類鐸受體 (toll-like receptor, TLR) 是一種細胞表面的接受器,可辨識外來抗原,產生先天免疫反應及誘導後天免疫反應。本實驗利用國立海洋生物博物館內圈飼的阿德利、極地、馬可羅尼及巴布亞,共四種企鵝血液樣本,使用雞隻各種特異性TLR 引子進行聚合酶連鎖反應,以檢測企鵝TLRs。確認產物位置後,純化DNA 進行轉型及定序,並利用生物資訊軟體分析其親源關係。本實驗於阿德利、極地、馬可羅尼及巴布亞企鵝身上增幅出TLR1、TLR5、TLR7 和TLR15,而巴布亞企鵝及極地企鵝也檢測出TLR4。遺傳距離矩陣結果顯示,企鵝間各TLRs 核酸相似度皆高於95%,而企鵝與雞的TLR1、TLR7、TLR15 相似度為84.4 ~ 93.5%,但TLR4、TLR5 與雞的相似度卻不到50%(39.7 ~ 43.7%)。四種企鵝皆無法順利以雞隻引子增幅出TLR21 核酸序列。由此得知,企鵝與雞TLRs 雖具有一定程度的相似度,但可能在漫長的演化的過程中某些TLRs 已出現較大的歧異度,另外也可能是序列相似度較低或非變異區不同導致企鵝與雞TLRs 差異。本實驗結果可在企鵝基礎免疫學、疾病預防及疫苗應用上提供一些參考。
Toll-like receptors (TLRs), a kind of membrane-spanning protein, can recognize antigens, generate innate immunity and induce adaptive immunity. The aims of this study are to determine the types of TLR inpenguins and to analyze the TLR phylogeny of chickens and penguins. In this study, blood samples were collected from Adélie, Chinstrap, Gentoo and Macaroni Penguins reared at the National Museum of Marine Biology and Aquarium. PCR reactions performed with specific primers for TLRs of chickens were employed to determine TLR types of penguins. After DNA purification, transformation and sequencing, the phylogeny was analyzed using bioinformatics software. TLR1, TLR5, TLR7 and TLR15 were amplified from four species of penguins, and TLR4 was detected only in Gentoo and Chinstrap penguins. The similarity in rates of nucleic acid of TLRs among penguins exceeded 95%. Between chickens and penguins, the similarity in rates of nucleic acid of TLR1, TLR7 and TLR15 were in the range of 84.4- 93.5%, and those of TLR4 and TLR5 were in the range of 39.7-43.7%. TLR21 was not detected in the four species of penguins studied. Although TLRs of penguins and chickens have a certain degree of similarity, some TLRs may show a larger degree of divergence in the long process of evolution. Moreover, low sequence similarity and difference in non-variable regions could also result in variations between TLRs of penguins and chickens. The results of this study can provide referencesfor further studies inpenguin immunology, disease prevention and vaccine applications.
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