Optimized CpG-oligodeoxynucleotide sequence for activation of Toll-like receptor 9 from companion animals

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恩達瑪(Undarmaa Tsengel) 查詢紙本館藏

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論文名稱

(Optimized CpG-oligodeoxynucleotide sequence for activation of Toll-like receptor 9 from companion animals)

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★ 結合TLR9與STING促進劑協同激活免疫反應並增強頭頸癌的抗腫瘤作用

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至系統瀏覽論文 (2027-1-28以後開放)

摘要(中)

類鐸受體9（TLR9）是哺乳類細胞上偵測細菌和病毒DNA的受體。此外，TLR9可以被人工合成的CpG-oligodeoxynucleotides（CpG-ODNs）激活。合成的CpG-ODN是一種有效的免疫激活劑。它們可以促進和加速抗原依賴和非依賴的免疫反應。由於其強大的免疫刺激活性，CpG¬¬¬-ODNs正被研究用於各種治療用途上，這些包括癌症治療和用作疫苗佐劑。CpG-ODN的結構特徵，包括其CpG-六聚體序列的類型、間距、核苷酸序列和長度，均能影響其免疫刺激活性和物種特異性的活性。狗和貓是人類最常見的伴侶動物。家狗和家貓與人類之間的共處是對彼此均有益處。由於相互之間的有利關係，伴侶動物已經演變成人類生活中一個不可或缺的部分，而近幾十年來，寵物的健康和福祉也越來越受到人們的關注。在本研究中，我們比較了狗和貓這二個TLR9的蛋白質結構，克隆了它們的TLR9 DNAs，及研究它們被CpG-ODNs激活的作用。狗和貓的TLR9的cDNA序列分別由3096個核苷酸和3093個核苷酸所組成。它們的蛋白質各有1032及1031個胺基酸，相似度為90.9%。我們使用TLR9的細胞激活試驗來研究了這兩個TLR9被各種序列不同的CpG-ODNs激活的情況。結果顯示，這兩個物種的TLR9被帶有GTCGTT六聚體序列的CpG-ODN大幅度的激活。進一步的研究顯示，5′-端的 TCG 核苷酸對 CpG-ODN 的活性起關鍵作用。另外，CpG-ODN的長度也是它們對狗和貓TLR9活性的關鍵因素。這項研究發現了優化CpG-ODNs作為伴侶動物免疫刺激劑的核苷酸序列特徵。

摘要(英)

Toll-like receptor (TLR) 9 is a cellular receptor for detecting bacterial and viral DNA in mammalian species. In addition, this TLR is activated by synthetic CpG-oligodeoxynucleotides (CpG-ODNs), which are an effective activator for boosting and expediting antigen dependent and independent immune responses. Because of their potent immune stimulatory activities, the CpG-ODNs are continually developed for therapeutic purposes, including cancer therapy, and utilized as a vaccine adjuvant. The structural features of CpG-ODNs, including their CpG-hexamer motif characters, spacing between motifs, nucleotide sequence, and range of CpG-ODN, impact its immunostimulatory and species-specific activities. The most common human companions are dog and cat. Domestic dogs and cats continue to cohabit with humans, benefiting from one another. Companion animals have evolved into a permanent part of human life due to mutual interest, and pet health and wellbeing have received increasing attention in recent decades. This study characterized these dog and cat TLR9 proteins and cloned their cDNA for investigation of their interaction with CpG-ODNs. The dog and cat TLR9 cDNA consist of 3096 and 3093 nucleotides, which encode 1032 and 1031 amino acid residues with 90.9% of identity between these two TLR9 proteins. Activation of these two TLR9s by various CpG-ODNs was investigated using cell-based TLR9 activation assays. CpG-ODN with a GTCGTT motif immensely activated TLR9s from these two species. Further studies indicated that the 5’-end TCG nucleotides might play a vital role in the activities of the CpG-ODNs. The nucleotide length is also crucial in CpG-ODN to trigger dog and cat TLR9 activations. This study discovered nucleotide sequence features for optimizing CpG-ODNs as an immune stimulatory agent in cats and dogs.

關鍵字(中)

★ 類鐸受體
★ CpG寡脫氧核苷酸
★ 先天免疫
★ 疫苗佐劑
★ 免疫反應

關鍵字(英)

★ Toll-like receptor
★ CpG oligodeoxynucleotides
★ Innate immunity
★ Vaccine adjuvant
★ Immune response

論文目次

中文摘要 i
Abstract ii
Acknowledgment iii
Contents iv
Introduction 1
Materials and Methods 7
Materials 7
Cell culture 7
Dog and cat spleen first strand cDNA libraries 7
PCR amplification of dog and cat TLR9 cDNAs 8
Restriction enzyme digestion 9
Subcloning of dog and cat TLR9 cDNAs into the mammalian expression vector 9
cDNA sequence analysis 10
Nucleotide and protein sequence analysis 10
Evolution tree analysis 11
Extracellular domain analysis 11
TLR9 activation assay 11
Statistical analysis 11
Results 12
Characterization of dog and cat TLR9s 12
Phylogenetic analysis of TLR9 from mammalian species 13
Molecular cloning of dog and cat TLR9 cDNAs and construction of their expression vector 13
Activation of dog and cat TLR9s by CpG-ODNs with different type of CpG-hexamer-motif. 14
Concentration dependent activation of dog and cat TLR9s by CpG-ODNs 15
CpG-dideoxynucleotides in the CpG-2007 required for activation of dog and cat TLR9s. 16
Activation of dog and cat TLR9 by different concentrations of CpG-2007 and CpG-2007-GC1. 17
Activation of dog and cat TLR9s by CpG-ODNs with different nucleotide length derived from CpG-2722 17
Discussion 18
Conclusion 23
References 24
Figures 31
Figure 1. Protein sequence alignment of human, dog, and cat TLR9s. 32
Figure 2. Protein structures of TLR9 ectodomains 34
Figure 3. Phylogenetic and protein identity analysis of mammalian TLR9s 35
Figure 4. Molecular cloning of dog TLR9 cDNA and construction of its expression vector 36
Figure 5. Molecular cloning of cat TLR9 cDNA and construction of its expression vector 37
Figure 6. Mammalian expression vector of dog TLR9 and cat TLR9 38
Figure 7. Schematic of TLR9 mediated CpG-ODN activation and luciferase assay procedure 39
Figure 8. Activation of human, dog, and cat TLR9s with CpG-ODNs containing distinct CpG-hexamer motif 41
Figure 9. Activation of dog and cat TLR9s by CpG-ODNs with different concentrations 43
Figure 10. CpG-dideoxynucleotides in the CpG-2007 required for activation of dog and cat TLR9s. 45
Figure 11. Concentration-dependent activation of dog and cat TLR9s by CpG-2007 and CpG-2007GC1. 47
Figure 12. Activation of dog and cat TLR9s by CpG-ODNs with different length derived from CpG-2007. 49
Tables 51
Table 1. Identification of TLR9 proteins from different species. 52
Supplementary figures 53
Supplementary figure 1. Nucleotide sequence and amino acid sequence of dog TLR9 54
Supplementary figure 2. Nucleotide sequence and amino acid sequence of cat TLR9 56
Supplementary figure 3. Protein sequence alignment of dog and cat TLR9s. 58

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指導教授

莊宗顯王健家(Tsung-Hsien Chuang Chien-Chia Wang)

審核日期

2022-1-21

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