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研究生:沈炳成
研究生(外文):Pign-chen Shen
論文名稱:傳染性華氏囊病毒VP2似病毒粒子免疫效能之探討
論文名稱(外文):Evaluation of the Immunogenicity of rVP2H Particles formed by Infectious Bursal Disease Virus Structural Protein VP2
指導教授:王敏盈
指導教授(外文):Min-ying Wang
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
中文關鍵詞:似病毒粒子擬尺蠖
外文關鍵詞:VLPTrichoplusia ni
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傳染性華氏囊病毒是造成傳染性華氏囊病變的病原,該病毒對幼雞具高度感染力。傳染性華氏囊病毒之結構蛋白VP2是誘發宿主免疫反應的主要抗原,本研究以重組昆蟲桿狀病毒感染擬尺蠖藉以生產重組蛋白rVP2H。
經擬尺蠖生產之rVP2H可自行組裝成似病毒粒子,本研究嘗試將rVP2H以口服及注射方式免疫無特定病原雞隻,評估應用rVP2H於次單位疫苗研發的可行性。雞隻保護試驗共分為3組,首先以20 μg由擬尺蠖生產的似病毒粒子抗原混合弗氏完全佐劑,經肌肉注射免疫雞隻,試驗証實注射蟲產似病毒粒子的雞隻能誘發高度中和抗體,並能完全對抗一個LD50強毒型傳染性華氏囊病毒的感染。
為了減低疫苗成本,第2組試驗分別以2 μg及0.2 μg低劑量的rVP2H混合弗氏完全佐劑,以肌內注射方式免疫無特定病原雞隻,免疫後兩週以10000 LD50強毒型傳染性華氏囊病毒進行保護力測定,結果証實雞隻接種0.2 μg rVP2H能完全抵抗病毒的感染,死亡率為零,以西方墨點法及瓊脂凝膠沈澱法檢測華氏囊,皆無病毒殘留。此外,由於利用固定化金屬離子親和性分析法純化rVP2H需額外吸收親和性樹脂成本,所以本研究亦使用硫酸沈澱方式直接鹽析擬尺蠖蟲液中的rVP2H,取2 μg的注射劑量混合弗氏完全佐劑以肌肉注射方式免疫無特定病原雞隻,免疫2週再以10000 LD50強毒型傳染性華氏囊病毒進行保護力測定,結果免疫雞隻死亡率為零,華氏囊中無偵測得殘留病毒,研究確定免疫2 μg硫酸銨鹽析之rVP2H即能完全保護雞隻。
第三組試驗著手於傳染性華氏囊病毒口服性次單位疫苗的研發,試驗中以等量未經純化及經固定化金屬離子親和性分析法純化的rVP2H,以餵食方式免疫無特病原雞隻,經保護力測定証實免疫純化rVP2H的雞隻具75 % 的保護力,免疫未純化rVP2H的雞隻保護力為25 ~ 50 %。
本研究中單一擬尺蠖約可生產5 mg rVP2H,經硫酸銨沈澱約可獲得4 mg rVP2H,該產量可提供2000隻雞隻注射免疫所需;若以固定化金屬離子親和性分析法純化,單蟲約可生產800 μg rVP2H,以單雞最低有效劑量0.2 μg估算,該產量可供給4000隻雞隻注射免所需。
The objective of the present study was to investigate the feasibility of rVP2H proteins produced by insect larvae to protect chickens against infectious bursal disease virus infection. Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious disease in young chickens.
The recombinant protein rVP2H has an extra six-histidine residue at the C- terminus of the VP2, a structural protein of IBDV and the primary host-immunogen containing the antigenic regions responsible for elicitation for neutralizing antibodies. The rVP2H self-assemble to form virus—like particles (VLPs) when expressed in Trichoplusia ni larvae which could be purified by either ammonium sulfate precipitation or IMAC (immobilized metal-ion affinity chromatography).
The protective efficacy of the purified rVP2H particles was evaluated using a chicken protection assay. VLPs administered intramuscularly with CFA to SPF (specific-pathogen-free) chickens induced higher neutralizing antibodies than commercial vaccine. At dpi 28, injecting VLP vaccine about 4-folds high than the VN titer induced by injecting commercial vaccine. All chickens injecting VLPs were protected from challenge; no virus in bursal was shed by AGP and Western blotting after challenge. Otherwise, the dosage of 20 μg、2 μg、0.2 μg rVP2H injected to SPF chickens provide the protection against the 10000-folds of LD50 IBDV challenge. No virus in bursal was detected by AGP and Western blotting, too.
The 2 μg rVP2H product from larvae treatment by 50 % ammonium sulfate precipitation absence IMAC purification be the subunit vaccine test. The SPF chickens injected the kinds of subunit vaccine induced the protection against 10000-folds of LD50 very virulent IBDV challenge. No virus in bursal was shed by AGP and Western blotting. Additionally, the recombinant protein rVP2H administered orally induced protection from LD50 very virulent IBDV challenge in 25 ~ 75 % in chickens.
Under the condition test, the low dosage of 0.2 μg purified rVP2H are immunogenic when administered intramuscularly to SPF chicken addition of CFA. The efficacy dosage of cheaper subunit vaccine prepared by ammonium sulfate precipitation is 2 μg.
In conclusion, the expression level of rVP2H is 5 mg per Trichoplusia ni. Following ammonium sulfate precipitation, approximately 4 mg / larvae was obtained. Thus, the amount of rVP2H (4mg) produced by one larvae was able to immunize 2000 chickens and protected them from infection of a strain of very virulent IBDV. On other hand, 800μg / larvae of rVP2H protein was obtained following the purification using IMAC, which was able to immunize 4000 chickens and protect them from infection of a strain of very virulent IBDV.
目錄
中文摘要………………………………………………………………………………1
英文摘要………………………………………………………………………………3
傳染性華氏囊病毒簡介…………………………………………..…………………..5
第一章 傳染性華氏囊病毒疫苗發展概說…………………………………………..7
第二章 擬尺蠖生產rVP2H…………………………………………………………14
壹、 前言………………………………………………………………………..14
貳、 材料與方法………………………………………………………………..17
一、 重組昆蟲桿狀病毒…………………………………………………..17
二、 擬尺蠖來源和感染…………………………………………………..17
三、 抗體的取得…………………………………………………………..18
四、 擬尺蠖體液的獲得………………………...…………………..…….18
五、 rVP2H的純化………………………………………………………..18
六、 SDS-PAGE…………………………………...………………………19
七、 銀染…………………………………………………………………..19
八、 西方點墨法…………………………………………………………..19
九、 酵素連結免疫吸附測定定量 rVP2H……………………………….20
十、 穿透式電子顥微鏡觀察法…………………………………………..21
十一、氯化銫梯度離心純化似病毒粒子………………………………..21
參、 結果………………………………………………………………………..22
一、 擬尺蠖的感染………………………………………………………..22
二、 蛋白表現量和感染時間之關係……………………………………..22
三、 大量生產rVP2H……………………………………………………..23
四、 以硫酸銨沈澱方式純化rVP2H……………………………………..24
五、 蟲體液黑化問題的克服………………...…………………………...24
六、 定量表現蛋白rVP2H………………………………………………..24
七、 穿透式電子顯微鏡觀察似病毒粒子………………...……………...25
八、 似病毒粒子的密度分析……………………………………………..25
肆、 討論………………………………………………………………………..26
一、 擬尺蠖體液黑化問題之探討………………………………………..26
二、 擬尺蠖生產rVP2H方式之探討…………………………………….27
三、 擬尺蠖生產rVP2H產能及成本之探討…………………………….29
第三章 疫苗試驗……………………………………………………………………31
壹、 前言………………………………………………………………………..31
貳、 材料與方法………………………………………………………………..33
一、 無特定病原雞隻及雞蛋的獲得……………………………………..33
二、 傳染性華氏囊病毒來源……………………………………………..33
三、 LD50攻毒劑量的測定……………………………………………….33
四、 雞隻免疫……………………………………………………………..34
五、 免疫雞隻血清之取得………..……………………………..………..35
六、 保護力測定…………………………………………………………..35
七、 華氏囊之均質化……………………………………………………..36
八、 AC-ELISA……………………………………………………………36
九、 雞胚胎細胞之製備…………………………………………………..38
十、 中和抗體試驗………………………………………………………..39
十一、瓊脂凝膠沈澱法檢測雞隻血清中anti-IBDV抗體………….……39
十二、瓊脂凝膠沈澱法檢測華氏囊中傳染性華氏囊病毒殘留情況…...40
十三、W/B assay檢測華氏囊中傳染性華氏囊病毒………………….…41
十四、最小顯著差異測驗法……………………………….……….…….42
參、 結果………………………………………………………………………..43
試驗第一組 擬尺蠖生產之rVP2H似病毒粒子免疫效能之探討………43
一、 檢測 rVP2H —IgG力價……………..…………………….…………43
二、 中和抗體試驗………………………………………………………..44
三、 保護力測定…………………………………………………………..44
a. 雞隻存活率及B/B ratios……………………………………………44
b. 檢測華氏囊中傳染性華氏囊病毒………………………………….45
試驗第二組 rVP2H 最低有效注射劑量評估………………………….46
一、檢測 rVP2H-IgG力價……………….…………….…………………46
二、以瓊脂凝膠沈澱法測定anti-rVP2H 抗體………………………….46
三、保護力測定…………………………………………………………..47
a. 雞隻存活率及B/B ratios……………………………………………47
b. 檢測華氏囊中傳染性華氏囊病毒………………………………….47
試驗第三組 口服免疫試驗………………………………………………48
保護力測定………………………………………………………………..48
a. 雞隻存活率及B/B ratios……………………………………………48
b. 檢測華氏囊中傳染性華氏囊病毒………………………………….48
肆、 討論………………………………………………………………………..50
一、 注射似病毒粒子免疫效能探討……………………………………..50
二、 施打未純化rVP2H及最小有效劑量之免疫效能探討…………….51
三、 口服rVP2H免疫效能之探討……………………………………….53
未來展望……………………………………………………………………………..54 參考文獻…………………………………………………..…………………………55
圖表…………………………………………………………..……………………....62
附錄………………………………………………..…………………………………87
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