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。;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.
