臺灣博碩士論文加值系統

白點症病毒 (White Spot Syndrome Virus, WSSV) 是甲殼類生物一個重要的病毒，對養殖蝦有很高的感染率與致死率。自從1993年於中國大陸爆發傳染以來，WSSV已成為世界養蝦產業的重大病源。目前對此疾病尚無藥物可以治療，因此養殖業將此病稱為「蝦癌」。凝集素 (lectin)，由於能和細胞表面的醣基結合造成凝集反應而得名。此外凝集素也可以辨識與許多的病源體的表面例如病毒外套膜上的醣基結合而與病源產生凝集反應防止病毒的感染，因此具有抗病的功能。所以凝集素是生物中先天免疫力 (innate immunity) 很重要的一部分。本研究即是以柳松菇凝集素 (Agrocybe aegerita lectin, AAL) 為對象，分析其抗WSSV感染的效用。首先選殖AAL基因cDNA全長序列，並以原核 (Escherichia coil) 的蛋白質表現系統，生產重組柳松菇凝集素 (rAAL)，再分析該重組蛋白對抗WSSV之活性。經由軟體分析AAL胺基酸序列其具有carbohydrate recognition domain (CRD) 及蛋白後修飾 (modifications motif) 辯識區域。此外，1997年Cooper等學者指出，凝集素胺基酸序列中若含有cysteine，則會降低凝集素之凝集活性。而AAL不具有cystine，推測其可能具有較佳之凝集活性。rAAL之分子量約為20 kDa，與軟體所預測之分子量大小相當。而以native gel的電泳分析顯示rAAL會形成具功能之雙聚合體 (dimer) 四級結構。利用E-coil大量表現並純化後的rAAL作為抗原生產抗體，以西方轉漬法分析，柳松菇之蛋白質萃取物，此抗體可高專一性的辨識出天然柳松菇凝集素。E-coli生產rAAL，以白蝦進行抗WSSV之活體實驗，結果發現以rAAL處理第五天,可降低20％累積死亡率,具有抑制白點症病毒之功效，值得進ㄧ步研究及應用，包含施用方式時機，劑量，頻率等。

White spot syndrome virus (WSSV) is a highly pathogenic and prevalent virus affecting crustacean. Disease caused by WSSV is the greatest challenge to word wide shrimp aquaculture. In this stuffy, full length cDNA of Agrocybe aegerita lectin (AAL) gene was cloned, recombinant AAL (rAAL) was expressed by prokaryotic (Escherichia coli) expression system and the activities of this recombinant protein including the anti WSSV effects were analyzed. Amino acids analysis showed that AAL with a carbohydrate recognition domain (CRD) and protein modification motif. AAL might be with strong agglutination activities for lacking cystine in its amino acids composition. Native gel electrophoresis demonstrated the rAAL with the ability for self-forming a dimmer structure. In agglutination test, rAAL agglutinated rabbit Red Blood Cell (RBC) at very low concentration (0.25ng/ml). The anti-WSSV assays performed on Litopenaeus vannamei showed an obvious effect. The rAAL treated shrimp groups were with greater than 20％ survival rate than the untreated ones in five days.

目錄

封面內頁
簽名頁
授權書　　　　　　　　　　　　　　　　　　　　　 　 iii
中文摘要　　　　　　　　　　　　　　　　　　　　 　 v
英文摘要　　　　　　　　　　　　　　　　　　　 　　 vi
誌謝　　　　　　　　　　　　　　　　　　　　　　 　 vii
目錄　　　　　　　　　　　　　　　　　　　　　 　　 viii
圖目錄　　　　　　　　　　　　　　　　　　　 　　　 xii

1.前言　　　　　　　　 　 1
1.1台灣養殖蝦之概況　　　　　　　　　　　　　 　 1
1.2白蝦之分類及特徵　　　　　　　　　　　　　 　 3
1.3蝦病原之簡介　　　　　　　　　　　　　　 　 3
1.4蝦之白點症　　　　　　　　　　　　　　 　 5
1.4.1蝦白點症之歷史背景　　　　　　　　　　　 5
1.4.2蝦白點症病毒之特性　　　　　　　　　　　 7
1.4.3蝦白點症病毒感染之宿主及地域分布　　　　 9
1.4.4蝦白點症病之傳撥途徑　　　　　　　　　　 10
1.4.5蝦白點症病之臨床症狀及病徵　　　　　　 10
1.4.6蝦白點症病之診斷及治療　　　　　　　　　 11
1.5蕈類之功能　　　　　　　　　　　　　　 　 12
1.6柳松菇　　　　　　　　　　　　　　 　 13
1.7凝集素　　　　　　　　　　　　　　 　 15
1.7.1凝集素之生理功能　　　　　　　　　　　　　 17
1.7.2凝集素之作用機制　　　　　　　　　　　　　 18
1.7.3醣蛋白之生合成及生理意義　　　　　　　 　 19
1.7.4凝集素之消化吸收　　　　　　　　　　　　　 20
1.7.5凝集素對細胞之胞飲作用　　　　　　　　　　 21
1.7.6凝集素對細胞凋亡之引響　　　　　　　　　　 22
1.7.7凝集素的功能及其運用　　　　　　　　　　　 25
2.材料與方法　　　　　　　　 　 28
2.1實驗材料　　　　　　　　　　　　　　 28
2.1.1柳松菇子實體　　　　　　　　　　　　　　 28
2.1.2實驗動物　　　　　　　　　　　　　　 28
2.1.3實驗養殖系統　　　　　　　　　　　　　　 28
2.2實驗流程　　　　　　　　　　　　　　 28
2.3實驗方法　　　　　　　　　　　　　　 29
2.3.1柳松菇凝集素譯讀區之選殖　　　　　　 　　 29
2.3.1.1柳松菇RNA 之萃取　　　　　　　　　 29
2.3.1.2柳松菇cDNA製備　　　　　　　　　 30
2.3.1.3柳松菇凝集素譯讀區序列擴增反應　　 31
2.3.1.4載體pGEN-T easy之構築　 　　　　 31
2.3.1.5柳松菇凝集素蛋白質特性之分析　 　 32
2.3.2柳松菇凝集素重組蛋白質之表現 　 32
2.3.2.1表現質體pET-28b之構築　　　 32
2.3.2.2以E-coil表現柳松菇凝集素重組蛋白　 32
2.3.2.3少量細胞先驅測試　　　　　　 33
2.3.2.4重組柳松菇凝集素水溶性測試　　　 　 34
2.3.2.5重組柳松菇凝集素表現量時序分析　　 34
2.3.3抗體的製備　　　　　　　　　　　　　　 34
2.3.4柳松菇凝集素蛋白質大量表現　　　　　　 　 34
2.3.5柳松菇凝集素重組蛋白質之純化　　　 35
2.3.6 rAAL蛋白質特性分析　　　　　　　　　　 　 36
2.3.6.1天然AAL分子量分析　　　　　　 　 36
2.3.6.2 Native gel 之分析　　　　　　　 36
2.3.6.3重組柳松菇凝集素之血球凝集活性分析 37
2.3.6.3.1 2％兔子紅血球懸浮液製備 　 37
2.3.6.3.2最小凝集活性測定　　　　 　 37
2.3.6.4重組松菇凝集素抗蝦白點症病毒活性分析 37
2.3.6.4.1重組松菇凝集素對白蝦毒性分析　 38
2.3.6.4.2白點症病毒液之製備 38
2.3.6.4.3白點症病毒液濃度測試　 　 38
2.3.6.4.4抗病毒試驗　　　　　　　　 39
3.結果與討論　　　　　　　　 　 40
3.1柳松菇凝集素譯讀區基因之選殖　　　　　　　　　　 40
3.1.1柳松菇凝集素譯讀區基因之分析　　　　　　　 40
3.1.2柳松菇凝集素胺基酸組成分析集蛋白質特性預測 40
3.1.3柳松菇凝集素糖類鍵結位置　　　　 41
3.2柳松菇凝集素重組蛋白之大量表現及純化 　 41
3.2.1重組柳松菇凝集素現量、水溶性時間點及測試 41
3.2.2柳松菇凝集素重組蛋白之大量表現及純化 42
3.3重組柳松菇凝集素之特性分析　　　　　　　　 　 42
3.3.1柳松菇凝集素重組蛋白與天然柳松菇凝集素之比 42
3.3.2柳松菇凝集素重組蛋白結合次單元之分析　 43
3.3.3重組柳松菇凝集素之血球凝集活性分析 　 43
3.3.4 白點症病毒濃度與累積死亡率測試分析 　 43
3.3.5重組柳松菇凝集素生物毒性之分析 　 44
3.3.6重組柳松菇凝集素抗病毒效果之分析 　　 　 44
4.結論　　　　　　　　 　 46
參考文獻 　 65

圖目錄

圖1. Alignment of Agrocybe aegerita lectin gene　　　　 　 47
圖2. Alignment of Agrocybe aegerita lectin　　　　　 　 48
圖3. Agrocybe aegerita lectin Amino acids composition　　　 49
圖4. Galectin carbohydrate binding motif 　 50
圖5. Alignment of Agrocybe aegerita lectin gene in pGEM-0612
easy and pET-AAL-27　　　　　　　　　　　 　 51
圖6. Determination of target protein solubility 52
圖7. Growth of standard E.coil expression cultures 53
圖8. Time-course analysis of protein expression 　 54
圖9. E. coil culture growth for preparative purification　　　 55
圖10. Western blot analysis rAAL expression (Anti-his) 56
圖11. Western blot analysis rAAL expression (Anti-rAAL) 57
圖12. Western Blot analysis native Agrocybe aegerita protein 58
圖13. Native gel analysis rAAL　 　 59
圖14. Haemagglutination activity of rAAL toward rabbit RBC 60
圖15. WSSV濃度與累積死亡率測試　 　 61
圖16. rAAL生物毒性測試　 　 62
圖17. rAAL抗WSSV效力及累積死亡率之分析　 63
圖18. BSA抗WSSV效力及累積死亡率之分析　 64

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