鸽Toll样受体7基因的克隆及其免疫生物学功能研究
发布时间：2019-07-09 12:20
【摘要】：天然免疫系统是机体防御病原体入侵的首道防线。病原微生物感染后,机体细胞通过模式识别受体(Pattern-recognition receptors, PRRs)识别病原相关分子模式(Pathogen-associated molecular patterns, PAMPs),然后激活下游信号通路,诱导炎性因子和I型干扰素的表达。Toll样受体7(Toll-like receptor 7, TLR7)位于细胞的内体上,能识别病毒的单链RNA及咪唑喹啉类成分,并诱导天然免疫应答,在抗病毒感染中具有重要作用。相较于哺乳动物TLR7,禽类TLR7的序列特征和生物功能还研究较少,目前主要集中在鸡、鸭和鹅等水陆禽类中,而飞禽如鸽TLR7的研究至今还未见报道。本研究旨在克隆和分析鸽TLR7的基因序列,挖掘其关键的配体识别位点,并探索其在天然免疫应答中的功能,从而为进一步研究鸽TLR7的结构特征和生物功能以及禽类的天然免疫积累理论依据。1.鸽TLR7基因的克隆、序列分析及其表达谱检测本研究根据禽类TLR7基因的保守序列设计出特异性引物,首次从大王鸽克隆TLR7基因。利用生物信息学软件分析其序列特征,鸽TLR7基因的开放阅读框为3144 bp,编码1047个氨基酸,结构预测表明其是一类典型的TLR,由N端的一个信号肽序列、15个LRR重复序列、一个LRR-CT和胞内TIR区组成。蛋白相互作用空间结构预测结果表明两个TLR7的膜内结构域形成典型的“m”形二聚体。糖基化和磷酸化位点预测表明鸽TLR7含有18个潜在的N-糖基化位点和38个磷酸化位点。鸽TLR7氨基酸序列与人、鼠、鸡、鸭TLR7的同源性分别为65.0%、62.2%、81.5%和83.8%。通过RT-PCR分析鸽不同组织中TLR7基因的表达差异,结果表明鸽TLR7在免疫相关组织尤其是脾脏和肝脏中具有较高的表达水平。2.鸽TLR7的免疫生物学功能及其配体识别位点的挖掘构建含有鸽TLR7的真核表达质粒pCMV-PiTLR7,转染HEK293T细胞后,Western blotting鉴定了TLR7的表达。利用NF-κB荧光素酶报告载体,在R848配体刺激后,鸽TLR7能够显著地介导NF-κB的活化,表明鸽TLR7是一类有功能的TLR。对鸽TLR7 LRR序列进行分析后,发现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饰,于是我们通过overlap-PCR的方法分别构建了这些位点缺失的TLR7突变体,并连接到pCMV真核表达载体上。利用NF-κB荧光素酶报告系统,结果显示这些插入序列的缺失完全废除了TLR7介导的NF-κB的活化,表明非经典LRR序列是TLR7发挥功能所必需的,这些序列可能是TLR7识别配体的关键位点。此外,通过qRT-PCR和ELISA方法检测了TLR7和细胞因子的表达,结果表明TLR7突变体均不能介导IL-8的产生,这与NF-κB活性检测实验结果是一致的；然而部分突变体(DellOIN10和De114IN15)可介导IFN-α和TNF-α的产生,同时,R848刺激后,鸽TLR7突变体的表达水平均有不同程度的上调,表明鸽TLR7突变体通过增加其表达量进而代偿其功能的缺陷。然而,鸽TLR7 De111IN15突变体尽管增加了其自身的表达量,仍不能有效地诱导IFN-α、IL-8和‘TNF-α的产生,表明关键LRR插入序列的缺失会导致假代偿的出现。3.鸽TLR7介导激动剂及新城疫病感染的天然免疫应答研究通过R848刺激鸽外周血单核细胞,应用荧光定量PCR的方法检测TLR7、IFN-γ、IL-6、 IL-8、CCL5和IL-10的mRNA水平,结果表明R848刺激12 h和24 h后前炎性细胞因子和相关抗病毒分子均显著上调,而TLR7的表达水平没有显著变化。分别使用新城疫LaSota疫苗株和R848激动剂肌肉注射大王鸽,通过qRT-PCR的方法检测了脾脏中TLR7及相关细胞因子的表达,结果表明R848肌肉注射组中TLR7的mRNA水平显著升高,LaSota感染组TLR7的表达在第三天时显著降低,而前炎性细胞因子和趋化因子在两个肌肉注射组中均显著上调,表明鸽TLR7介导的免疫应答与其自身的表达水平并没有必然联系。鸽TLR7能介导NF-κB的活化,显著增强前炎性细胞因子和相关抗病毒分子的产生,在天然免疫抗病毒感染中扮演了重要的作用。
文内图片：
图片说明：图１－２重组质粒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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S836
本文编号：2512144
文内图片：
图片说明：图１－２重组质粒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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S836
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