| Summary: | 乳酸菌（lactic acid bacteria；LAB）長久以來被廣泛應用於食品發酵。乳酸菌歸屬於安全菌種（generally recognized as safe；GRAS），近年來的研究開始朝向將乳酸菌開發成活體疫苗攜帶者。本實驗應用可分泌廣效性殺菌物質-洛德因（reuterin）的洛德乳酸桿菌（Lactobacillus reuteri），作為構築分泌表現載體及分泌異源性蛋白質的宿主，以來自L. lactis and B. subtilis的不同啟動子-訊號序列（promoter-signal sequence），構築6個不同的表現分泌載體，利用澱粉脢分解試驗，評估其在L. reuteri標準株DSM 20016中的表現能力。結果顯示，源自Lactococcus lactis subsp. cremoris nlp1蛋白質的Pnlp：SPnlp vector，在E. coli及L. reuteri中均有表現功能。此外，本實驗亦自野鼠（Rattus rattus）消化道中篩選出2株野生L. reuteri；以B. subtilis來源的植酸脢基因置換澱粉脢基因，成功地將具有表現分泌外來基因產物能力的重組質體，ESV-phy-nlp、ESV-phy-201及 ESV-phy-203，分別轉型至L. reuteri DSM 20016、野鼠胃部及大腸區段分離所得之2株野生L. reuteri中。以固體培養基進行植酸脢分泌測試結果顯示，僅有野鼠胃部及大腸區段來源野生L. reuteri轉型株具有分泌植酸脢的能力，L. reuteri DSM 20016轉型株不見植酸脢分泌現象。本實驗利用植酸脢活性試驗，定量分析帶有ESV-phy-nlp、ESV-phy-201及 ESV-phy-203之野生L. reuteri轉型株中的植酸脢結果顯示重組載體分泌植酸脢的效能確實受不同的啟動子-訊號序列的影響；且同一個重組質體，在不同來源之L. reuteri菌株中，分泌異源性蛋白的能力並不相同。本實驗成功自野鼠消化道分離得到L. reuteri菌株，並構築出以L. reuteri作為異源性蛋白攜帶者；期望瞭解洛德乳酸桿菌菌株間表現外來基因的差異性，作為日後研發本菌成為廣用性動物疫苗載體的參考基礎。 Lactic acid bacteria (LAB) have been used worldwide in the preparation of fermented foods. Their “generally recognized as safe”(GRAS)status has recently led them to be developed as potential live-vaccine vehicles. In this work, we use Lactobacillus reuteri, which was found to be unique in its ability of producing a broad-spectrum antimicrobial substance termed “reuterin”, as a host and focused on optimizing heterologous protein secretion and developing expression-secretion vector(ESV)for use in L. reuteri. Six L. reuteri ESVs were constructed in this study by employing promoter-signal sequences from L. lactis and B. subtilis and were screened by starch-degraded halo test. The DNA fragment, termed nlp being 570 bp in length with a standard promoter-signal sequence from L. lactis, was found to be functional in both E. coli and L. reuteri. We also isolated two strains of L. reuteri from Rattus rattus. With an aim of constructing an efficient ESV capable of expression and secretion of foreign gene products in different strain of L. reuteri, we replaced the original amylase gene in the ESVs with the phytase fragment from B. subtilis, resulting in the success construction of recombinant vector ESV-phy-nlp, ESV-phy-201, and ESV-phy-203, which were subsequently transformed into L. reuteri DSM 20016 and two strains of L. reuteri isolated from Rattus rattus stomach and Rattus rattus colon. Phytase like enzyme was found to be expressed on plates inoculated with L. reuteri of Rattus rattus stomach and colon origin, but not in L. reuteri DSM 20016. Furthermore, analysis of the medium grown with L. reuteri bearing pESV-201 and pESV-203, respectively, by a phytase specific method confirmed that the promoter-signal sequence is actually effect in the heterologous protein secretion and export in L. reuteri. The same vector construction also showed the different function in these 3 strains of L. reuteri. Hopefully, the work of this study may led to more understanding for developing L. reuteri as a live-vaccine vehicle. 中文摘要……………………………………………………………………Ⅰ 英文摘要……………………………………………………………………Ⅱ 目次…………………………………………………………………………Ⅲ 圖次…………………………………………………………………………Ⅴ 表次…………………………………………………………………………Ⅶ 第一章 緒言…………………………………………………………………01 第二章 文獻探討……………………………………………………………04 第三章 材料與方法…………………………………………………………24 第四章 結果…………………………………………………………………49 第五章 討論…………………………………………………………………86 參考文獻……………………………………………………………………91 附錄…………………………………………………………………………103
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