觀賞魚細胞株與錦鯉疱疹病毒症檢測方法之建立

Establishment and Characterization of Ornamental Fish Cell Lines and Development of Methods for Detection of Koi Herpesvirus Disease

林學廉(Shiue-Lian Lin)

指導教授 ： 陳秀男

國立臺灣大學/生命科學院/漁業科學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.00262

摘要

觀賞魚細胞株的建立對於觀賞魚病毒性疾病的研究是非常的重要，尤其是已經可以進行人工繁殖的觀賞魚品種，常會因高密度養殖方式，及國際貿易的流通，造成病毒性疾病迅速的滋生及廣泛的傳染，造成嚴重的經濟損失。
本論文首次成功地從海水觀賞魚的藍嘴神仙(Three-spot angelfish, Apolemichthys trimaculatus)的鰭組織，建立了TSAF細胞株。另從紅鯽( Red carp, Carassius auratus)的鰓、心臟、腎、頭腎、脾、卵等組織，建立了的RCF、RCG、RCH、RCK、RCHK、RCS、RCO等7種細胞株，同時也從紅鯽的頭腎細胞建立一株巨噬細胞RCHKM。另外，從錦鯉的鰭、鰓、心臟、鰾、卵等器官組織，成功地建立了錦鯉(Koi, Cyprinus carpio)的KF、KG、KH、KB、KO等5種細胞株。這些細胞都可以在20°C∼30°C、10% FBS-L15簡單的培養條件下不斷地繼代生長。
TSAF細胞株對於喜歡生長在30°C的EHVF，以及喜歡在18°C生長的IPNV、EVE、HCRV等病毒，都具有感受性，適合作為冷水性魚類和溫水性魚類的病毒檢測工具。RCO及RCHKM細胞株，以及KF、KG、及KB等細胞株，對KHV都具有感受性，會產生疱疹病毒的細胞病變，可以使用在大量增殖KHV的用途之上。因以細胞株是研究魚類的病毒學、病理學、分子生物學及環境檢測等的最佳工具。
從台灣南部錦鯉繁殖場發現一株KHV的變異品種，命名為NKV2，屬於錦鯉魚疱疹病毒的成員，但無法利用KHV Primer、CNGV primer、TK primer、Gray primer等檢測出來，卻可以用檢測鯉魚疱疹病毒的CyHV-1 Helicase primer及CyHV-1 Triplex primer檢測出來。
我們以KHV primer為藍本，設計開發出兩個新的primer，命名為NKV2-2K primer及NKV2-1.5K primer，都可以同時檢測出KHV及NKV2。另以TK primer為藍本，設計開發出新的primer，命名為NKV2-TK primer，可以利用NKV2的intron部份比KHV的intron較大的特性，同時檢測出KHV及NKV2，可作為PCR檢定錦鯉疱疹病毒的引子之用。

關鍵字

觀賞魚 ； 細胞株 ； 錦鯉疱疹病毒 ； 引子對

並列摘要

Establishing ornamental fish cell lines is essential for researching the viral diseases that affect these ornamental fish, particularly ornamental fish species that can be artificially bred. Typically, because of high-density breeding and the flow of international trade, these fishes cause rapid viral disease spread and transmission, resulting in severe economic losses.
This study reports the first successful cultivation of cell lines from the fin tissue of the marine ornamental fish species Apolemichthys trimaculatus, that is, the three-spot angelfish (TSAF). We successively cultivated 7 Carassius auratus or red carp (RC) cell lines from the fin (F), gill (G), heart (H), head kidney (HK), spleen (S), and ovum (O) tissues, yielding RCF, RCG, RCH, RCK, RCHK, RCS, and RCO cell lines. An RCHK macrophage (RCHKM) cell line was also cultivated from RC head kidney tissues. We also established 5 Cyprinus carpio (Koi) cell lines (KF, KG, KH, KB, and KO) from the fin, gill, heart, air bladder, and ovum tissues of koi. These cell lines can be continually reproduced and grown in simple cultivation conditions of 20°C-30°C and 10% FBS-L15.
TSAF cell lines were highly susceptible to the eel herpesvirus in Formosa (EHVF), which ideally grows in 30°C environments, and to the infectious pancreatic necrosis virus (IPNV), endogenous viral elements (EVEs), and hippeastrum chlorotic ringspot virus (HCRV), which ideally grow in 18°C environments. Therefore, TSAF cell lines are extremely appropriate as virus detectors for cold and warm water fishes. The RCO, RCHKM, KF, KG, and KB cell lines were all susceptible to koi herpesvirus (KHV), generating the cytopathic effect commonly presented in herpesvirus. Hence, these cell lines can be used to mass-proliferate KHV. Accordingly, cell lines are the optimal tools for researching fish virology, pathology, molecular biology, and conducting environmental testing.
A mutated KHV cell line, named NKV2, was identified at a koi breeding ground in Southern Taiwan. NKV2 cannot be detected using various KHV primers, carp interstitial nephritis and gill necrosis virus (CNGV) primers, thymidine kinase (TK) primers, and Gray primers, but can be detected using the carp pox cyprinid herpesvirus 1 (CyHV-1) helicase primer and CyHV-1 triplex primers, which are used to detect KHV.
We designed and developed two new primers (i.e., the NKV2-2K and NKV2-1.5K primers) based on KHV primer. The proposed primers can be employed to detect both KHV and NKV2. An additional primer (i.e., the NKV2-TK primer) was designed and developed based on TK primer. This primer can be used to detect both KHV and NKV2 according to the introns of NKV2 are larger compared with those of KHV. The developed primers can be adopted in polymerase chain reaction (PCR) to detect KHV and NKV2 in the same time.

並列關鍵字

ornamental fish ； cell line ； KHV ； PCR primer

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