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研究生:張家榮
研究生(外文):Jia-Rong Chang
論文名稱:金目鱸Latescalcarifer細胞株的建立及其應用
論文名稱(外文):Establishment of a continuous cell line derived from barramundi (Lates calcarifer ) and its applications
指導教授:郭村勇
指導教授(外文):Tsun-Yung Kuo
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:生物技術研究所碩士班
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:79
中文關鍵詞:金目鱸神經壞死病毒
外文關鍵詞:fish cell linenodavirus
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金目鱸Lates calcarifer(bloch)又名尖吻鱸,為硬骨魚綱 (Osteichthyes),鱸目(Perciformes),鋸蓋魚科(Centropomidae),Lates屬的熱帶及亞熱帶海域魚類,分佈於琉球至印度洋海域及沿岸之半淡鹹水域。為東南亞地區及台灣南部沿海的大宗養殖魚類。
神經壞死病毒Nervous Necrosis Virus (NNV)為二十面體、無套膜、直徑25~30 nm的RNA病毒,為Betanodavirus屬的魚類病毒。NNV主要感染稚魚,患魚因腦神經破壞導致不正常游動,且大量死亡,造成許多養殖魚種重大的損失。由於鱸魚幼苗對NNV具有極高的感受性,因此本論文建立了鱸魚的持續型細胞株,自金目鱸仔魚取下肝臟、腎臟及鰾進行初代及繼代培養,結果腎臟及肝臟已繼代90代以上,鰾則繼代超過150代,各細胞以龍膽石斑魚NNV進行感受性測試,結果發現僅鰾(LCGB)具高度敏感性,LCGB細胞株在28℃及32℃下增殖最快,在含5% FBS以上之L-15 medium 下,可維持細胞之繼代及培養。本文以該細胞株成功分離東星斑神經壞死病毒(PLNNV),並可增殖病毒,該病毒力價可達107 TCID50/mL。病毒經大量培養及純化後免疫BALB/c 小鼠,進行神經壞死病毒單源抗體之製備,經極限稀釋法獲得了3株單源抗體,此3株單源抗體,分別以西方轉印法及免疫單層細胞磷酸過氧化酶染色法(IPMA)確認為抗NNV之單源抗體,此外將東星斑神經壞死病毒外殼蛋白基因選殖入表現載體pQE30中,並送入大腸桿菌表現宿主M15後,可成功表現出NNV外殼基因重組蛋白,並經由西方轉印法證明本論文所製備之單源抗體皆為抗NNV外殼蛋白之單源抗體,以此單源抗體可作為未來進一步研究病毒的重要利器。
Lates calcarifer (bloch) belongs to the Lates genus, Centropomidae family, Perciformes order and Osteichthyes class. This tropical and sub-tropical fish is located in the coastal brackish water throughout Okinawa to the Indian Ocean. Lates calcarifer is massively cultured in Southeastern Asia and Southern Taiwan.
Nervous Necrosis Virus (NNV), a member of the Betanodaviridae, is an icosahedral, non-enveloped RNA virus 25-30 nm in diameter. NNV mainly infects juveniles, destroying the nerves of the brain, leading to abnormal swimming and high mortality of infected fish, causing devastating economic impact to the aquaculture industry.
Since the larvae of Lates calcarifier are highly susceptible to NNV, this research study developed primary and continuous cell lines from the liver, kidney, and the gas bladder of bloch. The liver and kidney cells were propagated over 90 passages while the gas bladder cell line was propagated over 150 passages, maintained in 5% FBS, L-15 medium, and grew quickly at both 28°C and 32°C. Epinephelus lanceolatus NNV was first used to test these different cell lines for susceptibility and results showed that Lates calcarifer gas bladder (LCGB) was highly susceptible. Then, wild type Plectropomus leopardus Nervous Necrosis Virus (PLNNV) was replicated in LCGB and the viral titer was 107 TCID50/mL. Large quantities of PLNNV was grown in LCGB, collected and purified, serving as the antigen for anti-NNV monoclonal antibodies production in BALB/c mice. After limiting dilution and screening, 3 different hybridomas secreting monoclonal antibodies against NNV were successfully produced. The 3 monoclonal antibodies were confirmed by Western blot and IPMA against purified NNV. Subsequently, cloning of PLNNV capsid gene and expression of recombinant proteins in E. coli was done in pQE30 vector and M15 host. A Western blot of the monoclonal antibodies against the recombinant protein proved all 3 monoclonal antibodies produced in this study were against the NNV capsid protein. These monoclonal antibodies are important tools to have for further researches on Nervous Necrosis Virus.
目錄

中文摘要……………………………………………………………………………i
英文摘要……………………………………………………………………………ii
目錄…………………………………………………………………………………iii
表目錄………………………………………………………………………………vii
圖目錄………………………………………………………………………………viii
第一章 緒言…………………………………………………………………………1
第二章 文獻探討……………………………………………………………………3
一、 初代細胞分離、培養……………………………………………………3
二、 培養細胞的鑑定…………………………………………………………3
三、 Piscinodavirus………………………………………………………5
四、 建立增殖Piscinodavirus 之細胞株………………………………6
五、 單株抗體…………………………………………………………………7
第三章 材料與方法…………………………………………………………………9
金目鱸腎臟、肝臟及鰾細胞株的建立
一、 金目鱸初代細胞的分離…………………………………………………9
二、 細胞的換液與繼代……………………………………………………10
三、 鱸魚肝臟、鰾及腎臟細胞之型態學觀察……………………………11
四、 鱸魚鰾細胞生長特性分析……………………………………………11

神經壞死病毒(NNV)之研究
一、 神經壞死病毒(PLNNV)之分離………………………………………13
二、 PLNNV 病毒力價測試…………………………………………………14
三、 PLNNV的大量培養……………………………………………………14
四、 PLNNV P5病毒液之濃縮與純化…………………………………… 15
五、 PLNNV TOTAL RNA之萃取…………………………………………16
六、 PLNNV RNA2外鞘蛋白基因選殖……………………………………17
1.病毒核酸萃取及RT-PCR………………………………………17
2.PCR產物電泳分析…………………………………………………18
3.PCR產物純化……………………………………………………18
4.載體DNA pQE30的製備…………………………………………19
5.限制酶切割反應(Restriction Enzyme Cleavage Reaction)…20
6.接合反應(Ligation)…………………………………………………21
7.勝任細胞(Competent cell)的製備及轉型作用(Transformation)…21
8.重組質體的挑選及確認………………………………………………22
9.重組載體的定序………………………………………………………22
七、 PLNNV RNA2 基因重組外鞘蛋白之表現……………………………………22
八、 PLNNV病毒液及其基因重組蛋白進行SDS-PAGE確認………………………23
PLNNV多株抗體之產製
一、 山羊之免疫及抽血………………………………………………………24
二、 PLNNV病毒及基因重組蛋白以山羊抗NNV
多株抗體進行Western blot確認………………………………………24
PLNNV單源抗體之產製
一、 老鼠之免疫……………………………………………………………26
二、 融合瘤之製作…………………………………………………………27
三、 以ELISA法篩選融合瘤………………………………………………28
四、 融合瘤之單株化………………………………………………………29
五、 單株抗體的產製…………………………………….………………30
以免疫學方法測定單株抗體與病毒及病毒基因重組外鞘蛋白之特異性
一、 PLNNV 病毒及表現的基因重組蛋白以
單株抗體進行Western blot 分析…………………………………30
二、 以免疫過氧化酶單層細胞分析法分析單株抗體與
PLNNV活病毒之特異性…………………………………………………31
第四章 結果……………………………………………………………………32
金目鱸腎臟、肝臟及鰾細胞株的建立
一、 鱸魚腎臟、肝臟及鰾初代細胞的分離…………………………….32
二、 鱸魚腎臟、肝臟及鰾細胞不同代數細胞型態觀察…………………32
三、 鱸魚鰾細胞生長特性分析……………………………………………33
四、 鱸魚腎臟、肝臟及鰾細胞對NNV之感受性分析……………………33
神經壞死病毒(NNV)之研究
一、 東星石斑神經壞死病毒(PLNNV)之分離……………………………34
二、 不同代數PLNNV病毒力價測試結果…………………………………34
三、 PLNNV RNA2 外鞘蛋白基因基因選殖及序列比對…………………34
四、 PLNNV病毒及基因重組蛋白SDS-PAGE確認………………………35
五、 PLNNV病毒及基因重組蛋白Western blot確認…………………35
六、 PLNNV單株抗體製備及其特異性分析………………………………35
七、 以免疫過氧化酶單層細胞分析法分析單株抗體與…………………36
PLNNV活病毒之特異性
第五章 討論………………………………………………………………………37
附錄……………………………………………………………………………………40
參考文獻………………………………………………………………………………64
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