臺灣博碩士論文加值系統

摘 要

石斑魚虹彩病毒基因體為140 Kb，有120個開放讀架 (ORF) 。以反向疫苗學 (Reverse vaccinology) 方式，利用石斑魚虹彩病毒打入魚體及小鼠體內所產生的抗體以西方轉漬分析，發現病毒蛋白質分子量為100 KDa、60 KDa和45 KDa的位置有明顯的訊號，由虹彩病毒基因體的序列，預測了五個目標基因，分別為GIV001L、GIV010R、GIV021L 、GIV042L及GIV045R，其中GIV045R是虹彩病毒主要的結構蛋白質(major capsid protein) 。選殖目標基因並於大腸桿菌表現系統，表現其重組蛋白，以製備虹彩病毒次單位疫苗。目前已構築GIV010R、GIV042L至表現載體pET20b(+)，以此重組蛋白來製備小鼠及紐西蘭大白兔多株抗體，免疫分析結果顯示經這些重組蛋白免疫後產生的抗體，可辨認到GIV病毒。
將所預測的次單位疫苗，腹腔內注射石斑魚苗進行免疫14天後，以力價109 TCID50 mL-1之GIV病毒液進行攻毒試驗。負對照組在第14天死亡率達100%，正對照組觀察24天期累計死亡率為20%，施打次單位疫苗的實驗組在攻毒後第13-16天死亡率達最高峰，持續到24天的累計死亡率為60%。以相對百分存活率表示，施打次單位疫苗實驗組約為40% ，顯著高於未施打疫苗的對照組。因此初步結果顯示這些次單位疫苗，具有保護石斑魚抵抗GIV 感染的效力。

Abstract

The genome of grouper iridovirus consists 140 Kb and encodes 120 open reading frames. Three putative protein with molecular weight of 100 KDa, 60 KDa and 45 KDa were detected after Western immunoblot analysis of the antibodies produced from fish and mice against the grouper iridovirus. Five candidate genes including GIV001L、GIV010R、GIV021L、GIV042L and GIV045R were selected from the grouper iridovirus genome. One of the candidate genes, the GIV045R gene encodes the major capsid protein of iridovirus. These putative target genes were cloned and their encoded protein were expression from the E. coil expression system. We have already constructed GIV042L and GIV010R in pET20b(+) expression vector. We produced polyclonal antibodies from mice and New Zealand rabbit that were immunized with the recombinant proteins expressed from Rosetta DE3 pLysS. Results obtained from Western immunoblot analysis indicate that these antibodies could recognize the grouper iridovirus particles.
The protection efficiency of these subunit vaccines against grouper iridovirus were estimated by injecting these recombinant protein into grouper fry and subsequently challenged with grouper iridovirus. After intraperitoneal immunizing with these recombinant proteins as subunit vaccines for 14 days, the immunizing grouper fry were challenged with 109 TCID50 mL-1 grouper iridovirus. The highest mortality of the sample group were reached at 13-16 days after virus challenge and the cumulative mortality of 60 % was found at the end of 24 days. On the contrary, the cumulative mortality of negative control group was 100% after 14 days grouper iridovirus infection and positive control group was 20% after 24 days. These subunit vaccine protection against grouper iridovirus infection is significant with 40% relative percentage of survival.
In summary, several putative and effective subunit vaccine were developed successfully from the grouper iridovirus genome. Preliminary results indicted that significant protection effects of these subunit vaccines were observed.

目錄
摘要………………………………………………………………………I
Abstract…………………………………………………………………III
第一章 前言……………………………………………………………1
第二章 文獻整理………………………………………………………3
第一節 虹彩病毒之簡介……………………………………………3
第二節 魚類虹彩病毒感染症相關疫情報導………………………6
第三節 反向疫苗之簡介………………………………………10
第四節 水產動物疫苗之簡介………………………………………12
第三章 材料與方法……………………………………………18
第一節 材料與藥品………………………………………………18
第二節 實驗方法…………………………………………………26
第三節 石斑魚虹彩病毒目標基因之重組蛋白表現與純化……34
第四節 病毒力價測定與目標抗原製備…………………………40
第五節 次單位疫苗保護效果之評估……………………………46
第四章 實驗結果………………………………………………………48
第五章 討論……………………………………………………………77
第六章 參考文獻……………………………………………………80

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