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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.author | 林淑卿 | zh_TW |
| dc.date.accessioned | 2021-07-01T08:21:01Z | - |
| dc.date.available | 2021-07-01T08:21:01Z | - |
| dc.date.issued | 1998 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76391 | - |
| dc.description.abstract | 近年來南台灣石斑養殖孵化場魚苗屢有病毒性神經壞死症（Viral nervous necrosis disease，NNV disease）疫情發生，造成極高的死亡率。為瞭解病毒基本特性以提供未來防治方法之參考，本篇報告利用本實驗室新建立的石斑魚鰭細胞株（GF-1 cell line）來分離罹患VNN症石斑魚苗體中的病原體，並做特性的分析，包括：以光學及電子顯微鏡做病理觀察，用PCR檢定病毒核酸，用螢光免疫染色法檢測病毒在感染細胞中出現的時間，並探討溫度與酸鹼值因數對此病毒在細胞株與活體中感染力之影響。 將石斑病魚分離出來的病毒，利用GF-1細胞增殖、純化並萃取核酸，以條紋?神經壞死症病毒（Striped Jack Nervous Necrosis Virus, SJNNV）鞘蛋白質基因的兩組核酸引子對，（F1，R3）及（F2，R3），進行PCR檢測，結果可得到兩個引子對的目標核酸片段T2及T4，故證實病原體是屬於魚類結病毒（Fish nodavirus），並命名為GNNV（Grouper nervous necrosis virus）。純化的GNNV在電子顯微鏡觀察下，為不具外套膜，直徑20-25nm之圓形至20面體顆粒；經核酸萃取與電泳分析，測到兩段RNA，分子量分別是1.02×103kDa及0.5×103kDa；純化GNNV在SDS-PAGE電泳分析下，可得到兩條主要蛋白質，分子量分別是為43kDa及41kDa。GNNV在GF-1細胞株及活體中的複製能力受溫度影響，最適複製溫度範圍是28 - 32℃，低溫則會降低病毒的複製量並延緩活體感染魚發病的時間。GNNV在強鹼的環境（pH10、pH12）下感染力受到的抑制最大。比對臺灣養殖石斑分離出來的GNNV與日本養殖石斑魚分離出來的RGNNV的T2核酸序列，相似度高達99％，顯示GNNV與日本分離之RGNNV屬於同一類海水魚NNV。本實驗結果除了建立GNNV的物化特性資料外，更證實了GF-1細胞無論在分離或增殖GNNV方面都有很好的效率。由GF-1細胞所複製出的GNNV，還能保有在活體內時的特性。 | zh_TW |
| dc.description.abstract | Mass mortalities caused by nervous necrosis virus (NNV) of hatchery-reared grouper larvae and juveniles have been found in southern Taiwan. In the present study, a cell line derived from the fin tissue of a grouper hybrid (Einephelus malabarius × Epinephalus akaara) and nominated as GF-1 cell line, is used for isolation and characterization grouper nervous necrosis virus (GNNV). The purified virus was examined by PCR using striped jack nervous necrosis virus (SJNNV) RNA2 specific primer sets (F1, R3) or (F2, R3), and the target fragments T2 and T4 were detected in the PCR products. Viruses after replication in GF-1 cells were purified and observed under electron microscope and follow by analysis by using electrophoretic technique. The purified virus has spherical to icosahedral morphology with diameter of 20-25 nm, and the buoyant density of the virus is 1.34 g/mL. The protein profile of purified virus revealed two major proteins with molecular weight of 43 kDa and 41 kDa, respectively. Viral genome extracted from the purified virus showed two separate RNAs with molecular weights of 1.02 × 103 kDa and 0.5 × 103 kDa, respectively. The results of in vitro and in vivo effect of temperature on GNNV infection indicated that the suitable temperature range for viral replication is 28-32℃, and lower temperature could decrease viral replication rate in vitro and delay the outbreak of mass mortality in vivo. GNNV was inactivated under a culture medium of pH 10 or pH 12. The T2 sequence similarity between GNNV and grouper virus RGNNV was about 99%. It is therefore suggested that these two viruses should be classified in the same group. All the properties of GNNV in vitro suggested that GNNV replicated in GF-1 cells sustained most similarities of GNNV in vivo, GF-1 cell line is recommended as an important tool for the studying NNV. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-01T08:21:01Z (GMT). No. of bitstreams: 0 Previous issue date: 1998 | en |
| dc.description.tableofcontents | 謝誌…………………………………………………………………………………………………………………………Ⅰ 目錄…………………………………………………………………………………………………………………………Ⅱ 摘要…………………………………………………………………………………………………………………………Ⅴ Abstract……………………………………………………………………………………………………………………Ⅵ 第一章 緒論………………………………………………………………………………………………………………1 1．1 石斑魚的簡介………………………………………………………………………………………………………1 1．2 石斑魚在台澎地區的養殖概況……………………………………………………………………………………1 1．3 石斑魚之病毒性疾病………………………………………………………………………………………………2 1．4 海水魚神經壞死症病毒（NNV）…………………………………………………………………………………2 1．4．1 海水魚神經壞死症病毒（Nervous Necrosis Virus, NNV）發現史………………………………………2 1．4．2 病癥……………………………………………………………………………………………………………3 1．4．3 NNV的特性與分類地位…………………………………………………………………………………………4 1．4．4 NNV的傳染途徑與感染病程……………………………………………………………………………………6 1．4．5．魚類細胞株對NNV的感受性…………………………………………………………………………………7 1．5 實驗目的……………………………………………………………………………………………………………7 第二章 材料與方法………………………………………………………………………………………………………9 2．1 材料…………………………………………………………………………………………………………………9 A．病毒……………………………………………………………………………………………………………………9 B．細胞株…………………………………………………………………………………………………………………9 C．未受GNNV感染的石斑魚苗……………………………………………………………………………………………9 2．2 病毒分離及鑑別方法：……………………………………………………………………………………………9 2．2．1 GF-1細胞對GNNV感受性之測試…………………………………………………………………………………9 2．2．1．1．GF-1細胞對GNNV感受性之光學顯微鏡觀察………………………………………………………………9 2．2．1．2 穿透式電子顯微鏡切片之檢驗……………………………………………………………………………10 2．2．2 病毒純化與負染色觀察………………………………………………………………………………………12 2．2．2．1 GNNV的純化…………………………………………………………………………………………………12 2．2．2．2 穿透式電子顯微鏡（TEM）負染色觀察…………………………………………………………………13 2．2．3 利用聚合酵素連鎖反應（PCR）檢驗純化之GNNV……………………………………………………………14 2．2．3．1 病毒核酸之萃取……………………………………………………………………………………………14 2．2．3．2 GNNV核酸反轉錄（Reverse Transcription）反應……………………………………………………15 2．2．3．3 聚合酵素反應（PCR）之檢測……………………………………………………………………………15 2．2．4 病毒力價測試…………………………………………………………………………………………………16 2．2．5 GNNV核酸分子量測定…………………………………………………………………………………………16 2．2．6 GNNV的核酸定序………………………………………………………………………………………………17 2．2．6．1 聚合酵素連鎖反應（PCR）………………………………………………………………………………17 2．2．6．1．神經壞死病毒基因（T2）與pCR-TOPO載體接合反應（Ligation）…………………………………17 2．2．6．3 重組質體的轉殖作用………………………………………………………………………………………18 2．2．6．4 點菌…………………………………………………………………………………………………………19 2．2．6．5 Mini preparation…………………………………………………………………………………………20 2．2．6．7 定序…………………………………………………………………………………………………………21 2．2．7 多源抗體的製備………………………………………………………………………………………………21 2．2．7．1 動物免疫……………………………………………………………………………………………………21 2．2．7．2 眼窩採血法…………………………………………………………………………………………………22 2．2．7．3 酵素連接免疫吸附（Enzyme Linked Immunosorbant Assay, ELISA）測定多源抗體效價…………23 2．2．8 GNNV SDS膠體電泳分析………………………………………………………………………………………24 2．2．9 膠體染色法……………………………………………………………………………………………………27 2．2．9．1 Coomassie Brilliant Blue R-250（CBR）蛋白質染色法……………………………………………27 2．2．9．2 醣蛋白質染色法（過碘酸－硝酸銀法）…………………………………………………………………28 2．2．10 GNNV感染GF-1細胞在不同感染時間之螢光抗體檢測（Fluorescent Antibody Test, FAT）…………30 2．3 環境因數對GNNV感染力之影響及病原性測試……………………………………………………………………32 2．3．1 酸鹼值對GNNV感染能力之影響………………………………………………………………………………32 2．3．2 溫度與GNNV在GF-1細胞增殖之關係…………………………………………………………………………32 2．3．2．1 不同溫度對GNNV在GF-1細胞增殖之光學顯微鏡觀察……………………………………………………32 2．3．2．2 GF-1細胞培養液之T4 PCR檢測……………………………………………………………………………32 2．3．2．3 CAV之蛋白質免疫轉印試驗………………………………………………………………………………33 2．3．3 溫度與石斑魚苗感染GNNV的關係……………………………………………………………………………34 2．3．3．1 GNNV感染石斑魚苗之現場觀察及樣品收集………………………………………………………………34 2．3．3．2 GNNV魚苗之PCR檢測………………………………………………………………………………………35 2．3．3．3 光學病理組織切片之檢驗…………………………………………………………………………………36 第三章 結果………………………………………………………………………………………………………………39 3．1 GF-1細胞株對GNNV的感受性………………………………………………………………………………………39 3．2 感染GNNV的GF-1細胞株之病理觀察………………………………………………………………………………39 3．3 GNNV的純化…………………………………………………………………………………………………………39 3．4 利用聚合酵素連鎖反應（PCR）檢驗GF-1細胞複製之GNNV……………………………………………………40 3．5 GNNV在GF-1細胞複製產量的測定…………………………………………………………………………………40 3．6 GNNV之核酸分子量測定……………………………………………………………………………………………40 3．7 GNNV T2核酸序列分析……………………………………………………………………………………………40 3．8 GNNV之多源抗體的製備……………………………………………………………………………………………41 3．9．GNNV蛋白質的定性分析…………………………………………………………………………………………41 3．9．1 神經壞死病毒之蛋白質圖譜…………………………………………………………………………………41 3．9．2 GNNV醣蛋白的測定……………………………………………………………………………………………41 3．9．3 用螢光抗體檢測在GF-1細胞株中GNNV蛋白質合成的時間…………………………………………………41 3．10 酸鹼值對GNNV感染力之影響……………………………………………………………………………………42 3．11 溫度與GNNV在GF-1細胞增殖之關係……………………………………………………………………………42 3．12 溫度與石斑魚苗感染GNNV之關係………………………………………………………………………………43 第四章 討論………………………………………………………………………………………………………………44 4．1 GF-1細胞株對GNNV的感受性………………………………………………………………………………………44 4．2 T2核酸序列比對……………………………………………………………………………………………………45 4．3 GNNV定性分析………………………………………………………………………………………………………45 4．4 酸鹼值對GNNV感染力之影響………………………………………………………………………………………46 4．5 溫度與GNNV在GF-1細胞增殖之關係………………………………………………………………………………46 4．6 溫度與石斑魚苗感染GNNV之關係…………………………………………………………………………………47 4．7 結論與展望…………………………………………………………………………………………………………48 參考文獻…………………………………………………………………………………………………………………49 附表與附圖………………………………………………………………………………………………………………53 Table 1．酸鹼值對GNNV感染能力之影響………………………………………………………………………………53 Table 2．溫度與GNNV在GF-1細胞增殖之關係…………………………………………………………………………54 Table 3．溫度與石斑魚苗感染GNNV的關係……………………………………………………………………………55 Table 4．石斑魚苗感染GNNV之PCR檢測………………………………………………………………………………56 Fig．1 感染GNNV的GF-1細胞之細胞病變………………………………………………………………………………57 Fig．2 利用穿透式電子顯微鏡觀察感染GNNV之GF-1細胞……………………………………………………………58 Fig．3 病毒的純化………………………………………………………………………………………………………59 Fig．4 GNNV PCR分析……………………………………………………………………………………………………60 Fig．5 GNNV核酸分子量分析……………………………………………………………………………………………61 Fig．6 T2核酸序列之比對………………………………………………………………………………………………63 Fig．8 GNNV在GF-1細胞株中蛋白質合成之專一性螢光抗體檢測……………………………………………………65 Fig．9 溫度與GNNV在GF-1細胞增殖之關係……………………………………………………………………………66 Fig．10 感染GNNV之石斑魚苗的光學病理組織切片…………………………………………………………………67 Fig．11 三種不同來源之GNNV的核酸比較……………………………………………………………………………68 | |
| dc.language.iso | zh-TW | |
| dc.title | 石斑魚神經壞死病毒的分離與定性 | zh_TW |
| dc.title | Isolation and Characterization of Grouper Nervous Necrosis Virus (GNNV) | en |
| dc.date.schoolyear | 86-2 | |
| dc.description.degree | 碩士 | |
| dc.relation.page | 78 | |
| dc.rights.note | 未授權 | |
| dc.contributor.author-dept | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 動物學研究所 | zh_TW |
| 顯示於系所單位： | 動物學研究所 | |
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