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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53550

Title: 開發石斑魚虹彩病毒主要外鞘蛋白之枯草桿菌表現系統並藉由活體探討其抗病毒活性
Development of expression system for major capsid protein of grouper iridovirus in Bacillus subtilis and in vivo trial for evaluation of antiviral activity
Authors: Lin, Po-Hung
林柏宏
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 石斑魚虹彩病毒;主要外鞘蛋白;枯草桿菌表現系統;重組蛋白;保護效果
Grouper iridovirus (GIV);major capsid protein;Bacillus subtilis expression system;recombinant protein;protective effect
Date: 2019
Issue Date: 2020-07-03T08:30:22Z
Abstract: 石斑魚為臺灣高經濟價值水產養殖魚種之一,然而石斑魚虹彩病毒 (Grouper iridovirus, GIV) 的感染對石斑魚養殖產業造成巨大威脅。GIV 是二十面體的雙股 DNA 病毒,其外殼主要外鞘蛋白 (major capsid protein, MCP) 是一個具潛力的抗原候選者。依據資料庫預測分析和 ELISA 的抗原抗體分析,本論文針對全長 MCP 以及兩片段 MCP a.a 82–180 和 MCP a.a 274–438,使用枯草桿菌 (Bacillus subtilis WB800N) 表現系統表現重組蛋白,並利用活體試驗來分析此三種重組蛋白的保護效果。首先選殖出 GIV 的全長 mcp 以及兩片段 mcp a.a 82–180 和 mcp a.a 274–438,接合到枯草桿菌的表現載體 pHT254,接著利用電穿孔法 (electroporation) 轉形至枯草桿菌,在 24℃ 以最終濃度 0.2 mM IPTG 誘導表現 16 小時。再經過蛋白質濃縮後得到重組蛋白,最後透過 SDS-PAGE 和西方墨點法的分析,確認成功建構了全長 mcp 以及兩片段 mcp a.a 82–180 和 mcp a.a 274–438 的三個枯草桿菌表現系統。接著將上述三種重組蛋白和所建構之三個枯草桿菌菌液,以點帶石斑魚 (體重為 4.0 ± 0.6 g 且體長為 4.5 ± 0.3 cm) 進行活體試驗。將三種重組蛋白先以兩種劑量 1 μg/fish (g) 和 2 μg/fish (g) 腹腔注射14 天後進行混料投餵 7 天作為追加免疫處理石斑魚;菌液則以混料投餵 21 天,免疫 21 天後進行 GIV 腹腔注射攻毒試驗。結果顯示,2 μg/fish (g) 組有較佳的保護效果,MCP a.a 82–180 組的相對活存率 (relative percent survival, RPS) 為 47%,全長 MCP 和 MCP a.a 274–438 分別為37% 和 27%,菌液投餵組以及 1 μg/fish (g) 組的相對活存率皆低於 10%。以上述結果顯示,利用枯草桿菌表現系統開發水產病毒重組蛋白疫苗是可行的對策,但如何提升保護效果是未來重要的工作。
Grouper is one of the high economic value aquaculture species in Taiwan. However, the grouper iridovirus (GIV) causes the deleterious effect in grouper aquaculture. GIV is an icosahedral double-stranded DNA virus and the major capsid protein (MCP) composing the GIV capsid is a potential antigen candidate. Based on the database predictive analysis and antigen-antibody analysis of ELISA, the full-length MCP and two fragments MCP a.a 82–180 and MCP a.a 274–438 were used and utilize Bacillus subtilis WB800N expression system to express recombinant proteins, and the in vivo trial was used to analyze the protective effect of three recombinant proteins. The mcp, mcp a.a 82–180 and mcp a.a 274–438 genes from GIV were subcloned into B. subtilis WB800N expression vector pHT254 to express (final concentration of 0.2 mM IPTG, 24°C, 16 hrs) and concentrate recombinant proteins in B. subtilis WB800N by using electroporation. Through the analysis of SDS-PAGE and western blot, it was confirmed that three B. subtilis WB800N expression systems were successfully constructed. The in vivo trial was used to analyze three recombinant proteins and three strains of B. subtilis WB800N bacterial fluid by using orange-spotted grouper (body weight is 4.0 ± 0.6 g and body length is 4.5 ± 0.3 cm). The three recombinant proteins were intraperitoneally injected in two doses 1 μg/fish (g) and 2 μg/fish (g), after 14 days, the booster immunization by feeding mixing feeds for 7 days; the B. subtilis WB800N bacterial fluid was fed with mixing feeds for 21 days. After 21 days, GIV intraperitoneal challenge trial was performed. The results suggest that the 2 μg/fish (g) groups have better protective effect. The relative percent survival of the MCP a.a 82–180 group is 47%, and the full-length MCP and MCP a.a 274–438 are 37% and 27%. The relative percent survival of B. subtilis WB800N bacterial fluid feeding groups and the 1 μg/fish (g) groups are all less than 10%. The above results suggest that the development of aquatic virus recombinant protein vaccine by using B. subtilis WB800N expression system is a feasible strategy, but how to improve the protective effect is an important mission in the future.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010533021.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53550
Appears in Collections:[水產養殖學系] 博碩士論文

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