Application of Lactoferrin in Shrimp Enhancing Resistance of White Spot Syndrome Virus Infection
詹雲翔、張雲祥
E-mail: [email protected]
ABSTRACT
White spot syndrome virus (WSSV) is one of the most important pathogens of crustaceans with high lethality in culture shrimp. So far there is no shrimp species showing resistance to this virus. Lactoferrin (LF) is a kind of iron-carrying glycoprotein existing commonly in human and other animals. LF is a member of transferring family with the functions of physiological regulation, bacteria and virus replication inhibition, lymphocytes differentiation, macrophage regulation, granulocytes proliferation, and helping in iron ion transportation and uptaking in guts. In this study LF was tested the ability in enhancing the anti-wssv infection in shrimp.
Some immuno-response indexs such as phenoloxidase activity, superoxide anion production and superoxide dismutase activity, and immune related genes expression were analized to elucidate the possible anti-viral mechanisms. By using the different concentration of LF treated shrimp indicated a lower down of the accumulate lethality and the use of 0.04 mg/g shrimp body weight showed the most effective dosage which could lower down the lethality rate to 59 % . The indicated immuno-response indexs in 0.01 and 0.04 mg/g body weight LF treated shrimp showed significant differences than non-treated shrimp (P
Keywords : Penaeus monodon ; Lactoferrin ; WSSV
Table of Contents
目錄 封面內頁 簽名頁 授權書 中文摘要iv 英文摘要vi 誌謝viii 目錄ix 圖目錄xii 表目錄xiii 附錄xiv 第一章前 言... 1 第二章文獻回顧... 3 2.1草蝦之介紹及養殖現況... 3
2.2WSSV... 5 2.3甲殼類免疫防禦機制及血球種類... 7 2.3.1細胞型免疫... 8 2.3.1.1甲殼類血球細胞作用因子... 9 2.3.2體液型免疫... 12 2.3.3甲殼類血球種類...
15 2.4LF... 17 2.4.1LF之簡介... 17 2.4.2LF之結構... 17 2.4.3LF之 分布... 18 2.4.4LF之接受器... 18 2.4.5LF之isoform... 19 2.4.6LF之生物功 能... 19 2.4.7LF之應用... 21 2.4.8LF抗病毒機制及抗病毒相關之研究... 22 2.5研究目 的... 24 第三章材料與方法... 26 3.1實驗材料... 26 3.1.1實驗動 物... 26 3.1.2實驗養殖系統... 26 3.1.3BLF與牛血清蛋白... 26 3.2實驗方 法... 27 3.2.1BLF處理對增進草蝦及白蝦抗WSSV能力的影響.... 27 3.2.1.1BLF對草蝦及白蝦之毒性分 析... 27 3.2.1.2草蝦之WSSV人工感染... 28 3.2.1.3BLF於草蝦抗WSSV實驗... 28 3.2.1.4BLF於白 蝦抗WSSV實驗... 29 3.2.1.5草蝦DNA之萃取... 29 3.2.1.6 2-step WSSV diagnostic PCR之檢測...
30 3.2.2BLF處理草蝦之免疫指標分析... 31 3.2.2.1血球細胞PO活性之測定... 32 3.2.2.2血球細胞O2－活性 之測定... 32 3.2.2.3血球細胞SOD活性之測定... 33 3.2.2.4統計分析... 34 3.2.3BLF處理對 草蝦免疫相關基因表現之分析... 34 3.2.3.1草蝦RNA之萃取... 36 3.2.3.2RT-PCR... 36 第 四章結果與討論... 38 4.1BLF對草蝦抗WSSV能力的影響... 38 4.1.1BLF對草蝦及白蝦之毒性分 析... 38 4.1.2以不同濃度之WSSV病毒液注射草蝦之存活率... 38 4.1.3以不同濃度之BLF注射草蝦後，以WSSV 10-6 病毒液 感染草蝦之累積死亡率... 39 4.1.4以不同濃度之BSA注射草蝦後，以WSSV 10-6病毒液 感染草蝦之累積 死亡率... 40 4.1.5 2-step WSSV diagnostic PCR之檢測... 41 4.1.6以不同濃度之BLF注射白蝦後，以WSSV 10-6 病毒液 感染白蝦之累積死亡率... 41 4.2BLF處理對之草蝦免疫指標分析... 42 4.3BLF處理對草蝦免疫相 關基因表現之分析... 44 第五章結論... 46 參考文獻... 71
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