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標題: 大腸桿菌表現之VP243次單位疫苗對雞傳染性華氏囊病之免疫反應及保護效力之評估
Immune responses and protection efficacy of subunit vaccines containing E. coli expressed VP243 in chickens against infectious bursal disease
作者: 劉旭哲
Liu, Hsu-Che
關鍵字: 大腸桿菌
infectious bursal disease
subunit vaccine
出版社: 微生物暨公共衛生學研究所
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摘要: 雞傳染性華氏囊病 (Infectious Bursal Disease; IBD) 係由雞傳染性華氏囊病病毒 (Infectious Bursal Disease Virus; IBDV) 所引起之高度傳染性免疫抑制疾病。該疾病多好發於三至六週齡之年幼雞隻,目前台灣對於雞傳染性華氏囊病的控制方法普遍於母雞產蛋前利用接種不活化疫苗或活減毒疫誘導雛雞之移行抗體,再以疫苗接種來控制,然而,活減毒疫苗本身具有病毒毒力回復的問題且仍會造成華氏囊之輕微損傷。隨著科技的進步,次單位疫苗成為疫苗發展上一個重要的突破,不僅比起活減毒疫苗更安全,且在製備上更為容易。本次試驗之目的為評估以大腸桿菌表現之IBDV的VP243蛋白於雞隻體內對雞傳染性華氏囊病之免疫反應及保護效力。首先將構築好之重組質體pTri-VP243轉形至大腸桿菌Rosetta中進行蛋白表現,大腸桿菌經由IPTG誘導蛋白表現後可以得到可溶及不可溶之VP243蛋白,再以酵素連結免疫吸附試驗 (enzyme-linked immunosorbent assay; ELISA)及西方墨點法 (western blotting) 進行蛋白分析確認,並於穿透式電子顯微鏡 (transmission electron microscope; TEM) 下觀察到VP243形成之類病毒顆粒 (virus-like particle; VLP) 。本次試驗總共進行四次動物實驗,以無特定抗原 (specific pathogen free; SPF) 的雞隻進行免疫。分別測試未經純化之可溶或不可溶VP243蛋白、經蔗糖密度離心純化之VP243蛋白以及與不同佐劑混合後之VP243蛋白免疫雞隻後產生之免疫反應及保護效力。雞隻於第一週及第三週齡時進行蛋白免疫,於每週進行採血分離血清,並於第四週以口腔餵食進行4×102 EID50 / 200 μl之強毒株IBDV (very virulent IBDV) 的攻毒,攻毒後四天進行犧牲及解剖。分離之血清以酵素連結免疫吸附試驗 (ELISA) 進行抗IBDV之抗體力價測定,而解剖後採其華氏囊,並依據華氏囊損傷程度進行保護力之判斷。結果顯示,無論是可溶或不可溶之VP243蛋白皆可以於雞隻體內誘導顯著的抗體揚升;VP243之可溶蛋白經過蔗糖初步純化或混合其他佐劑進行免疫之組別,經過攻毒後四天的保護力皆不甚理想,而雞隻免疫以佛氏佐劑混合之VP243可溶蛋白的組別有較佳之保護力,且其華氏囊損傷程度最為輕微。因此,實驗結果證明大腸桿菌表現之VP243確實可以形成類病毒顆粒,且該次單位疫苗確實可以有效的於雞隻產生免疫反應,並提供良好之保護力對抗強毒株IBDV之感染。
The avian infectious bursal disease virus (IBDV) causes infectious bursal disease (IBD) in young chickens and is an important pathogen in poultry industry worldwide. The IBD is commonly controlled by vaccination with attenuated and / or inactivated vaccines in Taiwan. Researches have shown that the VP243 polyprotein which was expressed in eukaryotic cell could form a subviral particle and could induce protective immune responses. The objective of this study is to evaluate the immune responses and protection efficacy of chickens vaccinated by subunit vaccines containing E. coli expressed VP243 protein against very virulent IBDV (vvIBDV) challenge. The pTri-VP243 plasmids were constructed by cloning vp243 gene into pTriEx-3 vector and transformed into E. coli for protein expression. The soluble and insoluble VP243 proteins in the E. coli lysate were collected by centrifuging after IPTG induction. Both soluble and insoluble proteins were confirmed and analyzed by Western blotting, and the Virus-like particle (VLP) was confirmed by transmission electron microscope (TEM). Four animal trials were conducted and specific pathogen free (SPF) chickens were used in this objective. Chickens were intramuscularly vaccinated twice at one and three weeks of age with various types of antigens such as soluble VP243, insoluble VP243, soluble VP243 purified by 30 % sucrose or soluble VP243 mixed with different adjuvants. The chickens of vaccinated and challenge control groups were challenged with 4×102 EID50 / 200 μl of vvIBDV orally at one week after the last vaccination and euthanatized at 5 days after challenge. Blood was collected from each chicken weekly and the anti-IBDV antibody titers were measured by ELISA. At the termination day, a bursal gross lesion score was given for each chicken according to the severity of bursa damage. Prior to challenge, the groups vaccinated with various vaccines containing VP243 proteins have significantly higher anti-IBDV ELISA titers than that in the control groups (p < 0.05). The chickens vaccinated with VP243 soluble proteins which mixed with Freund''s Adjuvant showed well protection against vvIBDV with the similar bursal gross lesion scores as that in negative control groups (p > 0.05). The results demonstrated that the VP243 protein was successfully expressed by E. coli and provided protection of chickens against vvIBDV.
其他識別: U0005-2308201209055800
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