石斑魚神經壞死病毒不活化及次單位疫苗之研發

Development of Inactivated and Subunit Vaccine Against Grouper Nervous Necrosis Virus

史歷棋(Shih Li-Chi)

指導教授 ： 柯冠銘

國立屏東科技大學/獸醫學院/動物疫苗科技研究所/碩士(2020年)

https://doi.org/10.6346/NPUST202000212

摘要

病毒性神經壞死症(Viral Nervous Necrosis; VNN)是全球70多種海水和淡水魚類中最嚴重的疾病之一。VNN的致病因子是神經壞死病毒(Nervous Necrosis Virus; NNV)屬於Nodaviridae家族中的Betanodavirus屬，為無封套膜的RNA病毒。台灣石斑魚感染的神經壞死病毒是屬於赤點石斑魚神經壞死病毒(Red-spotted Grouper Nervous Necrosis Virus; RGNNV)基因型，會導致魚類神經功能障礙，臨床上呈現異常的游泳行為，且感染時期為幼苗和幼魚免疫系統尚未成熟之時，導致幼苗和幼魚死亡率常高達100%，使水產養殖業遭受重大經濟損失。本研究目的在開發NNV不活化及次單位混合疫苗並制定最佳免疫程序，此外嘗試建立龍虎斑腦、鰓、脾、腎、鰭等細胞株，用於未來疫苗開發成功後可取代GF-1細胞以降低生產成本。本研究以GF-1細胞培養NNV，且以TCID50與Real-time PCR測定病毒力價，NNV經13代馴化後其病毒量可達109.5TCID50/mL，此外選殖NNV的外殼蛋白並以桿狀病毒表現系統表現蛋白，經SDS-PAGE與Western blot進行蛋白確認與定量分析，結果顯示蛋白表現量可達1,250 μg/mL。不活化混合次單位疫苗之製備選擇劑量為105TCID50/mL之不活化病毒與濃度分別為5 μg、10 μg與25 μg之蛋白進行混合並加入油質佐劑乳化成疫苗，動物實驗後以免疫後第21天採集之血清經中和試驗結果分析免疫劑量為105TCID50‧10μg之組別血清中和抗體濃度可達log213,具有發展成疫苗之潛力。龍虎斑腦、鰓、鰭細胞目前已能穩定培養，且以NNV感染測定細胞對病毒的感受性，細胞在受NNV感染後第三天即可觀察到明顯的CPE現象，經TCID50測定病毒力價可達108TCID50~109TCID50間，表示龍虎斑腦、鰓、鰭細胞具有培養並增殖NNV的潛力。

關鍵字

神經壞死病毒（NNV） ； β諾達病毒 ； 次單位疫苗 ； 不活化疫苗 ； 龍虎斑細胞株

並列摘要

Viral nervous necrosis (VNN) is one of the most serious diseases among more than 70 species of marine and freshwater fishes worldwide. The causative agent of VNN is nervous necrosis virus (NNV), a non-enveloped RNA virus, belonging to the genus Betanodavirus within the family Nodaviridae. Actually, the NNV spread out in the Taiwanese grouper population has been identified as the red-spotted grouper nervous necrosis virus (RGNNV) genotype, that causes abnormal swimming patterns, neurological dysfunction. The infection results in a high mortality up to 100% in seedlings and juveniles without mature immune system, thereby causing a significant economic loss in the grouper aquaculture industry.
In this study, we developed both inactivated and subunit vaccines against NNV and established an optimal immunization program for grouper. In addition, the brain, gill, spleen, kidney and fin cell lines from Epinephelus fuscoguttatus x Epinephelus lanceolatus were also established to replace GF-1 cell, thereby achieving cost-effective large-scale production of NNV. NNV was cultured in GF-1 cells and the virus titer was measured at TCID50 and real-time PCR. The results showed that the virus titer could reach 109.5 TCID50/ mL after 13 passages in GF-1 cells. On the other hand, the capsid protein gene of NNV was cloned and then expressed in the baculovirus expression system. Afterwards, SDS-PAGE and Western blot were undertaken to confirm and quantitate recombinant capsid protein. The results showed that the expressed protein amount reached 1,250μg/mL. Then, preparation of inactivated mixed subunit vaccine and the virus titer containing 105TCID50/mL was mixed respectively with 5μg, 10 μg and 25 μg recombinant protein and then emulsified with an oil adjuvant to produce different vaccine formulations in the present study. After the animal experiment, the serum collected on the 21st day after immunization was analyzed by the neutralization test. The results showed that the neutralizing antibody titers of 105TCID50‧10μg could reach log213, which has the potential to develop into a vaccine.The brain, gill and fin cell lines from Epinephelus fuscoguttatus x Epinephelus lanceolatus be able to culture and the susceptibility of cell lines, including brains, gills and fins, to NNV was analyzed and the CPE in cells could be observed three days after infection with NNV. The results showed that the virus titer ranged could be between 108 TCID50 and 109 TCID50 by TCID50. Therefore, the cell lines established in the present study possessed the capacities to cultivate and proliferate NNV.

並列關鍵字

Nervous necrosis virus (NNV) ； Betanodavirus ； Subunit vaccine ； Inactivated vaccine ； Epinephelus fuscoguttatus x Epinephelus lanceolatus cell line

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