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標題: Enhancement of Immunity and Protection Efficacy in Chickens against Infectious Bronchitis by DNA Prime-Protein Boost Vaccination
藉由 DNA prime-protein boost 之免疫策略提升雞隻對抗傳染性支氣管炎的免疫力及保護力
作者: 高翊綺
Yi-Chi Kao
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摘要: Infectious bronchitis virus (IBV) is a highly contagious chicken disease, making a severe economic impact on poultry industry. The IBV genome encodes four structural proteins, known as the spike (S) glycoprotein, the envelope (E) protein, the membrane (M) glycoprotein, and the nucleocapsid (N) protein. The objective of this study is to apply the strategy of the DNA prime-protein boost vaccination to enhance the immunity and protection efficacy in chickens against IBV challenge. Three DNA plasmids containing S1, M or N gene were constructed and demonstrated to express related proteins by immunofluorescence assay and Western blotting. Chickens were vaccinated prior to three weeks of age by using DNA vaccines, subunit vaccines and DNA prime-protein boost strategy in three trials, respectively. Chickens were challenged at 5 weeks of age and observed for one week. Sera were collected weekly and anti-IBV antibody titers were detected by commercial IBV ELISA kit. The kidney lesion score was given to each chicken from 0 to 3 according to the increasing severity. In Trial I, chickens receiving DNA plasmids containing pTriEx-S1+pTriEx-N, or pTriEx-S1+pTriEx-M+pTriEx-N had higher antibody titers besides the commercial killed vaccine before challenge. However, the best protection efficacy in chickens injected with three doses of DNA containing S1 gene had 100 % protection with lesion score of 0.8. In Trial II, neither S1 nor N protein could provide protection efficacy higher than 40%. In Trial III, home-made killed vaccine prepared by inactivating IBV with formaldehyde was also compared along with subunit proteins. Chickens receiving killed vaccines showed higher anti-IBV antibody titers. Chickens primed with DNA containing S1 gene at one week of age and boosted with S1subunit protein at three weeks of age showed the highest protection efficacy with 100 % protection. In conclusion, DNA prime-protein boost vaccination provided a new efficacious strategy in chickens against IBV infection.
雞傳染性支氣管炎病毒 (Infectious bronchitis virus, IBV) 是一種高度傳染性的病毒,雞感染此病毒會造成家禽業嚴重的經濟損失。IBV 有四個主要的結構蛋白,分別為棘突蛋白(Spike glycoprotein,S)、封套蛋白(Envelope protein,E)、膜蛋白(Membrane glycoprotein,M),核蛋白(Nucleocapsid protein,N)。本研究的目的是應用 DNA prime-protein boost 策略,以加強雞隻對抗 IBV 的免疫力和保護力。將 IBV 以反轉錄聚合酶鏈鎖反應分別增幅出 S1、M 和 N 三個基因片段,再進而選殖至可於真核及原核細胞表現之載體 pTriEx-3 上,構築出 pTriEx-S1、pTriEx-M和 pTriEx-N 重組質體。以間接螢光免疫分析確定轉染至 DF-1 細胞的各重組質體可被正確的表現出各自之重組蛋白,並以西方墨點法分析,確認含有各重組質體的 E.coli 亦可被誘導表現出重組蛋白,之後將製備之 DNA 疫苗和次單疫苗進行動物試驗。在雞隻 3 周齡以前,分別免疫 DNA 疫苗和次單疫苗與使用 DNA prime- protein boost 策略,共進行三次動物實驗;在雞隻 5 周齡時進行攻毒,並於攻毒後一周進行犧牲。每周採集血液樣本以市售 ELISA 試劑檢測抗 IBV 抗體。腎臟損傷評分按照嚴重程度給予,分數從 0 (正常)到 3 (腫大及尿酸鹽沉積)。第一次動物實驗結果顯示除了免疫市售不活化疫苗之外,免疫 pTriEx-S1+ pTriEx-N 及pTriEx-S1+ pTriEx-M+ pTriEx-N 組別的雞隻,在攻毒前能檢測到較高的抗體力價;然而,最好的保護功效表現在雞隻免疫 pTriEx-S1 重組質體組,其腎臟病變指數為 0.8 且保護力為 100%。第二次動物實驗結果顯示免疫 S1 與 N 次單位疫苗提供之保護力只有 40%。在第三次動物實驗將自製不活化 IBV 疫苗與次單位疫苗相比較,結果顯示免疫自製不活化 IBV 疫苗的雞隻表現出較高的抗 IBV 抗體力價;雞隻先免疫 S1 DNA 疫苗再使用 S1 次單位疫苗進行補強的組別有最高的保護效果(100%)。因此,首次免疫 S1 DNA 疫苗再使用 S1 次單位疫苗補強,可以提升雞隻對抗 IBV 的免疫力及保護效力。實驗結果證實,DNA prime-protein boost 策略是個可以有效提升雞隻對抗 IBV 感染之免疫策略。
文章公開時間: 2018-07-16
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