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標題: 環境友善型食品級乳球菌口服疫苗系統之研發
Development of environment-friendly food-grade Lactococcus lactis systems as oral vaccine
作者: 李珮瑄
Pei-Hsuan Lee
關鍵字: Enterovirus 71;non-genetically modified environmental- friendly cell surface display system;recombinant VP1e-anchor motif fusion protein;live L. lactis vector;gram-positive enhancer matrix(GEM);腸病毒 71 型;非基改環境友善型表層展示系統 重組VP1e-anchor motif;融合蛋白質;乳球菌活載體;革蘭氏陽性菌加強基質
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Enterovirus is a general term for a group of viruses which contained many different types and has widely distributed all over the world. Enterovirus 71 (EV71) commonly causes Hand, Foot and Mouth Disease (HFMD) in children, in severe
cases even result in neurological and cardiorespiratory complications. EV71infection have been recently reported as Asian-Pacific regionale epidemics. Human beings are the only known hosts and source of infection. Currently, there are still no approved antiviral drugs or vaccines against EV71 infection yet. This study developed systems based on a concept of non-genetically modified environmental- friendly cell surface display. The Escherichia coli T7 expression
system express large amount of EV71 capsid protein VP1 epitopes(VP1e) fused with an anchor motif (designated as recombinant VP1e-anchor motif fusion protein). About 1.7mg/L of the purified recombinant VP1e-anchor motif fusion protein was obtained after purification. The purified fusion protein was anchored onto the outer surface of live L. lactis vector or gram-positive enhancer matrix(GEM). Results showed that the maximum binding capacity was about 105 molecules purified VP1e-anchor motif fusion protein /cell. Besides, the binding capacity of GEM was better than live L. lactis vector. The stabilities of bound fusion protein were evaluated by storage at three different temperatures (-80°C, 4 °C, and room temperature [20–30 °C]) during 6 months. The results showed that GEM-bound VP1e-anchor motif fusion protein was more stable than live L. lactis vector-bound. To confirm the stability, the microscopy analysis of the purified fusion protein was exmined and the fusion proteins definitely anchored on GEM. The non-genetically modified L. lactis (live or GEM) as vaccine carrier, can avoid the risk of genetically modified microorganisms (GMMs) to apply in human oral consumption. In another part of this study, the recombinant VP1e was expressed by L. lactis expression system. The constitutive expression vector (pNZSASVP1e) can
constitutively expressed recombinant VP1e(rVP1e) yielding 102.5μg/L. Another acid-inducible system (MpHI)( pNZAUS-SacBAVP1e) was constructed and which was expected to improve the production of rVP1e. Unfortunately, the secretion level
was not improved. Both expression levels were too low to examine the stability of rVP1e mixed with GEM. The VP1e was proved to be a good vaccine candidate, because anti-VP1
antiserum was successfully achieved previously. In the future, the improvement and evaluation of non-genetically modified cell surface display system as oral vaccine is
highly worthwhile to develope. To express the rVP1e by GRAS or food-grade system may provides safer and convenient tool to develop L. lactis-based food-grade oral vaccine.

腸病毒是一群病毒的總稱,型別繁多且廣泛分布於全球,其中以腸病毒 71型(human enterovirus 71)最容易引起手足口病及神經系統等相關之嚴重併發症,為亞太地區地方性的流行性傳染疾病,而人類是其已知的唯一宿主及感染源。目前並無任何有效的疫苗可供預防及治療。本論文利用非基改環境友善(environmental friendly)型表層展示(cell surface
display)系統之概念,以大腸桿菌表現系統大量表現融合有錨定功能性區塊(anchor motif)之腸病毒 71 型外鞘蛋白質 VP1 抗原決定部位VP1e,以最適純化條件純化重組融合蛋白質,經濃縮定量後可得到1.7mg/L 之重組 VP1e-anchor motif融合蛋白質,並將其分別與乳球菌活載體(live L. lactis vector)及革蘭氏陽性菌加強基質(gram-positive enhancer matrix, GEM)進行共置培養,結果證實重組VP1e-anchor motif 融合蛋白質能結合於細胞表面。並利用西方墨點法及免疫墨點法定量分析測得其最大結合能力(Maximum binding capacity),結果顯示每個細胞約可與 105 分子之重組 VP1e-anchor motif 融合蛋白質結合。此外,GEM 的結合能力較乳球菌活菌載體好。一般疫苗之儲存溫度為 4°C,少數為-20°C 以下,若經長時間之存放且不失活,即為一高穩定性且具良好品質之疫苗。因此本論文另進行穩定性測定,將其置於-80°C、4°C 及室溫(20~30°C)三種不同溫度下存放數星期至數個月後,以蛋白質電泳及西方墨點法觀察重組 VP1e- anchor motif 融合蛋白質是否仍結合於細胞壁/GEM 上。結果顯示結合至 GEM 之重組VP1e- anchor motif 融合蛋白質較乳球菌活菌載體穩定。最後以掃描式電子顯微鏡(SEM)及免疫螢光顯微鏡(Immunofluorescence microscopy)分析照相確認。透過運用非基因工程改造乳酸菌株作為疫苗攜帶者(vaccine carrier),可免除消費者對基因改造菌株於應用上之安全疑慮。此外,本論文亦利用乳酸菌表現系統分泌表現重組 VP1e 蛋白質。以本實驗室先前構築之持續型表現載體(pNZSASVP1e)表現重組 VP1e,但經純化後僅得到 102.5μg/L 之重組 VP1e,於是以 MpHI 酸誘導系統另構築一誘導型表現載體(pNZAUS-SacBAVP1e),然而其胞外重組蛋白質之表現量甚低,因此皆無法由持續型及誘導型表現系統純化出足夠之重組 VP1e 蛋白質與 GEM 混合。本實驗室先前已經動物實驗成功製備出VP1特異性抗體,證實該蛋白質極具疫苗發展之潛力。未來可針對非基改表層展示系統進行動物試驗及疫苗效價評估。為避免全株基改微生物之諸多爭議,未來期待能將基改技術應用於安全級/食品級微生物系統,開發食品級口服乳酸菌疫苗,對人類有更多貢獻。
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