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- Identification and cDNA Cloning of α2-macroglobulin (α2-M) and Its Expression Following Vibrio alginolyticus Injection in Scalloped Spiny Lobster Panulirus homarus
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題名 | Identification and cDNA Cloning of α2-macroglobulin (α2-M) and Its Expression Following Vibrio alginolyticus Injection in Scalloped Spiny Lobster Panulirus homarus=波紋龍蝦α2巨球蛋白分子選殖及注射溶藻弧菌的表現 |
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作者 | 胡文聰; 林永慶; 黃千倫; 陳建初; | 書刊名 | 臺灣水產學會刊 |
卷期 | 41:4 2014.12[民103.12] |
頁次 | 頁299-314 |
分類號 | 439.66 |
關鍵詞 | 波紋龍蝦; α2巨球蛋白; 溶藻弧菌; 即時定量聚合酶鏈反應; Panulirus homarus; α2-macroglobulin; Vibrio alginolyticus; Quantitative real-time RT-PCR; |
語文 | 英文(English) |
中文摘要 | 本研究選殖波紋龍蝦Panulirus homarus血球中α2巨球蛋白的cDNA序列。α2巨球蛋白的全長序列共4,809個鹼基對,開放式讀框為4,380個鹼基對,5端及3端未轉譯區長度分別為72個及357個鹼基對。預測可轉譯為1,460個胺基酸,推測轉譯蛋白質分子量為161.2kDa,等電點為5.73。波紋龍蝦的α2巨球蛋白具有三個功能性區域,分別為1) 蛋白質誘引區,位於第678號胺基酸到第784號胺基酸。2) thioester domain,位於第979號胺基酸到第985號胺基酸,序列為GCGEQNM。3) receptor-binding domain,位於第1219號胺基酸到第1460號胺基酸。波紋龍蝦的α2巨球蛋白序列與淡水長臂大蝦相似度為63.4%,草蝦為62.0%,白蝦為62.0%,斑節蝦為62.1%,鋸緣青蟹為46.4%,與鱟的相似度為43.8%,與非洲鈍緣蜱相似度為44.1%。α2巨球蛋白經即時定量聚合酉每 鏈反應證明只存在於血球。波紋龍蝦在受到溶藻弧菌攻擊後,α2巨球蛋白在第6小時的表現量開始上升,在第24小時達到最高,顯示α2巨球蛋白會受到病原誘發而啟動。 |
英文摘要 | The full-length sequence of α2-macroglobulin (α2-M) was cloned from the haemocytes of scalloped spiny lobster Panulirus homarus by reverse transcription polymerase chain reaction (RT-PCR), cloning and sequencing of overlapping PCR and the rapid amplification of cDNA ends (RACE) method. The α2-M cDNA consists of 4,809 bp with an open reading frame (ORF) of 4,380 bp, a 72-bp 5'-untranslated region (UTR), and a 357-bp 3'-UTR. The predicted molecular mass of the translated protein (1,460 amino acids) is 161.2 kDa, with an estimated pI of 5.73. The P. homarus α2-M sequence contains putative functional domains including a bait region (678-784), a GCGEQNM (979-985) thioester domain, and a receptorbinding domain (1219-1460). Sequence comparison and phylogenetic analyses show that α2-M deduced amino acid sequence of P. homarus has similarity to that of freshwater prawn Macrobrachium rosenbergii (63.4%), tiger shrimp Penaeus monodon (62.0%), white shrimp Litopenaeus vannamei (62.0%), kuruma shrimp Marsupenaeus japonicus (62.1%), mud crab Scylla serrata (46.4%), and American horseshoe crab Limulus polyphemus (43.8%), and soft tick Ornithodoros moubata (44.1%), respectively. The α2-M transcript was mainly expressed in haemocytes. Quantitative real-time RT-PCR analysis showed that α2-M mRNA transcript in haemocytes of P. homarus increased significantly in 6, 12, and 24 h, then slightly declined, but still maintained significantly higher in 48 and 72 h post Vibrio alginolyticus injection indicating an activation of innate response. |
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