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標題: Development of food grade spore display system as oral vaccine against EV71 and evaluation of vaccination effect in BALB/c mice
作者: Han-Sheng Lin
關鍵字: 腸病毒71型;枯草桿菌;非重組孢子表層展示;重組蛋白質rCotZ-VP1e;口服疫苗;Enterovirus 71;Bacillus subtilis;Non-recombinant spore-surface display;recombinant CotZ-VP1e;Oral vaccine
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本實驗室先前已構築出腸病毒外鞘蛋白 VP 1 之N端表位蛋白序列VP1e (VP1序列胺基酸1-81) ,並證實能夠引發專一性免疫反應,除此之外,本實驗室先前以重組rVP1e 與來自Lactobacillus acidphilus ATCC4356之表層蛋白融合 (rVP1e-anchor)。利用GRAS級/食品級的乳酸菌Lactococcus lactis MG1363作為革蘭陽性加強介質(gram-positive enhancer matrix; GEM),將兩者共製培養,製作出結合 rVP1e 的 GEM (rVP1e-GEM)之口服疫苗,也成功誘導專一性IgA。
而本論文中探討利用非基改孢子表層展示之概念,利用枯草桿菌孢子作為疫苗載體,吸附重組抗原rVP1e,製成口服疫苗,並藉由動物實驗觀察其免疫效果。本論文首先構築乳酸菌、枯草桿菌和大腸桿菌為表現宿主,表現重組蛋白rCotZ-VP1e,其中以大腸桿菌表現量最佳,經濃縮純化後可得1.8 mg/ml。接著將蛋白與孢子共置培養,並利用免疫墨點法定量分析其最大結合能力(Maximum binding capacity)。結果證實重組蛋白rCotZ-VP1e可被孢子吸附,最佳吸附環境緩衝液酸鹼值為pH 4,最適吸附孢子為枯草桿菌3A16 ( Bacillus subtilis 3A16 )益生孢子,最大結合能力為以蛋白質濃度200 μg與枯草桿菌3A16孢子於酸鹼值pH 4下共置吸附,其最大結合量約55.8±3μg之rCotZ-VP1e結合在孢子上,相當於每個孢子結合6.25×105分子數的rCotZ-VP1e蛋白質(6.25×105 molecules/spore)。
最後以動物實驗測試疫苗效果,一共四組介入,分別是控制組、孢子組、腹腔注射組(rCotZ-VP1e)以及口服疫苗組(rCotZ-VP1e-spore),觀察小鼠抗體產生情形。結果顯示,腹腔注射組之小鼠抗體有顯著上升。而在小鼠抗體 IgA 的部分,口服疫苗組與腹腔注射組皆有產生,口服疫苗組略高腹腔注射組。由結果顯示口服疫苗組確實有誘發腸道的免疫反應。

The Human enterovirus 71 is a major cause of hand-foot-and-mouth disease(HFMD) in children below 6 years old. Over the last decade, HFMD has become endemic in the Asia Pacific region. The VP1 capsid protein was reported to be the main neutralizing epitopes of EV71. The recombinant N- terminal VP1 (rVP1e) was proved to induce immune response in our previous study. Prebiotic spore surface display is a potent strategy for safe oral vaccine.
In this study, we constructed a fusion expression system of spore coat protein CotZ and VP1e. The recombinant CotZ-VP1e (rCotZ-VP1e) was successfully expressed and purified by Eschrichia coli and food grade host Lactococcus lactis, Bacillus subtilis, respectively.The expression level of Eschrichia coli is better than Lactococcus lactis and Bacillus subtilis. The rCotZ-VP1e was purified and bound to spore surfaces of a B. subtilis prebiotic strain. The optimal binding condition of rCotZ-VP1e on B. subtilis WB800 spore surface was examined to be under pH 4. In this condition, the absorption rate of protein to spore was about 55.4 % which indicated 2.49×105 molecules/spore. The optimal binding condition of rCotZ-VP1e to B. subtilis 3A16 prebiotic spore was about 27.9% (6.25× 105 molecules/spore).
The vaccination effects of rCotZ-VP1e -spore by oral route and rCotZ-VP1e by injection route were evaluated in animal trials.The specific antibody IgG 1, IgG 2a and sIgA were examined. As the results , the specific antibody IgG1 and IgGa were induced by injection route. There are significant difference between oral immunization groups and control groups in the specific antibody, IgA. Results suggested that the rCotZ-VP1e bound spore can be seen as a potent oral vaccine.
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