【摘要】：Toll樣受體(Toll like receptors TLRs)是哺乳動(dòng)物具備的天然免疫分子,屬于模式識(shí)別受體(pattern recognition receptor PRR),在識(shí)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(lái),在哺乳動(dòng)物體內(nèi)相繼發(fā)現(xiàn)了13種Toll樣受體(TLR1-TLR13),人體內(nèi)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(xì)菌DNA中含有的CpG序列是TLR-9目前唯一明確識(shí)別的病原相關(guān)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(dòng)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(dòng)物免疫系統(tǒng)正是通過識(shí)別CpG特征結(jié)構(gòu)來(lái)識(shí)別微生物DNA并產(chǎn)生針對(duì)抗原的保護(hù)性免疫反應(yīng)。目前關(guān)于CpG序列如何在抗過敏反應(yīng),抗腫瘤反應(yīng)中,抗自身免疫反應(yīng)中發(fā)揮作用已成為基礎(chǔ)及臨床醫(yī)學(xué)研究中的熱點(diǎn)。作為CpG序列在體內(nèi)的唯一受體,自發(fā)現(xiàn)以來(lái)也受到了研究者的關(guān)注。 盡管許多實(shí)驗(yàn)已證實(shí)TLR-9可以介導(dǎo)CpG激活多種免疫細(xì)胞,但是關(guān)于TLR-9蛋白的特征,如何與CpG結(jié)合并介導(dǎo)后續(xù)免疫反應(yīng)目前仍有許多未知需要進(jìn)一步研究。本研究擬對(duì)人TLR-9(hTLR-9)進(jìn)行克隆、表達(dá)并制備其相關(guān)的多克隆抗體。同時(shí)構(gòu)建轉(zhuǎn)染有hTLR-9基因的HEK293細(xì)胞模型,這也達(dá)到了對(duì)hTLR-9進(jìn)行真核表達(dá)的目的。在此基礎(chǔ)上,擬利用轉(zhuǎn)染的細(xì)胞作為免疫原,以原核表達(dá)的hTLR-9 N-末端作為篩選抗原,制備相應(yīng)的抗hTLR-9 N—末端的單克隆抗體。本實(shí)驗(yàn)結(jié)果獲得了抗hTLR-2單、多克隆抗體,構(gòu)建成功穩(wěn)定轉(zhuǎn)染有hTLR-9基因的HEK293細(xì)胞模型,為進(jìn)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(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

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相關(guān)期刊論文 前1條

1 劉艷君;廣譜模式識(shí)別分子Toll-like receptor 2的研究進(jìn)展[J];免疫學(xué)雜志;2002年03期

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本文編號(hào)：2203469