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標題: 以禽類傳染性支氣管炎病毒為研究模式評估開發乳酸菌為黏膜性疫苗之運輸載體
Development of Lactic Acid Bacteria as Mucosal Vaccine Delivery Vehicles Using the Study Model of Avian Infectious Bronchitis Virus
作者: 許愛萍
Hsu, Ai-Ping
關鍵字: lactic acid bacteria
mucosal vaccine
infectious bronchitis virus
出版社: 獸醫學系暨研究所
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摘要: 乳酸菌 (lactic acid bacteria) 為革蘭氏陽性、可發酵碳水化合物、以乳酸為主要代謝產物之益生菌。多項研究已證實其對於腸道免疫平衡維持具有貢獻,且因其與身俱來的生物佐劑性質,因而被開發作為黏膜性疫苗運輸載體。來自於 Lactococcus lactis 之自溶素蛋白 AcmA’ 具有藉由其 C 端LysM 功能區段以外源性方式結合到乳酸菌表面之能力,因此本研究利用 AcmA’ 中的第一個 LysM 來執行抗原性蛋白外源性結合到乳酸菌表面之憑藉。參考文獻於傳染性支氣管炎病毒 (IBV,infectious bronchitis virus) 之棘蛋白上設計四段抗原決定位區域 (命名為 AB 、 EF 、 GH 、 I 片段) ,與 LysM 共同構築於表現質體上,並利用大腸桿菌系統表現 C 端帶有 AcmA’ 融合抗原蛋白-- AB-AcmA’ 、 EF-AcmA’。 由於 mammalian reovirus 的 σC 基因已被證實具有免疫促進之特質,為測試 avian reovirus 的 σC 是否同樣具有此功能,因此亦將 σC 之基因序列構築於 GH 、 I 之 DNA 片段與 AcmA’ 之間,以表現融合蛋白 GH-σC-AcmA’ 、 I-σC-AcmA’ 。 經 whole cell ELISA 以及西方墨點法證實,重組蛋白 σC-AcmA’ 、 AB-AcmA’ 、 EF-AcmA’ 、 GH-σC-AcmA’ 、 I-σC-AcmA’ 可外源性地結合到乳酸菌 Enterococcus faecium 、 Lactobacillus salivarius 、 Lactococcus lactis 的表面。實驗中一併調查此三株乳酸菌於小雞腸道中停泊、持續存在之情形,於七日的觀察發現,乳酸菌 E. faecium 的投予,其自小雞腸道中排出之菌量最少,且於小雞個體間腸段分佈之菌數變異小,因此選定 E. faecium 作為後續評估乳酸菌性疫苗黏膜免疫效益之運輸載體。抗原蛋白的選擇上,以在大腸桿菌中蛋白質表現量最高的 EF-AcmA’ 來探討乳酸菌性黏膜性疫苗之可行性,並評估 σC 對於 EF-AcmA’ 之專一性免疫反應有無助益。實驗結果證實,結合 EF-AcmA’ 之 E. faecium 以黏膜路徑免疫小鼠可誘發專一性的免疫反應,並且 σC 可能具有免疫促進性的特質。由於實驗動物數量有限,且動物個體之間免疫反應程度差異,關於乳酸菌載體之免疫成效、以及 σC 之免疫促進潛能,仍需進一步探究。
The lactic acid bacteria (LAB) are Gram-positive fermentative microorganisms known primarily for their roles as probiotics. Several lines of evidences have proved that LAB contribute to immunity homeostasis in the intestines and have biological adjuvanticity. Very recently, many studies have focused on developing LAB for the application of mucosal vaccine vehicles. LysM of AcmA', the autolysin of Lactococcus lactis that could exogenously bind onto the peptidoglycan of LAB, was chosen as an anchor molecule for docking the desire antigenic proteins to LAB. On the basis of literature reviews, four epitopes located at various regions of IBV (Infectious bronchitis virus) spike protein were identified and were successfully expressed as fusion proteins: downstream of each epitopes was tagged with AcmA' or AcmA' along with sigmaC (σC), the outer capsid protein of avian reovirus that was chosen for its potential ability of M cells adhesion. Results of whole cell ELISA and western blot analysis have evidenced that the recombinant proteins, σC-AcmA', AB-AcmA', EF-AcmA', GH-σC-AcmA', and I-σC-AcmA' were able to bind onto LAB Enterococcus faecium, Lactobacillus salivarius, Lactococcus lactis in trans. The distribution and colonization of LAB were also tested in chicken model; among the three LAB analyzed, E. faecium was the best candidate vector for the high consistence performance between individual animals and was used for further immunization studies. For evaluating the efficacy of LAB-based mucosal vaccination, EF-AcmA' was adopted as model antigen in combination with or without σC. The results demonstrated mucosal vaccination with EF-AcmA' bound E. faecium could elicit production of IgA specific for EF epitope in mice. Furthermore, high IgA titer as observed in two out of three mice vaccinated with EF-AcmA' in the presence of σC, suggesting σC may play a part in immune regulation. However, due to the limited animal size, the properties of immunity enhancement and the M cell targeting of σC should be further characterized.
其他識別: U0005-2306200821130900
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