【摘要】：Toll樣受體(Toll like receptors TLRs)是哺乳動物具備的天然免疫分子,屬于模式識別受體(pattern recognition receptor PRR),在識別和抵御各種病原微生物感染中發(fā)揮著著重要作用。1997年以來,在哺乳動物體內相繼發(fā)現(xiàn)了13種Toll樣受體(TLR1-TLR13),人體內目前發(fā)現(xiàn)有10種Toll樣受體(hTLR1-hTLR10),皆屬于工型跨膜受體。TLR-9是典型的Toll家族成員,細菌DNA中含有的CpG序列是TLR-9目前唯一明確識別的病原相關分子模式(pathogen associated molecular patterns PAMPs)。CpG序列即以非甲基化胞嘧啶鳥嘌呤二核苷酸(CpG)為核心的回文序列,該序列在脊椎動物基因組的出現(xiàn)頻率大大低于在原核生物、病毒基因組的出現(xiàn)頻率,且CG多發(fā)生甲基化。脊椎動物免疫系統(tǒng)正是通過識別CpG特征結構來識別微生物DNA并產生針對抗原的保護性免疫反應。目前關于CpG序列如何在抗過敏反應,抗腫瘤反應中,抗自身免疫反應中發(fā)揮作用已成為基礎及臨床醫(yī)學研究中的熱點。作為CpG序列在體內的唯一受體,自發(fā)現(xiàn)以來也受到了研究者的關注。 盡管許多實驗已證實TLR-9可以介導CpG激活多種免疫細胞,但是關于TLR-9蛋白的特征,如何與CpG結合并介導后續(xù)免疫反應目前仍有許多未知需要進一步研究。本研究擬對人TLR-9(hTLR-9)進行克隆、表達并制備其相關的多克隆抗體。同時構建轉染有hTLR-9基因的HEK293細胞模型,這也達到了對hTLR-9進行真核表達的目的。在此基礎上,擬利用轉染的細胞作為免疫原,以原核表達的hTLR-9 N-末端作為篩選抗原,制備相應的抗hTLR-9 N—末端的單克隆抗體。本實驗結果獲得了抗hTLR-2單、多克隆抗體,構建成功穩(wěn)定轉染有hTLR-9基因的HEK293細胞模型,為進一步研究hTLR-9-CpG系統(tǒng)提供了重要的制劑。
[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.
【學位授予單位】：第一軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R392

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相關期刊論文 前1條

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本文編號：2203469