臺灣博碩士論文加值系統

點帶石斑魚 (Epinephelus coioides) 為臺灣養殖漁業當中相當重要的養殖魚種之一，但其養殖過程中，幼苗時期常因疾病，如病毒性神經壞死症 (viral nervous necrosis, VNN) 感染的關係，造成高達百分之百的死亡率。而生物體用來對抗疾病發生主要為靠自身的免疫作用來進行。硬骨魚類是目前被發現為最古老具有適應性免疫反應的物種，意即魚類也如同高等脊椎動物一般，可針對不同的病原發展出專一性的免疫物質，也如同脊椎動物一般可以應用疫苗或免疫促進劑來進行疾病的防治。但在胚胎或幼兒時期，由於免疫系統仍屬於發育階段，若太早接受外來抗原，則將容易形成免疫耐受性 (immune tolerance) 的產生而造成無法免疫的現象。故為瞭解魚苗階段何時可以接受外來抗原以引起免疫反應，為在魚苗階段使用疫苗或免疫促進劑等產品重要的資訊。免疫相關細胞在進行特異性分化時，基因重組為一種重要的現象，重組的目的在於造成基因的多樣性，以因應不同的抗原。在執行這個重組現象時有兩個重組活化蛋白 (recombination activating gene 1 and 2 proteins, RAG1 及 RAG2 ) 扮演重要的角色。本研究的目的即為選殖點帶石斑魚重組活化蛋白基因 rag1 及 rag2 ，並探討此這些基因在點帶石斑魚魚苗階段與免疫球蛋白基因之表現情形，以了解特異性免疫反應在點帶石斑魚魚苗階段可能發育的情形。在這兩個基因的full-length cDNA的研究中，利用 RT-PCR 及 RACE 的方式，成功從胸腺中選殖出點帶石斑魚 rag1 及 rag2 全長，以及從頭腎中選殖出 IgM 之部分序列，所穫得之 rag1 cDNA全長為 3653 bp，內含轉譯讀窗 (open reading frame, ORF) 為 3216 bp，可轉譯為1071個胺基酸。rag2 cDNA全長為 1875 bp，ORF 為 1602 bp，可轉譯為533個胺基酸。偵測魚苗發育時期，在 rag1 的部分，在第二週時表現量開始上升，在第三週時表現量達到最大之後，在第四週時表現量較第三週為低；在 rag2 的部分，則第二週時表現量開始上升，在第三週時表現量達到最大，在第四週結束後表現量降低；在 IgM 的部分，到第22天時才有穩定且大量的表現。在偵測不同組織部分，在 rag1 的部分可以偵測到其在胸腺、頭腎及體腎有表現；在 rag2 的部分可以偵測到其在胸腺及頭腎有表現；在 IgM 的部分可以偵測到其在胸腺、頭腎、體腎、脾臟、腸及胰臟有表現，但在腦及肝臟則不表現。因此，本篇論文除了證實胸腺及頭腎在點帶石斑魚為重要的免疫器官之外，更提供了給予疫苗或是免疫促進劑在時間上的參考點。

Orange-spotted grouper (Epinephelus coioides) is a fish species with a high economic importance in the aquaculture industry in Taiwan. The high mortalities observed throughout early development such as viral nervous necrosis (VNN) causes the highest mortalities up to 100% always occur among 1-month-old larvae with total body lengths of 2.0 cm. Teleost is the oldest species has the adaptive immune system. However, there is a risk of inducing immunological tolerance if fish that are immunised at a very early age before they are immunocompetent. Thus, it is important to establish the earliest time that grouper can be vaccinated or give immunopotentiating agent. Recombination activating genes, rag1 and rag2, encode components of the recombinase involved in V(D)J recombination. During B lymphocyte development, the variable region of Ig gene is assembled by the recombination of multiple V, D, J segments, which can generate a vast array of immunoglobulin M (IgM) to against numerous antigen. IgM produced by B lymphocytes is also an important gene that can be used in the study of the ontogenesis of the immune system, as it is the first formed antibody of the primary humoral component of the acquired immune system in fish species. These genes are expressed together in this study in order to understand the expression profile of them. The genes, rag1 and rag2, of E. coioides were cloned and sequenced the open reading frames. The full-length cDNA of rag1 and rag2 were 3653 and 1875 base pairs (bp) long respectively. The lengths of rag1 and rag2 open reading frame were 3216 and 1602 bp encoding 1071 and 533 amino acids with the molecular weight of putative protein were about 117 and 58 kDa. Subsequently, the rag1, rag2 and IgM mRNA expression level in ontogeny of fish and different organs was evaluated by reverse transcriptase PCR (RT-PCR). The result showed the expression level of rag1, rag2 and IgM mRNA raised after 13, 13 and 22 dpf of fries respectively. This data was suggested that orange-spotted grouper at this stage might possess mature immunity and is able to produce immunoglobulin. The expression of rag1 was observed in thymus, head kidney and trunk kidney. The expression of rag2 was observed in thymus and head kidney. The expression of IgM was observed in thymus, head kidney, trunk kidney, spleen, intestines and pancreas. This data forms the basis for a proposal that the thymus and head kidney of teleost species play an essential developmental role in lymphopoiesis and thus can be regarded as a primary lymphoid organ.

摘要 III
Abstract V
目錄 VIII
表目錄 XI
圖目錄 XII
縮寫表 XIV
第一部分、研究背景 1
1.1 水產養殖的重要性 1
1.2 水產養殖在臺灣 1
1.3 台灣的石斑魚養殖--點帶石斑魚 (Epinephelus coioides) 2
1.4 魚類的免疫系統 4
1.4.1 免疫系統 4
1.4.2 魚類的免疫系統 5
1.5 免疫球蛋白 (immunoglobulin, Ig) 和重組活化蛋白基因 6
1.6 魚苗發育 10
研究目標 11
第二部分、材料方法 12
2.1 生物材料 12
2.1.1 實驗動物部分 12
2.1.2 菌種部分及質體部分 12
2.2 退化性及專一性引子部分 12
2.3 反應試劑組 13
2.4 其他藥品與試劑 13
2.5 使用物品 14
2.6 製備瓊脂膠體 14
2.7 樣本採集 14
2.8 抽取組織total RNA 15
2.9 選殖點帶石斑魚的 rag1 及 rag2 基因片段 15
2.9.1 引子的設計 15
2.9.2 藉由電泳膠分析聚合��鏈鎖反應之產物 16
2.9.3 利用膠體抽出 (Gel-extraction) 的方式純化PCR產物 16
2.9.4 在pGEM��- TEasy質體上藉由黏合反應 (ligation) 建構含有 rag1 及 rag2 基因的質體 17
2.9.5 培養、保存大腸桿菌 (E. coli) 及製備大腸桿菌之勝任細胞 (competent cell) 17
2.9.6 轉型已建構 rag1 及 rag2 基因的質體到大腸桿菌之勝任細胞 18
2.9.7 定序 18
2.10 藉由 RACE (rapid amplification of cDNA ends) 取得 rag1 及 rag2 mRNA全部序列 19
2.10.1 試驗材料的準備及 total RNA 的萃取 19
2.10.2 特異性引子的聚合�○s鎖反應 20
2.10.3 建構含 rag1 及 rag2 mRNA 全長之質體 20
2.11 分析在石斑魚魚苗不同發育階段及不同臟器 rag1 , rag2 及 IgM 基因的表現 21
2.11.1 點帶石斑魚(E. coioides) 魚苗收集 21
2.11.2 mRNA 從點帶石斑魚(E. coioides)不同時期魚苗及不同臟器抽出 22
2.11.3 以RT-PCR偵測 rag1 、 rag2 及 IgM 在不同魚苗時期及不同臟器各基因的表現 22
第三部分、研究結果 23
3.1 有關點帶石斑魚 rag1 及 rag2 基因選殖的結果 23
3.2 點帶石斑魚魚苗時期不同時間點基因表現部分 25
3.3 點帶石斑魚不同組織中基因表現部分 25
第四部分、討論 27
4.1 比對找到之基因部分 27
4.2 基因表現的部分 31
參考文獻 33
圖十一、以點帶石斑魚與不同物種之 RAG1 (胺基酸序列) 所繪出之演化樹圖。 54
圖十二、以點帶石斑魚與不同物種之 RAG2 (胺基酸序列) 所繪出之演化樹圖。 55
附錄 67
附錄一、常用溶液配方。 67
附錄二、設計的IgM引子所夾出之胺基酸相對位置。 69
附錄三、 V(D)J 重組基本機制。 70
附錄四、 RAG1 及 RAG2 蛋白質結構基本圖示。 71
附錄五、 B細胞與T細胞發育過程。 72
附錄六、 pGEM��-T Easy Vector 圖譜。 73

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