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研究生:李志旗
研究生(外文):Lei Chi Kei
論文名稱:傳染性華氏囊病基因疫苗的研發
論文名稱(外文):Development of DNA vaccine for infectious bursal disease
指導教授:吳昭良
指導教授(外文):C. L. Wu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:46
中文關鍵詞:傳染性華氏囊基因疫苗傳染性華氏囊病毒
外文關鍵詞:infectious bursal diseaseDNA vaccineIBDIBDVchickensVP2
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中文摘要
傳染性華氏囊病在雞群中是很嚴重的疾病,它是由傳染性華氏囊病毒引起。當雞群被病毒感染後,華氏囊中未成熟的B淋巴細胞會產生淍亡的現象,使產生免疫抑制。接種疫苗是目前主要的預防方法。 在過去的研究中利用VP2的重組蛋白能有效的使雞產生中和抗体,所以在本研究中嘗試利用含VP2基因的質体當作基因疫苗,使雞對傳染性華氏囊病毒產生保護的效果。把VP2基因接到質体pTCY, 重組後得到pTCYVP2,VP2便可以透過pTCY上的啟動子而表現。由於在接種pTCYVP2基因疫苗後產生中和抗体的效價不足,所以,當病毒感染雞群時沒有產生保護的效果。或許本實驗的策略要作出修正以達到保護的目的。此外,在實驗的過程中發現兩種有趣的現象。第一,是利用細胞培養的方法証明質体可以表現VP2基因,於是利用螢光蛋白EGFP和VP2基因作融合, 當有螢光表現時就可得知VP2基因有被表現。所以利用兩個質体pTCY-VP2-EGFP-fl 和 pTCY-VP2-EGFP-hl。當觀察螢光的時候 pTCY-VP2-EGFP-hl只在細胞質有螢光,而pTCY-VP2-EGFP-fl在細胞質和核中同樣有螢光的產生,原因目前還不確定。第二,利用大腸桿菌產生VP2-EGFP蛋白時,意外地發現大腸桿菌可以直接利用EGFP基因 5端的 ribosomal binding site和下游的GTG作為始點做出有功能的螢光蛋白。可能這序列影響VP2-EGFP的轉譯而導致VP2-EGFP不能被表現。

Abstract
Infectious bursal disease (IBD) is a serious disease in chickens. The pathogen of the disease is infectious bursal disease virus (IBDV). When chickens are infected by IBDV, immature B-lymphocytes in the bursa of Fabricius undergo apoptosis, which result in immunosuppression. The prevention of IBD is vaccination. In this study, a DNA vaccine based on the VP2 protein was used to immunize chickens against IBD. The viral protein VP2 can produce neutralizing antibody. The VP2 cDNA was subcloned into pTCY, an eukaryotic expression vector, to generate pTCY-VP2 driven by the rat beta-actin promoter as a vector for vaccination. Chickens immunized with VP2 DNA vaccine induced anti-IBDV antibodies, including low-titer neutralizing antibody. However, these chickens died when challenged with virulent IBDV, indicating that the immunization scheme should be modified. In the results from the experiments involving the expression of VP2-EGFP in chicken embryo fibroblasts or in E. coli, we found two interesting phenomena. First there was different localization of EGFP in cells when N-terminally fused with different fragments of VP2. The green fluorescence was localized exclusively in the cytoplasm in cells transfected with pTCY-VP2-EGFP-hl, whereas the fluorescence distributed in both the cytoplasm and nucleus in cells transfected with pTCY-VP2-EGFP-fl or EGFP. The reason for discrepancy in the localization of different EGFP fusion protein is unclear. Secondly, we found that there was a ribosomal binding site at the 5’ end of the EGFP gene and a GTG codon which many serve as a translational intiation site. These sequences may interfere the translation of the VP2-EGFP fusion protein, which resulted in the difficulty in producing VP2-EGFP fusion protein.

Contents
Abstract………………………………………………………..I
Chinese abstract……………………………………………….II
Acknowledgement……………………………………………III
Contents………………………………………………………IV
Figure contents………………………………………………...V
Table content………………………………………………….VI
Abbreviation………………………………………………….VII
Introduction…………………………………………………….1
Materials………………………………………………………..9
Results…………………………………………………………14
Discussion……………………………………………………..18
Conclusion…………………………………………………….20
Reference……………………………………………………...21
Figure & Table………………………………………………...27
Autobiography………………………………………………...46

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