鴿Toll樣受體7基因的克隆及其免疫生物學(xué)功能研究
發(fā)布時間：2019-07-09 12:20
【摘要】：天然免疫系統(tǒng)是機體防御病原體入侵的首道防線。病原微生物感染后,機體細胞通過模式識別受體(Pattern-recognition receptors, PRRs)識別病原相關(guān)分子模式(Pathogen-associated molecular patterns, PAMPs),然后激活下游信號通路,誘導(dǎo)炎性因子和I型干擾素的表達。Toll樣受體7(Toll-like receptor 7, TLR7)位于細胞的內(nèi)體上,能識別病毒的單鏈RNA及咪唑喹啉類成分,并誘導(dǎo)天然免疫應(yīng)答,在抗病毒感染中具有重要作用。相較于哺乳動物TLR7,禽類TLR7的序列特征和生物功能還研究較少,目前主要集中在雞、鴨和鵝等水陸禽類中,而飛禽如鴿TLR7的研究至今還未見報道。本研究旨在克隆和分析鴿TLR7的基因序列,挖掘其關(guān)鍵的配體識別位點,并探索其在天然免疫應(yīng)答中的功能,從而為進一步研究鴿TLR7的結(jié)構(gòu)特征和生物功能以及禽類的天然免疫積累理論依據(jù)。1.鴿TLR7基因的克隆、序列分析及其表達譜檢測本研究根據(jù)禽類TLR7基因的保守序列設(shè)計出特異性引物,首次從大王鴿克隆TLR7基因。利用生物信息學(xué)軟件分析其序列特征,鴿TLR7基因的開放閱讀框為3144 bp,編碼1047個氨基酸,結(jié)構(gòu)預(yù)測表明其是一類典型的TLR,由N端的一個信號肽序列、15個LRR重復(fù)序列、一個LRR-CT和胞內(nèi)TIR區(qū)組成。蛋白相互作用空間結(jié)構(gòu)預(yù)測結(jié)果表明兩個TLR7的膜內(nèi)結(jié)構(gòu)域形成典型的“m”形二聚體。糖基化和磷酸化位點預(yù)測表明鴿TLR7含有18個潛在的N-糖基化位點和38個磷酸化位點。鴿TLR7氨基酸序列與人、鼠、雞、鴨TLR7的同源性分別為65.0%、62.2%、81.5%和83.8%。通過RT-PCR分析鴿不同組織中TLR7基因的表達差異,結(jié)果表明鴿TLR7在免疫相關(guān)組織尤其是脾臟和肝臟中具有較高的表達水平。2.鴿TLR7的免疫生物學(xué)功能及其配體識別位點的挖掘構(gòu)建含有鴿TLR7的真核表達質(zhì)粒pCMV-PiTLR7,轉(zhuǎn)染HEK293T細胞后,Western blotting鑒定了TLR7的表達。利用NF-κB熒光素酶報告載體,在R848配體刺激后,鴿TLR7能夠顯著地介導(dǎo)NF-κB的活化,表明鴿TLR7是一類有功能的TLR。對鴿TLR7 LRR序列進行分析后,發(fā)現(xiàn)LRR2、LRR11、LRR13和LRR14的第15位以及LRR10的第10位均存在氨基酸的插入修飾,于是我們通過overlap-PCR的方法分別構(gòu)建了這些位點缺失的TLR7突變體,并連接到pCMV真核表達載體上。利用NF-κB熒光素酶報告系統(tǒng),結(jié)果顯示這些插入序列的缺失完全廢除了TLR7介導(dǎo)的NF-κB的活化,表明非經(jīng)典LRR序列是TLR7發(fā)揮功能所必需的,這些序列可能是TLR7識別配體的關(guān)鍵位點。此外,通過qRT-PCR和ELISA方法檢測了TLR7和細胞因子的表達,結(jié)果表明TLR7突變體均不能介導(dǎo)IL-8的產(chǎn)生,這與NF-κB活性檢測實驗結(jié)果是一致的；然而部分突變體(DellOIN10和De114IN15)可介導(dǎo)IFN-α和TNF-α的產(chǎn)生,同時,R848刺激后,鴿TLR7突變體的表達水平均有不同程度的上調(diào),表明鴿TLR7突變體通過增加其表達量進而代償其功能的缺陷。然而,鴿TLR7 De111IN15突變體盡管增加了其自身的表達量,仍不能有效地誘導(dǎo)IFN-α、IL-8和‘TNF-α的產(chǎn)生,表明關(guān)鍵LRR插入序列的缺失會導(dǎo)致假代償?shù)某霈F(xiàn)。3.鴿TLR7介導(dǎo)激動劑及新城疫病感染的天然免疫應(yīng)答研究通過R848刺激鴿外周血單核細胞,應(yīng)用熒光定量PCR的方法檢測TLR7、IFN-γ、IL-6、 IL-8、CCL5和IL-10的mRNA水平,結(jié)果表明R848刺激12 h和24 h后前炎性細胞因子和相關(guān)抗病毒分子均顯著上調(diào),而TLR7的表達水平?jīng)]有顯著變化。分別使用新城疫LaSota疫苗株和R848激動劑肌肉注射大王鴿,通過qRT-PCR的方法檢測了脾臟中TLR7及相關(guān)細胞因子的表達,結(jié)果表明R848肌肉注射組中TLR7的mRNA水平顯著升高,LaSota感染組TLR7的表達在第三天時顯著降低,而前炎性細胞因子和趨化因子在兩個肌肉注射組中均顯著上調(diào),表明鴿TLR7介導(dǎo)的免疫應(yīng)答與其自身的表達水平并沒有必然聯(lián)系。鴿TLR7能介導(dǎo)NF-κB的活化,顯著增強前炎性細胞因子和相關(guān)抗病毒分子的產(chǎn)生,在天然免疫抗病毒感染中扮演了重要的作用。
文內(nèi)圖片：
圖片說明：圖１－２重組質(zhì)粒ｐＣＭＶ－ＰｒｒＬＲ７酶切筵定逡逑Ｆｉｇ．邋ＭＰＣＲ邋ａｍｐｌｉｆｉｃａｔｉｏｎ邋ｏｆｐｉｇｅｏｎＴＬＲ７邋ｇｅｎｅ邋雌邋１－２邋Ｅｎｚｙｍｅ邋ｄｉｇｅｓｔｉｏｎ邋ｏｆ邋ｐｌａｓｍｉｄ逡逑
[Abstract]:The natural immune system is the first line of defense for the body to defend the invasion of the pathogen. After the infection of the pathogenic microorganisms, the body cells recognize the pathogen-associated molecular patterns (PMPs) by pattern recognition receptors (PRRs), and then activate the downstream signal pathway to induce the expression of inflammatory and I-type interferon. The Toll-like receptor 7 (TLR7) is located on the inner body of the cell, can identify the single-stranded RNA of the virus and the medetomas-like component, and induce the natural immune response, and has an important role in the anti-viral infection. Compared with the mammalian TLR7, the sequence characteristics and the biological functions of the poultry TLR7 are less, and the present invention is mainly concentrated on the surface and surface poultry such as the chicken, the duck and the goose, and the research of the flying birds such as the pigeon TLR7 has not been reported to date. The purpose of this study is to clone and analyze the gene sequence of the pigeon TLR7, to explore its key ligand recognition site and to explore its function in the natural immune response, so as to further study the structural characteristics and biological functions of the pigeon TLR7 and the theoretical foundation for the natural immunity accumulation of the poultry. The cloning, sequence analysis and expression profile of the TLR7 gene of the pigeon were studied. The specific primers were designed according to the conserved sequence of the avian TLR7 gene, and the TLR7 gene was cloned for the first time. Using bioinformatics software to analyze its sequence characteristics, the open reading frame of the pigeon TLR7 gene is 3144 bp, encoding 1047 amino acids, and the structural prediction shows that it is a typical TLR, consisting of one signal peptide sequence at the N end,15 LRR repeating sequences, one LRR-CT and an intracellular TIR region. The predicted results of the spatial structure of the protein interaction indicate that the membrane inner domain of the two TLR7 forms a typical "m"-shaped dimer. The predicted glycosylation and phosphorylation site predicted that the pigeon TLR7 contained 18 potential N-glycosylation sites and 38 phosphorylation sites. The homology of the amino acid sequence of pigeon TLR7 to human, mouse, chicken and duck TLR7 was 65.0%, 62.2%, 81.5% and 83.8%, respectively. The expression of TLR7 in different tissues of the pigeon was analyzed by RT-PCR. The results showed that the high level of expression of the TLR7 in the immune-related tissues, especially in the spleen and the liver. The eukaryotic expression plasmid pCMV-PiTLR7 of the pigeon TLR7 was constructed and the expression of TLR7 was identified by Western blotting. After the R848 ligand was stimulated with the NF-VIB luciferase reporter vector, the activation of the NF-VIB was significantly mediated by the pigeon TLR7, indicating that the pigeon TLR7 was a functional TLR. After the analysis of the LRR sequence of the pigeon TLR7, the insertion and modification of the amino acids were found in the 15th position of LRR2, LRR11, LRR13 and LRR14, and the 10th position of the LRR10, so that the TLR7 mutant with the deletion of these sites was constructed by the method of overcap-PCR, and ligated to the eukaryotic expression vector of pCMV. Using the NF-VIB luciferase reporter system, the results show that the deletion of these insertion sequences completely abolishes the activation of the TLR7-mediated NF-VIB, indicating that the non-classical LRR sequence is necessary for TLR7 to function as a key site for the TLR7 identification ligand. In addition, the expression of TLR7 and cytokines was detected by the qRT-PCR and the ELISA method, and the results showed that both the TLR7 mutants could not mediate the production of IL-8, which was consistent with the results of the test of the activity of NF-EMAB; however, some of the mutants (DellOIN10 and De114IN15) could mediate the production of IFN-1 and TNF-1, while at the same time, After the stimulation of R848, the expression level of the mutant of the pigeon TLR7 was up-regulated, indicating that the mutant of the pigeon TLR7 could compensate for its function by increasing its expression. however, that mutant of the pigeon TLR7 De111IN15, The results showed that the deletion of the key LRR insertion sequence could lead to the occurrence of false decompensation.3. The study of the natural immune response of the pigeon TLR7-mediated agonist and the new city's disease was stimulated by R848 to stimulate the peripheral blood mononuclear cells of the pigeon, and the mRNA levels of TLR7, IFN-1, IL-6, IL-8, CCL5 and IL-10 were detected by the method of fluorescence quantitative PCR. The results showed that the pro-inflammatory cytokines and related anti-viral molecules were significantly up-regulated at 12 h and 24 h after the stimulation of R848, while the level of TLR7 expression did not change significantly. The expression of TLR7 and related cytokines in the spleen was detected by qRT-PCR. The results showed that the mRNA level of TLR7 in the group of R848 was significantly increased, and the expression of TLR7 in the LaSota group was significantly lower in the third day. The pro-inflammatory cytokines and chemokines are up-regulated in both intramuscular injection groups, indicating that the immune response mediated by the pigeon TLR7 is not linked to its own level of expression. The pigeon TLR7 can mediate the activation of NF-B. The production of pro-inflammatory cytokines and related anti-viral molecules is remarkably enhanced, and plays an important role in the natural immune anti-viral infection.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S836
本文編號：2512144
文內(nèi)圖片：
圖片說明：圖１－２重組質(zhì)粒ｐＣＭＶ－ＰｒｒＬＲ７酶切筵定逡逑Ｆｉｇ．邋ＭＰＣＲ邋ａｍｐｌｉｆｉｃａｔｉｏｎ邋ｏｆｐｉｇｅｏｎＴＬＲ７邋ｇｅｎｅ邋雌邋１－２邋Ｅｎｚｙｍｅ邋ｄｉｇｅｓｔｉｏｎ邋ｏｆ邋ｐｌａｓｍｉｄ逡逑
[Abstract]:The natural immune system is the first line of defense for the body to defend the invasion of the pathogen. After the infection of the pathogenic microorganisms, the body cells recognize the pathogen-associated molecular patterns (PMPs) by pattern recognition receptors (PRRs), and then activate the downstream signal pathway to induce the expression of inflammatory and I-type interferon. The Toll-like receptor 7 (TLR7) is located on the inner body of the cell, can identify the single-stranded RNA of the virus and the medetomas-like component, and induce the natural immune response, and has an important role in the anti-viral infection. Compared with the mammalian TLR7, the sequence characteristics and the biological functions of the poultry TLR7 are less, and the present invention is mainly concentrated on the surface and surface poultry such as the chicken, the duck and the goose, and the research of the flying birds such as the pigeon TLR7 has not been reported to date. The purpose of this study is to clone and analyze the gene sequence of the pigeon TLR7, to explore its key ligand recognition site and to explore its function in the natural immune response, so as to further study the structural characteristics and biological functions of the pigeon TLR7 and the theoretical foundation for the natural immunity accumulation of the poultry. The cloning, sequence analysis and expression profile of the TLR7 gene of the pigeon were studied. The specific primers were designed according to the conserved sequence of the avian TLR7 gene, and the TLR7 gene was cloned for the first time. Using bioinformatics software to analyze its sequence characteristics, the open reading frame of the pigeon TLR7 gene is 3144 bp, encoding 1047 amino acids, and the structural prediction shows that it is a typical TLR, consisting of one signal peptide sequence at the N end,15 LRR repeating sequences, one LRR-CT and an intracellular TIR region. The predicted results of the spatial structure of the protein interaction indicate that the membrane inner domain of the two TLR7 forms a typical "m"-shaped dimer. The predicted glycosylation and phosphorylation site predicted that the pigeon TLR7 contained 18 potential N-glycosylation sites and 38 phosphorylation sites. The homology of the amino acid sequence of pigeon TLR7 to human, mouse, chicken and duck TLR7 was 65.0%, 62.2%, 81.5% and 83.8%, respectively. The expression of TLR7 in different tissues of the pigeon was analyzed by RT-PCR. The results showed that the high level of expression of the TLR7 in the immune-related tissues, especially in the spleen and the liver. The eukaryotic expression plasmid pCMV-PiTLR7 of the pigeon TLR7 was constructed and the expression of TLR7 was identified by Western blotting. After the R848 ligand was stimulated with the NF-VIB luciferase reporter vector, the activation of the NF-VIB was significantly mediated by the pigeon TLR7, indicating that the pigeon TLR7 was a functional TLR. After the analysis of the LRR sequence of the pigeon TLR7, the insertion and modification of the amino acids were found in the 15th position of LRR2, LRR11, LRR13 and LRR14, and the 10th position of the LRR10, so that the TLR7 mutant with the deletion of these sites was constructed by the method of overcap-PCR, and ligated to the eukaryotic expression vector of pCMV. Using the NF-VIB luciferase reporter system, the results show that the deletion of these insertion sequences completely abolishes the activation of the TLR7-mediated NF-VIB, indicating that the non-classical LRR sequence is necessary for TLR7 to function as a key site for the TLR7 identification ligand. In addition, the expression of TLR7 and cytokines was detected by the qRT-PCR and the ELISA method, and the results showed that both the TLR7 mutants could not mediate the production of IL-8, which was consistent with the results of the test of the activity of NF-EMAB; however, some of the mutants (DellOIN10 and De114IN15) could mediate the production of IFN-1 and TNF-1, while at the same time, After the stimulation of R848, the expression level of the mutant of the pigeon TLR7 was up-regulated, indicating that the mutant of the pigeon TLR7 could compensate for its function by increasing its expression. however, that mutant of the pigeon TLR7 De111IN15, The results showed that the deletion of the key LRR insertion sequence could lead to the occurrence of false decompensation.3. The study of the natural immune response of the pigeon TLR7-mediated agonist and the new city's disease was stimulated by R848 to stimulate the peripheral blood mononuclear cells of the pigeon, and the mRNA levels of TLR7, IFN-1, IL-6, IL-8, CCL5 and IL-10 were detected by the method of fluorescence quantitative PCR. The results showed that the pro-inflammatory cytokines and related anti-viral molecules were significantly up-regulated at 12 h and 24 h after the stimulation of R848, while the level of TLR7 expression did not change significantly. The expression of TLR7 and related cytokines in the spleen was detected by qRT-PCR. The results showed that the mRNA level of TLR7 in the group of R848 was significantly increased, and the expression of TLR7 in the LaSota group was significantly lower in the third day. The pro-inflammatory cytokines and chemokines are up-regulated in both intramuscular injection groups, indicating that the immune response mediated by the pigeon TLR7 is not linked to its own level of expression. The pigeon TLR7 can mediate the activation of NF-B. The production of pro-inflammatory cytokines and related anti-viral molecules is remarkably enhanced, and plays an important role in the natural immune anti-viral infection.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S836
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