臺灣博碩士論文加值系統

蝦類養殖產業是水產養殖中重要的一部分，每年有高達220億美金的產值。但養蝦產業卻常受到疾病侵擾，導致大量的損失。蝦的白點病(white spot disease, WSD)尤為嚴重，WSD典型病徵是在病蝦的頭胸部、附肢及體表出現白色斑點，且在病徵出現後短短10天內，死亡率高達90-100%。WSD是由白點病毒(white spot syndrome virus, WSSV)所導致。超過40個WSSV病毒蛋白已經被了解，包含結構和非結構蛋白。其中，VP28是WSSV主要的包膜蛋白，被認為在病毒感染細胞的過程中，扮演重要的角色。
WSD發現20多餘年，一直沒有有效的解決辦法。美國Transcending Biotechnologies Inc. 研發了單株抗體AP-1，能夠和WSSV的VP28緊密結合，阻擋病毒感染細胞的過程，進而防止蝦類受到WSSV的感染。但單株抗體利用動物細胞生產的成本太高，不適合大規模應用在養殖現場。先前冠群生技公司利用AP-1的變異片段(variable fragment)製成了單鏈抗體sAP-1，完整保留了AP-1的抗原結合位，成功在大腸桿菌原核表現系統中大量表現。但在先前的研究中發現了sAP-1的抗病毒效果不如AP-1。合理懷疑，具有兩個抗原結合位的AP-1，比只有一個抗原結合位的sAP-1，能跟病毒結合的更牢固，因此具有更好的抗病毒效果。
因此，實驗嘗試在sAP-1蛋白後端接上亮胺酸拉鏈( leucine zipper )胺基酸序列，使sAP-1形成二聚體dsAP-1，進而提升抗病毒效果。以利後續應用微生物製造的dsAP-1在大規模養殖中，防止WSSV的感染。
在ELISA的結果中發現，dsAP-1跟sAP-1相比，具有較好的WSSV結合能力。而在攻毒實驗的結果中，dsAP-1組比起sAP-1組，具有較高的存活率。顯示sAP-1形成二聚體dsAP-1後，抗白點病毒的能力有些提升的趨勢。

Shrimp farming is an important part of aquaculture industry, with output value reaching 22 billion U.S. dollar annually. However, pathogens always devastate shrimp farming, causing enormous economic losses. White spot disease (WSD) is one of the most potent and widespread disease, with the typical syndrome white spots on the cuticle of the shrimp cephalothorax, carapace and appendage. WSD can cause up to 90-100% mortality within 10 days after symptoms appear. WSD is caused by white spot syndrome virus (WSSV). More than 40 viral proteins of WSSV were identified and studied, including structural and non-structural proteins. One of them, VP28, is a major envelope protein of WSSV, may play an important role in the infection process of WSD.
WSD has been found for more than 20 years, but there are still no available treatment measures. Transcending Biotechnologies Inc. (U.S.A.) developed a monoclonal antibody, AP-1. AP-1 can bind to VP28 tightly and interfere the interaction between WSSV and host cell, thus preventing shrimp from infection of WSSV. However, the production cost of monoclonal antibody is too high to be practically utilized for shrimp farming. The variable fragment of AP-1, which contains complete antigen-binding site, was synthesized as the single chain peptide, sAP-1. sAP-1 was successfully expressed in huge quantity by E. coli expression system. However, previous studies found that the anti-WSSV activity of monomeric sAP-1 is weaker than that of the dimeric AP-1. We speculate that AP-1 with two antigen-binding site may bind to VP28 more tightly than sAP-1 with only one antigen-binding site, thus AP-1 has stronger anti-WSSV ability.
For those reasons, this study aims to modify sAP-1 by adding leucine zipper after its C-terminus, making two sAP-1 monomers forming dimmer, dsAP-1. The anti-WSSV efficacy of ds-AP-1 is proposed to be improved. The dsAP-1 may be used at large scale shrimp farming to prevent WSSV infection.
The result of ELISA showed that the WSSV binding ability of dsAP-1 was slightly better than that of sAP-1. WSSV viral challenge also showed that the survival rate of dsAP-1 group was better than sAP-1 group at the same amount of protein. The dimerized sAP-1 has better anti-WSSV activity than sAP-1.

致謝 i
中文摘要 ii
ABSTRACT iii
CONTENTS v
LIST OF FIGURES vii
LIST OF TABLES viii
Chapter 1 Introduction 1
1.1 Shrimp farming 1
1.2 White Spot Syndrome Virus 2
1.3 Neutralization antibody & Single chain antibody fragment 4
1.4 Single chain antibody fragment dimerization 7
Chapter 2 Materials and methods 9
2.1 Experimental animals and WSSV 9
2.2 Expression and purification of proteins 9
2.3 Biochemical analysis of recombinant proteins 14
2.4 Analysis of neutralizing activity 15
2.5 WSSV viral challenge assay 16
Chapter 3 Results 18
3.1 Proteins expression 18
3.2 Biochemical analysis of recombinant proteins 19
3.3 Analysis of neutralizing activity 19
3.4 WSSV viral challenge assay 20
Chapter 4 Discussion 22
4.1 Proteins expression 22
4.2 Biochemical analysis of recombinant proteins 23
4.3 Analysis of neutralizing activity 24
4.4 WSSV viral challenge assay 24
Chapter 5 Conclusion 26
References 27
Figures 43
Tables 61

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