臺灣博碩士論文加值系統

蝦類白點症病毒 (White spot syndrome virus, WSSV) 在九0年代初期首次出現於東南亞一帶，由於它可感染的甲殼類宿主種類相當廣泛，且在感染後七到十天便可造成養殖對蝦近百分之百的死亡率，因此疫情擴迅速擴散至全球，所到之處均造成蝦類繁養殖場極大的的經濟損失，迄今仍是危害全球養蝦產業的重要疾病之一。蝦類白點症病毒目前已知具有約四十餘種的病毒蛋白，其中約有二十二種屬於膜蛋白，負責構成一般認為與病毒感染能力具有正相關的套膜，而其中的兩種套膜蛋白 (VP26 and VP28) 便佔了蝦類白點症病毒套膜蛋白總量約三分之二，VP28更在文獻中被指出是套膜上含量最為豐富的病毒蛋白。不僅如此，以多株抗體或單株抗體AP-1中和VP28後亦可降低蝦類感染及死亡率，因此被認為是在蝦類白點症病毒感染宿主的過程中不可或缺的病毒蛋白。由於先前研究提及VP28可結合蝦細胞表面，故找尋宿主細胞與病毒蛋白VP28結合之受體可釐清病毒感染的途徑。過去的研究曾以E. coli表現VP28進行草蝦血球細胞膜蛋白之結合試驗，希望找尋與其結合之受體蛋白，結論指出蝦Rab7蛋白可能扮演此角色，但此實驗之設計及結論可能有兩個問題。一、E. coli屬於原核生物，缺乏真核生物的轉譯後修飾，因此表現所得的VP28相較於分離自感染白點症病毒蝦隻的VP28推測有結構上的差異；二、文獻指出真菌、綠猴 (Vero) 腎臟細胞與人類子宮頸腫瘤細胞 (HeLa cell) 之Rab7蛋白不存在細胞膜面上，也不參與向細胞內運輸的液泡形成，而Rab7蛋白在物種間的保留性極高，因此出現於蝦體細胞膜與VP28結合之可能性不大。有鑑於此，本實驗將VP28基因同源重組到桿狀病毒表現系統 (Baculovirus expression system) 後感染昆蟲細胞株sf-9製備VP28蛋白，再以E. coli表現選殖自草蝦的Rab7蛋白，與VP28單株抗體AP-1進行競爭性酵素連結免疫吸附分析 (Compete enzyme-link immunosorbent assay, Compete ELISA)，結果發現Rab7與單株抗體AP-1對VP28之結合無競爭現象，而已知AP-1處理的草蝦在WSSV攻毒實驗中所顯示的存活率顯著高於未處理組別，由這兩項結果得知，Rab7無法與AP-1競爭結合VP28的抗原決定基位，因此推測Rab7並非位於蝦細胞最早與WSSV VP28接觸引發感染機制的蛋白，而Rab7在WSSV感染徑中之角色尚待進一步釐清。

White spot syndrome virus (WSSV) was first emerged in south Asia in the early 1990’s. It has a wide range of hosts among crustaceans and causes up to 100% mortality within 7 to 10 days in cultured shrimps. It was spread out all over the world rapidly. Until now, white spot syndrome has become one of the most serious viral diseases of cultivated shrimp and causes considerable economic losses to the shrimp farming industry worldwide. Currently, there are about 40 structural proteins of WSSV being identified, of which 22 are envelope proteins constructing the infection-related structure. VP28 and VP26 are the most abundant structural proteins observed in the viral envelope, accounting for approximately two-thirds of the envelope proteins. Furthermore, in neutralization experiments, anti-VP28 polyclonal or monoclonal antibody such as AP-1 can protect shrimp from WSSV infection and reduce mortality, therefore it has been considered to be the most important viral protein in WSSV infection. In order to find out the VP28 receptor on shrimp cell, E. coli expressed VP28 was used to run binding test in Penaeus monodon hemocyte in pervious study. The Rab7 protein was thought to directly bind VP28. However, there are some problems about this study. First, E. coli belongs to prokaryotes which lack posttranslational modification as in eukaryotes, hence there would have some structural differences between VP28 of WSSV and that expressed by E. coli. Second, in previous study, Rab7 does not involve in vesicle formation and inner transportation in yeast, vero cell and HeLa cell, and even does not exist on cell membrane. In addition Rab7 is highly conserved in function domain and gene sequence among species. Therefore, the probability is very low for Rab7 to bind VP28 on shrimp cell membrane. In this study, VP28 gene was homologous recombined to the baculovirus and infected insect cell line (sf-9) to express VP28 viral protein. The E. coli expressed Rab7 and anti-VP28 monoclonal antibody, AP-1, were used for compete enzyme-link immunosorbent assay (Compete ELISA). Results showed that Rab7 did not compete with AP-1 on binding VP28. The survival rate of AP-1 treated shrimps was significantly higher than those AP-1 untreated ones after WSSV challenge. Taken together, Rab7 may not directly involve in WSSV infection on the plasma membrane, and the role of Rab7 needs further studies.

中文摘要 ........................................... I
Abstract ........................................... III
目錄 ............................................... V

一、前言 ........................................... 1
1. 蝦類養殖概況 .................................... 1
2. 蝦類白點症病毒 .................................. 2
2.1 白點症病毒之病徵與危害 ......................... 3
2.2 白點症病毒之內外部型態特徵與分類 ............... 5
2.3 蝦類白點症病毒結構蛋白 ......................... 7
2.3.1 白點症病毒套膜蛋白VP28 ....................... 7
2.4 蝦類白點症的防治 ................................ 10
3. 蝦類白點症病毒受體蛋白 ........................... 13
4. Rab蛋白 .................................................. 14
5. 桿狀病毒蛋白表現系統 ............................. 15
6. 研究動機與目的 ................................... 17

二、材料與方法 ...................................... 18
1. 實驗材料 ......................................... 18
1.1 生物性材料 ...................................... 18
1.2 反應試劑 ........................................ 19
1.3 儀器與器材 ...................................... 25
2. 實驗方法 ......................................... 26
2.1 草蝦Rab7基因選殖與蛋白表現 ...................... 26
2.1.1 草蝦total RNA之萃取 ........................... 26
2.1.2 反轉錄酶-聚合酶連鎖反應 ....................... 26
2.1.3 RT-PCR產物膠體電泳分析萃取 .................... 27
2.1.4 構築載體 ...................................... 27
2.1.5 轉型作用 ...................................... 28
2.1.6 鹼裂解法抽取小量質體DNA與定序.................. 28
2.1.7 草蝦Rab7蛋白表現 .............................. 29
2.2 桿狀病毒表現系統製備蝦白點症病毒蛋白VP28......... 30
2.2.1 細胞培養與病毒蛋白表現 ........................ 30
2.3 聚丙醯硫胺膠體電泳 (SDS PAGE) 與西方墨點法進行蛋白質定性 ........ 30
2.3.1 聚丙醯硫胺膠體電泳之蛋白樣本製備 .............. 31
2.3.2 聚丙醯硫胺膠體電泳 ............................ 31
2.3.3 蛋白質轉印 .................................... 32
2.3.4 西方墨點法進行草蝦Rab7蛋白定性 ................ 32
2.3.5 西方墨點法進行白點症病毒蛋白VP28定性 .......... 32
2.4 競爭性酵素連結免疫吸附分析 (ELISA) .............. 33
2.4.1 Rab7蛋白與VP28單株抗體AP-1競爭性酵素連結免疫吸附分析 ....... 34
2.5 實驗流程 ........................................ 35

三、試驗結果 ........................................ 36
1. 草蝦Rab7基因選殖與蛋白表現 ....................... 36
2. 桿狀病毒表現系統製備白點症病毒蛋白................ 36
3. 西方墨點法進行蛋白質定性 ......................... 37
4. 競爭性酵素連結免疫吸附分析 ....................... 37
四、討論 ............................................ 38
五、總結............................................. 40
六、參考文獻 ........................................ 41
七、圖表 ............................................ 51

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