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研究生:陳慧如
論文名稱:石斑魚泳鰾細胞株之建立與其在石斑魚虹彩病毒研究之應用
論文名稱(外文):Establishment of a continuous cell lines from swim bladder of grouper (Epinephelus coioides) and its application on study of TGIV (grouper iridovirus of Taiwan)
指導教授:周信佑周信佑引用關係
指導教授(外文):Chou, Hsin-Yiu
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2001
畢業學年度:90
語文別:中文
論文頁數:55
中文關鍵詞:石斑魚虹彩病毒細胞培養
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取石斑魚的泳鰾以L15培養基進行初代培養,觀察到類上皮組織及類纖維狀細胞由組織碎塊中游離長出。經體外繼代培養50代後,隨著繼代次數的增加,部分細胞開始出現堆疊生長之現象,同時也有染色體異數性的發生。計算100個細胞的染色體數目,結果細胞染色體數目在39到67之間,以56~60與61~65兩個區域為主,而又以58、63兩種最多。此外對血清需求量也逐漸降低,添加5%、10% 和15% 血清濃度的培養基對50代細胞生長的影響沒有差異。由不同培養溫度細胞增殖的結果推定,此細胞的最適生長溫度為25~28℃。以下將此株化細胞簡稱為SB細胞。
台灣石斑虹彩病毒(TGIV)在多種已知魚類細胞上增殖情形不佳,因此本論文針對TGIV進行了病毒在SB細胞之特性研究。結果在SB細胞繼代25代的病毒,力價仍維持107.5 TCID50/ml,且SB增殖之TGIV可穩定保存於4℃、-80℃及液態氮中達一年之久,顯示SB細胞可作為TGIV之有效培養系統。另外由病毒的生長曲線,發現TGIV為一連續性釋放的病毒,但病毒不易被釋出,有大部分留在細胞內。利用電子顯微鏡術觀察TGIV在SB細胞內之感染情形,發現大小約為170 ± 10nm病毒主要在細胞質內增殖,但也可在細胞核內觀察到病毒粒子的存在。病毒可能由細胞膜、胞內管狀構造和液泡狀構造芽出,具外套膜之病毒顆粒大小約為220 ± 10nm。進一步以SB細胞大量增殖病毒、經濃縮、Freon萃取和氯化銫連續梯度離心,可得到純化之病毒粒子。
進而比較TGIV在SB、BF-2和GF三種細胞之感染情形,結果發現SB細胞質內TGIV粒子數量多且構造完整;破碎的BF-2細胞內病毒粒子零星分布,而GF細胞增殖TGIV的情形最差,僅有少數幾顆類似空病毒粒子的構造出現在細胞質內。而且TGIV的力價會隨著在BF-2細胞繼代次數的增加而明顯下降,繼代十次後力價僅有101.5 TCID50/ml;更者在GF細胞繼代七次後,已完全測不到病毒力價。
目 錄
摘要 ………………………………………………………………………I
前言 ………………………………………………………………………1
文獻整理 …………………………………………………………………3
一、本省海水魚的養殖現況和疾病問題………………………………3
二、虹彩病毒之簡介……………………………………………………4
三、魚類虹彩病毒感染症………………………………………………6
四、魚類細胞株之開發與在魚類病毒研究上之應用…………………8
材料與方法………………………………………………………………11
一、石斑魚泳鰾細胞株之建立與培養 ………………………………11(一)、實驗材料………………………………………………………11
(二)、研究方法………………………………………………………12
1、初代培養 …………………………………………………………12
2、細胞之繼代培養 …………………………………………………12
3、株化細胞染色體數目之分析 ……………………………………13
4、培養溫度對石斑魚泳鰾細胞生長之影響 ………………………13
5、血清濃度對石斑魚泳鰾細胞生長之影響 ………………………14
6、群落形成率 ………………………………………………………14
二、石斑魚虹彩病毒在石斑魚泳鰾細胞之特性研究…………………14
(一)、實驗材料………………………………………………………14
(二)、研究方法………………………………………………………15
1、TGIV在石斑魚泳鰾細胞連續繼代之穩定性 ……………………15
2、TGIV之保存溫度 …………………………………………………15
3、TGIV之增殖曲線 …………………………………………………15
4、以電子顯微鏡術觀察TGIV在石斑魚泳鰾細胞之感染情形 ……16
5、TGIV病毒之純化 …………………………………………………17
6、純化TGIV的負染觀察 ……………………………………………18
三、TGIV在不同細胞株之感染力與病原性之比較 …………………18
(一)、實驗材料………………………………………………………18
(二)、研究方法………………………………………………………18
1、TGIV在不同細胞株細胞病變效應 ………………………………18
2、TGIV在不同細胞株連續繼代穩定性 ……………………………19
3、不同細胞株增殖之TGIV感染實驗 ………………………………19
結果………………………………………………………………………20
一、石斑魚泳鰾細胞株之建立與培養 ………………………………20
1、石斑魚泳鰾細胞初代培養 ………………………………………20
2、泳鰾初代培養細胞之繼代 ………………………………………20
3、株化細胞染色體數目之分析 ……………………………………20
4、培養溫度對石斑魚泳鰾細胞生長之影響 ………………………20
5、血清濃度對石斑魚泳鰾細胞生長之影響 ………………………21
6、群落形成率 ………………………………………………………21
二、石斑魚虹彩病毒在石斑魚泳鰾細胞之特性研究 ………………21
1、TGIV在石斑魚泳鰾細胞連續繼代之穩定性 ……………………21
2、TGIV之保存溫度 …………………………………………………22
3、TGIV之增殖曲線 …………………………………………………22
4、以電子顯微鏡術觀察TGIV在石斑魚泳鰾細胞之感染情形 ……22
5、TGIV病毒之純化 …………………………………………………23
6、純化TGIV的負染觀察 ……………………………………………23
三、TGIV在不同細胞株之感染力與病原性之比較 …………………24
1、TGIV在不同細胞株感染情形之觀察 ……………………………24
2、TGIV在不同細胞株連續繼代穩定性 ……………………………24
3、不同細胞株增殖之TGIV感染實驗 ………………………………25
討論………………………………………………………………………26
參考文獻…………………………………………………………………33
圖…………………………………………………………………………41
表…………………………………………………………………………55
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