神經壞死病毒為一種魚類乙型野田病毒，可廣泛感染海水硬骨魚類，造成魚苗和未成熟魚隻嚴重的病毒性神經壞死症和病毒性腦及視網膜病變。本實驗利用病毒覆蓋蛋白結合分析法來探討神經壞死病毒的感染機制，結果鑑定出點帶石斑魚腦細胞株的細胞核蛋白，組蛋白H3.3，會與神經壞死病毒顆粒結合。組蛋白H3.3為組蛋白H3的變異體之ㄧ，此蛋白與組蛋白H2A、H2B以及H4組成一個八聚體的結構提供DNA纏繞形成核小體。點帶石斑魚的組蛋白H3.3含有136個胺基酸，其分子量為15,327 Da，與其他物種的組蛋白H3.3具有極高序列相似性。本實驗利用遠西墨點法確認點帶石斑魚組蛋白H3.3和神經壞死病毒外套蛋白會結合。此結合區域位於點帶石斑魚組蛋白H3.3的35-97胺基酸片段與神經壞死病毒外套蛋白的195-338 胺基酸片段。利用免疫細胞染色法在病毒感染或神經壞死病毒外套蛋白基因轉染的點帶石斑魚腦細胞中，可以觀察到組蛋白H3.3和神經壞死病毒外套蛋白共同座落在細胞中。神經壞死病毒外套蛋白的進入細胞核以及會和組蛋白H3.3結合的特性，暗示神經壞死病毒外套蛋白在病毒感染石斑魚腦細胞後可能扮演一個調控宿主基因表現的角色。

Nervous necrosis virus (NNV), a piscine betanodavirus, is the causing agent of viral nervous necrosis and viral encephalopathy and retinopathy in larvae and juveniles of a wide range of marine teleost species. The mechanism of NNV infection was investigated using a virus overlay protein binding assay (VOPBA), which resulted in the identification of a nuclear protein, histone H3.3, from grouper brain cells. The histone H3.3 is the variant of histone H3 which gets together with H2A, H2B and H4 to form a histone octamer for DNA wrapped binding in nucleosome. The orange-spotted grouper histone H3.3 gene encodes a protein of 136 amino acids with a molecular mass of 15,327 Daltons, and shows higher similarity to other species histone H3.3. The binding of orange-spotted grouper histone H3.3 and NNV coat protein was confirmed by Far-western blotting. The binding regions of orange-spotted grouper histone H3.3 and NNV coat protein were identified to 35 to 97 and 195 to 338 amino acids, respectively. The co-localization of histone H3.3 and NNV coat protein was observed on grouper brain cells through NNV infection and NNV coat protein gene transfection by immunocytostaining. The nuclear entry of NNV coat protein and binding between histone H3.3 and NNV coat protein suggest that NNV coat protein may serves as a modulator to control the expression of host genes post NNV infection in grouper brain cells.

中文摘要 Ⅰ
英文摘要 Ⅱ
目錄 Ⅳ
圖目次 Ⅵ
表目次 Ⅶ

第一章、前言
1.1神經壞死病毒 ( nervous necrosis virus, NNV） 1
1.2病毒覆蓋蛋白結合法(virus overlay protein binding assay , VOPBA) 2
1.3組蛋白 ( histone ) 3
1.4研究動機 7
第二章、材料與方法
2.1 GB3細胞之培養和NNV病毒之增殖與純化 8
2.2蛋白質定量法(Bradford protein assay) 9
2.3細胞膜結合蛋白之萃取 10
2.4 Virus overlay protein binding assay (VOPBA) 11
2.5 LC/MS/MS,MASCOT 16
2.6石斑魚腦細胞( GB3 )組蛋白H3.3基因之選殖 16
2.6.1石斑魚腦細胞RNA萃取 16
2.6.2 cDNA之合成 17
2.6.3引子設計與聚合酶鏈鎖反應( polymerase chain reaction, PCR ) 18
2.6.4限制酶剪切和連接反應( ligation ) 20
2.6.5 轉型作用( transformation ) 21
2.6.6 小量質體DNA之製備和核苷酸序列定序確定 22
2.7 重組蛋白之表現 23
2.7.1 重組蛋白之表現與純化 23
2.7.2 蛋白再摺疊(refolding) 25
2.8 遠西墨點法(Far-western blotting) 26
2.9免疫細胞染色法 27
2.9.1神經壞死病毒感染 27
2.9.2神經壞死病毒外套蛋白基因之轉染 27
2.9.3免疫染色 28
第三章、 結果
3.1病毒覆蓋蛋白結合分析 (virus overlay protein binding assay , VOPBA) 30
3.2點帶石斑魚組蛋白H3.3的特徵 30
3.3重組點帶石斑魚組蛋白H3.3之表現 31
3.4遠西墨點 31
3.5免疫細胞染色 32
第四章、討論 34
第五章、參考文獻 38








圖目次

Figure 1: Detection of the NNV binding proteins of GB3 cell by VOPBA 46

Figure 2: Nucleotide and deduced amino acid sequences and specific features of orange-spotted grouper histone H3.3 47

Figure 3: Comparison of amino acid sequence of orange-spotted grouper histone H3.3 with that of other species. 48

Figure 4: Schematic map of serial deletions of recombinant orange-spotted grouper histone H3.3 protein. 49

Figure 5: Expression of recombinant orange-spotted grouper histone H3.3 protein. 50

Figure 6: Purification of denatured recombinant orange spotted grouper histone H3.3 protein by Ni-NTA column 51

Figure 7: Renatured recombinant orange-spotted grouper histone H3.3 1-136 aa protein. 52

Figure 8: Far-western blot of NNV serial deletion recombinant coat proteins by histone H3.3 53

Figure 9: Far-western blot of histone H3.3 serial deletion recombinant proteins by NNV coat protein 54

Figure 10: Localization of histone H3.3 and NNV coat protein analyzed by immunofluorescence staining. 55

Figure 11: Localization of histone H3.3 and recombinant EGFPC1-NNVCP analyzed by immunofluorescence staining. 56




表目次

Table 1: MASCOT search result of MP-15 protein by LC/MS/MS analysis. 57
Table 2: Primers for constructing recombinant plasmids. 58

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