臺灣博碩士論文加值系統

細胞遭受病毒感染後誘導第一型干擾素的表現，而此分泌的干擾素透過其受體調控其他細胞的訊息傳遞，進一步誘導該細胞具有ISRE (Interferon-Stimulated Response Element) 序列啟動子的下游基因表現。被誘導表現的下游基因以Mx基因研究最清楚。Mx蛋白質具有GTPase功能區，能將GTP水解成GDP，藉此去磷酸化來抑制病毒組成在細胞內的輸送，和病毒的組合。本實驗以石斑魚Mx基因 (cDNA) 為模版，設計引子對，利用聚合酶鏈反應增幅Mx基因開放讀架DNA片段，並將此DNA片段經限制酶修剪後，接進pET20b (+) 表現載體，並轉形至大腸桿菌表現出含有679個胺基酸組成的Mx重組蛋白質。IPTG誘導表現的Mx重組蛋白質不溶於水，利用變性溶液溶解重組蛋白質後，以可以認識重組蛋白質C端6-His胺基酸的鎳親和性管柱進行層析純化。所得純化之Mx重組蛋白質在SDS-PAGE蛋白質電泳分析下為72 kDa和推測的分子量一致。此外將此純化的Mx重組蛋白質進行N端胺基酸序列分析，所得10個胺基酸序列也與預期的重組蛋白質序列一致，證實石斑魚Mx重組蛋白質表現成功。進一步將此純化的重組蛋白質混合佐劑後，接種於小鼠，製備多株抗體。此多株抗體可以西方墨點和免疫細胞化學染色法辨認三型石斑魚Mx蛋白質。

Type I interferons are expressed by virus-infected cells. The secreted interferons can regulate the signal transduction of other cells via specific receptors and induce the downstream genes, whose promoter has ISRE (Interferon-Stimulated Response Element) sequences. Among these interferon induced genes, Mx gene is the most studied one. The Mx protein has a GTPase domain, which hydrolyzes GTP into GDP. This dephosphorylation will inhibit the intracellular trafficking and package of viruses. In this study, we designed specific primers, and amplified the Mx open reading frame by PCR as using the grouper Mx gene (cDNA) as template. The amplifed DNA fragment was cleaved by restriction enzymes and inserted into pET20b (+) vector. The obtained Mx expression plasmid was transformed into E. coli to express recombinant Mx protein with 679 amino acid residues. The IPTG-induced insoluble recombinant protein was dissolved in a denatured solution and purified by a nickel-nitrilotriacetic acid (Ni-NTA) agarose column affinity chromatography via 6-histidines on its C-terminal. By SDS-PAGE analysis, the molecular mass of the purified recombinant protein was 72 kDa, consistent with our expectation. Furthermore, as subjected to the automated N-terminal sequencing, the sequence of ten amino acid residues of the purified recombinant Mx protein were identical to the expected sequence. The polyclonal anti-Mx antiserum was prepared from mice immunized with the adjuvant-emulsified recombinant Mx protein. By using the Western blotting hybridization and immunocytochemistry, the obtained antiserum was proved to recognize three types of grouper Mx proteins.

謝誌 ----------------------------------------------------------------------------------I
中文摘要 --------------------------------------------------------------------------Ⅱ
英文摘要 --------------------------------------------------------------------------Ⅲ
目錄 --------------------------------------------------------------------------------Ⅳ
一、前言 -----------------------------------------------------------------------------1
1. 石斑魚之簡介 -----------------------------------------------------------1
2. 魚類神經壞死病毒 -----------------------------------------------------2
3. Mx 之簡介 ---------------------------------------------------------------4
4. 大腸桿菌表現系統 ------------------------------------------------------7
5. 實驗目的 -----------------------------------------------------------------8
二、材料與方法 --------------------------------------------------------------------9
1. 載體質體製備 -----------------------------------------------------------9
2. Insert的製備 -----------------------------------------------------------10
3. 連接反應(Ligation) ----------------------------------------------------13
4. 勝任細胞(Competent cell)之製備 -----------------------------------13
5. 轉形作用 ----------------------------------------------------------------14
6. 小量質體DNA之製備(Small-scale preparation of plasmid DNA) ----------------------------------------------------------------------------15
7. 質體DNA之鑑定(Identification of plasmid DNA) ---------------16
8. SDS-PAGE 蛋白質電泳分析 ---------------------------------------16
9. 蛋白質之定量分析 ----------------------------------------------------18
10. 重組Mx蛋白質誘導表現 --------------------------------------------19
11. 重組Mx蛋白質的純化 ----------------------------------------------19
12. Mx多株抗體製備 -----------------------------------------------------21
13. 西方墨點法（Western Blotting）--------------------------------------21
14. Mx多株抗體效價測試 -----------------------------------------------23
15. GB細胞感染NNV病毒 ----------------------------------------------23
16. GB細胞poly I：C誘導 ----------------------------------------------24
17. GB細胞轉染 -----------------------------------------------------------24
18. 免疫細胞化學染色(immunocytochemistry) -----------------------25
三、結果 --------------------------------------------------------------------------27
1. Mx表現質體的構築與重組蛋白質的表現 -------------------------27
2.純化與分析 ---------------------------------------------------------------27
3. 多株抗體的製備 --------------------------------------------------------28
4. 石斑魚腦細胞株的轉染 -----------------------------------------------28
5. 石斑魚與石斑魚腦細胞株Mx蛋白質的測試 --------------------28
四、討論 ---------------------------------------------------------------------------29
五、參考文獻 ---------------------------------------------------------------------32
六、圖表 ---------------------------------------------------------------------------41
圖一.石斑魚10-3 Mx重組蛋白質表現載體之架構 -----------------41
圖二.石斑魚11-2 Mx重組蛋白質表現載體之架構 ------------------42
圖三.石斑魚12-2 Mx重組蛋白質表現載體之架構 -----------------43
圖四.IPTG誘導石斑魚10-3，11-2 Mx表現載體表現出truncated 蛋白質 --------------------------------------------------------------------------44
圖五.IPTG濃度對誘導表現石斑魚12-2 Mx重組蛋白質之影響 -45
圖六. 誘導時間對誘導表現石斑魚12-2 Mx重組蛋白質之影響 -46
圖七. 石斑魚12-2 Mx重組蛋白質之誘導表現與純化 -------------47
圖八.石斑魚12-2 Mx重組蛋白質的N端十個胺基酸序列 --------48
圖九. 石斑魚Mx多株抗體效價測試 ----------------------------------49
圖十.轉染三型石斑魚Mx基因的石斑魚腦細胞GB3 西方墨點分析 ----------------------------------------------------------------------------------50
圖十一. 轉染三型石斑魚Mx基因的石斑魚腦細胞GB3免疫細胞化學分析 --------------------------------------------------------------------51
圖十二.感染NNV石斑魚腦細胞GB3西方墨點分析 -------------53
圖十三.以poly I:C處理的石斑魚腦細胞GB3西方墨點分析 ---54
圖十四.石斑魚組織西方墨點分析 -------------------------------------55

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