人Toll样受体-9的表达及其抗体制备
发布时间：2018-08-25 16:30
【摘要】：Toll样受体(Toll like receptors TLRs)是哺乳动物具备的天然免疫分子,属于模式识别受体(pattern recognition receptor PRR),在识别和抵御各种病原微生物感染中发挥着着重要作用。1997年以来,在哺乳动物体内相继发现了13种Toll样受体(TLR1-TLR13),人体内目前发现有10种Toll样受体(hTLR1-hTLR10),皆属于工型跨膜受体。TLR-9是典型的Toll家族成员,细菌DNA中含有的CpG序列是TLR-9目前唯一明确识别的病原相关分子模式(pathogen associated molecular patterns PAMPs)。CpG序列即以非甲基化胞嘧啶鸟嘌呤二核苷酸(CpG)为核心的回文序列,该序列在脊椎动物基因组的出现频率大大低于在原核生物、病毒基因组的出现频率,且CG多发生甲基化。脊椎动物免疫系统正是通过识别CpG特征结构来识别微生物DNA并产生针对抗原的保护性免疫反应。目前关于CpG序列如何在抗过敏反应,抗肿瘤反应中,抗自身免疫反应中发挥作用已成为基础及临床医学研究中的热点。作为CpG序列在体内的唯一受体,自发现以来也受到了研究者的关注。 尽管许多实验已证实TLR-9可以介导CpG激活多种免疫细胞,但是关于TLR-9蛋白的特征,如何与CpG结合并介导后续免疫反应目前仍有许多未知需要进一步研究。本研究拟对人TLR-9(hTLR-9)进行克隆、表达并制备其相关的多克隆抗体。同时构建转染有hTLR-9基因的HEK293细胞模型,这也达到了对hTLR-9进行真核表达的目的。在此基础上,拟利用转染的细胞作为免疫原,以原核表达的hTLR-9 N-末端作为筛选抗原,制备相应的抗hTLR-9 N—末端的单克隆抗体。本实验结果获得了抗hTLR-2单、多克隆抗体,构建成功稳定转染有hTLR-9基因的HEK293细胞模型,为进一步研究hTLR-9-CpG系统提供了重要的制剂。
[Abstract]:Toll like receptor (Toll like receptors TLRs) is a natural immune molecule in mammals. (pattern recognition receptor PRR), a pattern recognition receptor, plays an important role in the identification and resistance of various pathogenic microbial infections. Thirteen Toll like receptors (TLR1-TLR13) have been found in mammalian bodies. Ten Toll like receptors (hTLR1-hTLR10) have been found in human body, all of which belong to transmembrane receptors. TLR-9 is a typical member of Toll family. The CpG sequence contained in bacterial DNA is the only known pathogen-related molecular pattern (pathogen associated molecular patterns PAMPs). CpG sequence of TLR-9, that is, the palindrome sequence with non-methylated cytosine guanine dinucleotide (CpG) as the core. The frequency of this sequence in vertebrate genome is much lower than that in prokaryote, and the frequency of CG methylation is much higher than that in prokaryote. The vertebrate immune system recognizes the characteristic structure of CpG to recognize microbial DNA and produce protective immune response against antigens. At present, how to play the role of CpG sequence in anti-allergic reaction, anti-tumor reaction and anti-autoimmune reaction has become the focus of basic and clinical medical research. As the only receptor of CpG sequence in vivo, researchers have also paid attention to it since its discovery. Although many experiments have confirmed that TLR-9 can mediate CpG to activate a variety of immune cells, there are still many unknown questions about the characteristics of TLR-9 protein, how to bind to CpG and mediate the subsequent immune response. The aim of this study was to clone human TLR-9 (hTLR-9), express and prepare polyclonal antibodies. At the same time, the model of HEK293 cells transfected with hTLR-9 gene was constructed, which achieved the purpose of eukaryotic expression of hTLR-9. On this basis, the transfected cells were used as immunogen and the hTLR-9 N-terminal expressed in prokaryotic cells as screening antigen to prepare the corresponding monoclonal antibodies against the N-terminal of hTLR-9. The results showed that the single and polyclonal antibodies against hTLR-2 were obtained, and a stable HEK293 cell model with hTLR-9 gene was successfully constructed, which provided an important preparation for the further study of hTLR-9-CpG system.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
本文编号：2203469
[Abstract]:Toll like receptor (Toll like receptors TLRs) is a natural immune molecule in mammals. (pattern recognition receptor PRR), a pattern recognition receptor, plays an important role in the identification and resistance of various pathogenic microbial infections. Thirteen Toll like receptors (TLR1-TLR13) have been found in mammalian bodies. Ten Toll like receptors (hTLR1-hTLR10) have been found in human body, all of which belong to transmembrane receptors. TLR-9 is a typical member of Toll family. The CpG sequence contained in bacterial DNA is the only known pathogen-related molecular pattern (pathogen associated molecular patterns PAMPs). CpG sequence of TLR-9, that is, the palindrome sequence with non-methylated cytosine guanine dinucleotide (CpG) as the core. The frequency of this sequence in vertebrate genome is much lower than that in prokaryote, and the frequency of CG methylation is much higher than that in prokaryote. The vertebrate immune system recognizes the characteristic structure of CpG to recognize microbial DNA and produce protective immune response against antigens. At present, how to play the role of CpG sequence in anti-allergic reaction, anti-tumor reaction and anti-autoimmune reaction has become the focus of basic and clinical medical research. As the only receptor of CpG sequence in vivo, researchers have also paid attention to it since its discovery. Although many experiments have confirmed that TLR-9 can mediate CpG to activate a variety of immune cells, there are still many unknown questions about the characteristics of TLR-9 protein, how to bind to CpG and mediate the subsequent immune response. The aim of this study was to clone human TLR-9 (hTLR-9), express and prepare polyclonal antibodies. At the same time, the model of HEK293 cells transfected with hTLR-9 gene was constructed, which achieved the purpose of eukaryotic expression of hTLR-9. On this basis, the transfected cells were used as immunogen and the hTLR-9 N-terminal expressed in prokaryotic cells as screening antigen to prepare the corresponding monoclonal antibodies against the N-terminal of hTLR-9. The results showed that the single and polyclonal antibodies against hTLR-2 were obtained, and a stable HEK293 cell model with hTLR-9 gene was successfully constructed, which provided an important preparation for the further study of hTLR-9-CpG system.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 刘艳君;广谱模式识别分子Toll-like receptor 2的研究进展[J];免疫学杂志;2002年03期
，本文编号：2203469
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2203469.html
最近更新
教材专著
