉(zhuǎn)錄組篩選得到的LjTLR1、LjTLR3、LjTLR5基因片段,利用RACE PCR技術(shù)獲得三個(gè)TLR基因cDNA序列全長(zhǎng),利用不同軟件進(jìn)行序列生物信息學(xué)分析,進(jìn)一步利用實(shí)時(shí)熒光定量PCR技術(shù)分析TLR基因的組織表達(dá)分布。鑒于TLR蛋白對(duì)PAMP的抵抗作用,利用哈維弧菌(Vibrio harveyi)和無乳鏈球菌(Streptococcus agalactiae)注射刺激花鱸,研究三個(gè)TLR基因在面對(duì)不同病菌刺激后的表達(dá)差異。利用原位雜交技術(shù)進(jìn)一步確定病菌對(duì)TLR基因的表達(dá)影響。利用細(xì)胞轉(zhuǎn)染技術(shù)將三個(gè)TLR基因的真核重組質(zhì)粒轉(zhuǎn)入人胚腎293T(HEK-293T)細(xì)胞中,以期初步探究三個(gè)TLR蛋白的細(xì)胞定位。具體研究結(jié)果如下:(1)LjTLR1、LjTLR3、LjTLR5基因cDNA全長(zhǎng)分別為2755、3265和3907bp,開放閱讀框分別包含2484、2769和2676bp。LjTLR1蛋白理論分子量大小和等電點(diǎn)分別是93.55 kDa和6.56,LjTLR3的是103.85 kDa和8.73,LjTLR5的是101.15 kDa和6.10。三個(gè)基因都具有典型的TLR蛋白結(jié)構(gòu)域:LRR、LRRCT、跨膜區(qū)和TIR結(jié)構(gòu)域,只是LRR的數(shù)量有所不同。多重序列比對(duì)結(jié)果顯示,LjTLR1、LjTLR3、LjTLR5氨基酸序列與其他魚類的相似性更高。系統(tǒng)進(jìn)化樹也顯示,三個(gè)TLR基因在進(jìn)化關(guān)系上與魚類更為接近,而與哺乳動(dòng)物有一定的遺傳距離。(2)組織分布表達(dá)結(jié)果顯示,LjTLR1、LjTLR3、LjTLR5在檢測(cè)組織中均有表達(dá),并且在免疫組織中具有高表達(dá)水平。LjTLR1在頭腎、腸和肝臟中表達(dá)較高,在肌肉中表達(dá)最低;LjTLR3在脾臟、頭腎和肝臟中表達(dá)較高,在心臟中表達(dá)最低;LjTLR5在頭腎、肌肉和腸中表達(dá)較高,在肝臟中表達(dá)最低。(3)經(jīng)哈維弧菌和無乳鏈球菌刺激后,LjTLR1、LjTLR3、LjTLR5在免疫組織中均有顯著表達(dá)上調(diào),在肌肉中表達(dá)上調(diào)比例較平穩(wěn),而且哈維弧菌的刺激程度要高于無乳鏈球菌。經(jīng)哈維弧菌刺激后,頭腎中三者表達(dá)均顯著上調(diào),均在6h開始顯著的表達(dá)上調(diào),LjTLR1、LjTLR3、LjTLR5的最高表達(dá)量分別出現(xiàn)在48h、48 h和24 h;脾臟中三者表達(dá)均顯著上調(diào),均在6 h開始表達(dá)上調(diào),LjTLR1、LjTLR3、LjTLR5基因的最高表達(dá)量分別出現(xiàn)在48 h、48 h和24 h。肝臟中三者表達(dá)均顯著上調(diào),均在6 h開始表達(dá)上調(diào),LjTLR1、LjTLR3、LjTLR5基因的最高表達(dá)量分別出現(xiàn)在72 h、24 h和24 h。經(jīng)無乳鏈球菌刺激后,頭腎中三者表達(dá)均顯著上調(diào),均在6 h開始表達(dá)上調(diào),LjTLR1、LjTLR3、LjTLR5基因的最高表達(dá)量分別出現(xiàn)在72 h、96 h和24 h;脾臟中三者表達(dá)均顯著上調(diào),均在6 h開始表達(dá)上調(diào),LjTLR1、LjTLR3、LjTLR5基因的最高表達(dá)量分別出現(xiàn)在48 h、48 h和24h;肝臟中三者表達(dá)均顯著上調(diào),均在6 h開始表達(dá)上調(diào),LjTLR1、LjTLR3、LjTLR5基因的最高表達(dá)量分別出現(xiàn)在24 h、6 h和6 h。在肌肉中三者均在免疫后期才開始出現(xiàn)較顯著的表達(dá)上調(diào)。(4)原位雜交結(jié)果顯示,在脾臟和頭腎中,PBS處理后僅監(jiān)測(cè)到少量陽性信號(hào),而經(jīng)不同菌刺激后LjTLR1、LjTLR3、LjTLR5的陽性信號(hào)明顯多于PBS對(duì)照組。在經(jīng)不同處理的同一組織中,哈維弧菌的LjTLR1、LjTLR3、LjTLR5陽性信號(hào)要明顯強(qiáng)于無乳鏈球菌。這也就進(jìn)一步證明了LjTLR1、LjTLR3、LjTLR5在面對(duì)細(xì)菌刺激時(shí)的表達(dá)特征,均出現(xiàn)了顯著的表達(dá)上調(diào)。(5)本文成功構(gòu)建了真核重組表達(dá)質(zhì)粒,LjTLR1、LjTLR3、LjTLR5三個(gè)TLR重組蛋白在HEK-293T細(xì)胞中成功表達(dá)。三個(gè)TLR重組蛋白定位均在細(xì)胞質(zhì)和膜附近,說明三者可能都屬于典型的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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