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|Authors: ||Liu, Hsin-I|
|Contributors: ||NTOU:Department of Aquaculture|
Grouper;Iridovirus;Major capsid protein;recombinant protein vaccine;DNA vaccine
|Issue Date: ||2018-08-22T06:05:54Z
|Abstract: ||石斑魚虹彩病毒造成石斑魚苗場極大的損失，目前分離出的病毒株主要為隸屬於巨大細胞病毒屬 (megalocytivirus) 的臺灣石斑虹彩病毒 (grouper iridovirus of Taiwan, TGIV) 以及隸屬蛙病毒屬 (ranavirus) 的石斑虹彩病毒 (grouper iridovirus, GIV)。為了發展有效控制石斑虹彩病毒的策略，本論文探討溫度(物理性調控)及新式口服疫苗 (生物性調控)抑制病毒感染的功效。研究指出溫度對於兩種虹彩病毒致病性有相當的的影響，無論是TGIV或是GIV，死亡率最高的是常溫25℃的組別，高溫的32℃的死亡率反而最低，且病毒的MCP基因表現會明顯受到抑制，因此調控溫度是可以有效控制疾病發展的重要因子。接著我們進一步開發疫苗進行生物性調控，我們選殖出TGIV以及GIV的主要外鞘蛋白全基因序列 (major capsid protein, MCP)，轉殖入原核生物的表現載體pGS21a，製作兩種虹彩病毒主要外鞘重組蛋白，經測試以18℃、0.5~1 mM IPTG誘導表現12小時，可以得到GIV以及TGIV MCP的不可溶重組蛋白。將重組蛋白免疫兔子，製作TGIV-MCP以及GIV-MCP的多株抗體。經血清學鑑定分析結果顯示，TGIV-MCP以及GIV-MCP的多株抗體並無法互相辨識，推測兩種主要外鞘蛋白沒有共同的抗原辨識位。接著將兩種MCP重組蛋白製作成乳化疫苗，以腹腔注射免疫石斑魚，於免役後14天進行攻毒試驗，結果顯示，兩種MCP乳化重組蛋白疫苗可有效誘發石斑魚產生專一性抗血清，且對病毒保護效果可達七成以上，說明了MCP蛋白可以做為對抗虹彩病毒的候選抗原。但重組蛋白純化過程以及儲存方法需耗費過多成本，因此我們進一步開發虹彩病毒MCP DNA疫苗。我們以含有EGFP的pcDNA3建構兩種真核細胞表現MCP基因的載體，分別製作不同病毒DNA 疫苗。接著我們分別進行in vitro (GF-1細胞) 以及in vivo (斑馬魚受精卵) 的DNA疫苗表現測試，結果無論是再in vivo或是in vitro同樣皆可觀察到綠色螢光蛋白的表現，顯示我們建構的兩種MCP表現質體可以有效的表現出目標蛋白，接著將表現質體進行三重相乳化包埋，製成口服DNA疫苗。將石斑魚苗經乳化DNA疫苗灌食口服免疫後，進行腹腔注射攻毒試驗，在攻毒後14天，統計相對存活率，結果顯示出pcDEGm有50%的保護效果，pcDETm有68%的保護效果。測試免疫魚體內的病毒增殖情形，結果顯示，病毒基因的表現量有下降，且針對MCP的抗體力價於免疫後7天以及14天皆有上升。藉由pcDETm跟pcDEGm製作DNA疫苗免疫過的石斑魚，可以有效提高抵抗TGIV以及GIV病毒的感染，最後與市售GIV不活化疫苗比較其保護效果，我們開發的雙價DNA口服疫苗抗GIV的效果雖不如GIV不活化疫苗注射的效果，但對TGIV的保護效果優於GIV不活化疫苗，且混料投餵的方便度也優於腹腔注射的方式，如果可以更進一步提升其保護效果，未來仍有商業化的潛力。|
Fish iridoviruses cause systemic diseases with high mortality in various species of wild and farm-raised fish, resulting in severe economic losses. So far, the two most prominent iridoviruses isolated in Taiwan are grouper iridovirus (GIV) and grouper inridovirus of Taiwan (TGIV), belonging to the Renavirus and Megalocytivirus genus, respectively. To develop efficient strategy against iridoviral infection, the temperature effect on the pathogenicity of both GIV and TGIV was investigated in this study; in addition, a novel oral vaccine has been developed to counteract the infection in grouper larvae. The study on temperature effect showed that the pathogenicity of both GIV and TGIV were affected by temperature. The mortality of both viruses occurred at 25℃, whereas the mortality significantly declined at higher temperature (32℃). Furthermore, the expression of the viral major capsid protein (MCP, a late gene) echoed this pattern. These results suggest that manipulation of water temperature can be a potential strategy against GIV and TGIV infections in grouper. A novel oral DNA vaccination strategy has also been successfully developed in this study. Prior to the development of the vaccine, the antigenicity of MCP was first verified both in vitro and in vivo. The MCP genes from GIV and TGIV were subcloned into prokaryotic expression vector pGS21a to express recombinant MCP proteins in E. coli. The purified recombinant MCP proteins were then used to immunize rabbits to generate polyclonal antisera. By western blotting, no cross reactivity was observed in the GIV MCP and TGIV MCP antisera, indicating that no common epitope shared by GIV MCP and TGIV MCP. The antigenicity of MCPs was subsequently verified by immunizing grouper larvae with emulsified recombinant MCPs, followed by viral challenge. The production of specific antibodies was confirmed in the vaccinated fish. Furthermore, the relative percent survival (RPS) reached 70% in the fish vaccinated with the recombinant proteins. These results demonstrated that GIV and TGIV MCPs are effective antigens. To generate DNA vaccine, the GIV and TGIV MCP genes were subcloned into a eukaryotic expression vector pcDNA3-EGFP to generate plasmids pcDEGm and pcDETm respectively. The expression of EGFP-MCP fusion proteins was confirmed in GF-1 cells transfected with pcDEGm or pcDETm, as well as in zebrafish eggs injected with the plasmids. Oral DNA vaccines against GIV and TGIV were prepared by emulsifying the plasmids with a formula established previously. Grouper larvae were immunized with the oral vaccines and infected with the viruses at 14 days post-vaccination. The production of specific antibodies increased at as early as 7 days post-vaccination. The RPS reached 50% for the pcDEGm vaccine and 68% for the pcDETm vaccine. In addition, the expression of MCP genes was suppressed in the vaccinated fish. These data clearly demonstrate the efficacy of the oral DNA vaccines, indicating future commercialization of the vaccine for the grouper industry.
|Appears in Collections:||[水產養殖學系] 博碩士論文|
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