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研究生:陳夏津
研究生(外文):HsiaChinChen
論文名稱:利用重組加州苜蓿夜蛾核多角體病毒在擬尺蠖幼蟲表現傳染性華氏囊病病毒結構蛋白rVP2H之研究
論文名稱(外文):Expression of Chimeric Virus-Like Particles, rVP2H, Derived from Infectious Bursal Disease Virus Structural Protein, in Trichoplusia ni Larvae Infectedwith Recombinant Autographa California MNPV
指導教授:王敏盈
指導教授(外文):MingYinWang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:71
中文關鍵詞:傳染性華氏囊病蛋白似病毒粒子加州苜蓿夜蛾核多角體病毒傳染性華氏囊病病毒固定化金屬離子親和性色層分析法擬尺蠖
外文關鍵詞:infectious bursal diseasevirus-like particlesAutographa California multiple nucleopolyhedrovirusinfectious bursal disease virusImmobilized metal-ion affinity chromatographyTrichoplusia ni
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傳染性華氏囊病(infectious bursal disease;簡稱IBD)為雞隻的高度傳染性疾病,此病會導致其免疫力嚴重下降,造成高度死亡率,對養雞業而言為一重大損失。本研究的目的為針對蛋白似病毒粒子(virus-like particles;簡稱VLPs)疫苗,發展一有效且經濟的生產方式。首先利用加州苜蓿夜蛾核多角體病毒(Autographa California multiple nucleopolyhedrovirus;簡稱AcMNPV)為表現載體,在其核多角體蛋白啟動子下游構築一C端具六個組氨酸(histidine)的VP2蛋白遺傳密碼,在蟲體中表現傳染性華氏囊病病毒(infectious bursal disease virus;簡稱IBDV)結構蛋白。目前,以三齡末擬尺蠖(Trichoplusia ni)幼蟲感染重組AcMNPV。結果顯示在大於30%蟲體死亡率的時候,即感染後96小時後,可以西方墨點法及抗體三明治酵素連結免疫吸附測定法(antibody-sandwich enzyme-linked immunoabsorbent assay;簡稱Antibody-Sandwich ELISA)偵測到明顯的rVP2H外源蛋白,且rVP2H蛋白的表現量會隨蟲體累加死亡率的增加而提高,但死亡率若大於50%,約感染後120-144小時,rVP2H蛋白會開始產生降解現象。所以在蟲體大量死亡之初,即感染後96至120小時,應立即收蟲,可得到較高且完整的rVP2H蛋白產量。另外,當病毒效價愈高,蟲體在感染過程中,愈能達到一定的死亡率,進而提高rVP2H蛋白產量。以目前所構築的病毒載體而言,當病毒效價大於109 pfu/ml時,在感染後96-120小時可達到最高產量。此條件下,每批次蟲的rVP2H蛋白產量在0.1-0.8 mg rVP2H蛋白/每克蟲。為有效的從蟲體中純化出rVP2H蛋白,我們找到兩種較可行的純化方法。一為先經50%硫酸銨沉澱分離rVP2H蛋白,透析去除蟲體色素及鹽類,再以固定化金屬離子親和性色層分析法(Immobilized metal-ion affinity chromatography;簡稱IMAC)純化病毒結構蛋白。其純化結果可得到99%的純度與40%的回收率。另一經透析去除蟲體色素後,再以IMAC純化,可得到90%的純度與55%的回收率。且以這兩種純化方式純化rVP2H蛋白,均可得到直徑為20 nm的VLPs。以間接酵素連結免疫吸附測定法(indirect enzyme-linked immunoabsorbent assay;簡稱Indirect ELISA)比較vvIBDV與經IMAC純化的蟲體中rVP2H蛋白,其自然結構的差異性。由於兩者吸光值均有明顯的變化,表示抗原性相似。

Infectious bursal disease (IBD) is a highly contagious disease in chickens. It results in a severe immunosuppression and causes a high ratio of mortality in chickens. The disease causes heavy financial losses for the poultry industry. The purpose of this research is to develop an efficient and inexpensive method for the expression of the virus-like particles (VLPs) vaccine. We used Autographa California multiple nucleopolyhedrovirus (AcMNPV) as expression vector, and constructed a VP2 protein of infectious bursal disease virus (IBDV) with additional six histidine residues at its C-terminus under polyhedrin promoter. We infected the late 3rd instar Trichoplusia ni larvae with recombinant AcMNPV. Results revealed when mortality was more than 30% (ie 96 hours postinfection), we could use Western blotting analysis and antibody-sandwich enzyme-linked immunoabsorbent assay (Antibody-Sandwich ELISA) detecting significant rVP2H protein. The expression of rVP2H protein would be higher when larval cumulative mortality was higher. But when mortality was more than 50% (ie 120 to 144 hours postinfection), rVP2H protein started degrading. The optimal harvest time was 96 to 120 hours postinfection. When virus titer was higher, the mortality could reach enough level and enhanced rVP2H protein expression. For the construct virus vector in our laboratory, when virus titer was more than 109 pfu/ml, rVP2H protein could reach the highest quantity during 96 to 120 hours postinfection. Quantity of rVP2H protein in each batch larva was 0.1 to 0.8 mg rVP2H protein/g larvae. To purify rVP2H protein from larvae efficiently, we found two fine purification processes. One was separated by 50% ammonium sulfate precipitation, followed purifying with immobilized metal-ion (Ni+2) affinity chromatography (IMAC) after dialysis. We could get 99% purity and 40% efficiency. The other was purified by IMAC after dialysis. We could get 90% purity and 55% efficiency. The two methods both could form VLPs with size of 20 nm diameter. Comparing with native vvIBDV and VLPs structure, both could be well recognized by monoclonal mouse anti-rVP2H antibody with indirect enzyme-linked immunoabsorbent assay (Indirect ELISA). It suggested both conformation of the epitope was the same.

第一章 緒言 ------------------------------------------------------- 1
第二章 研究目的、背景及文獻探討 ------------------------------ 4
第一節 傳染性華氏囊病的歷史、流行病學、宿主及症狀----- 4
第二節 傳染性華氏囊病毒的分類與形態和物理化學特性----- 5
第三節 傳染性華氏囊病毒的增殖和致病機轉------------------ 6
第四節 傳染性華氏囊病毒的核酸結構及蛋白特性-------------7
第五節 傳染性華氏囊病的診斷及疫苗開發--------------------9
第六節 桿狀病毒表現系統------------------------------------11
第七節 以幼蟲為表現宿主------------------------------------14
第八節 實驗目的 ---------------------------------------------18
第三章 材料與方法 -----------------------------------------------20
第四章 結果與討論 -----------------------------------------------28
第五章 結論與未來展望 ------------------------------------------51
參考文獻 -----------------------------------------------------------52

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