Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66408
標題: 洛德乳酸桿菌表現分泌載體—pNlp1及p203的分子特性分析
Molecular characterization of expression-secretion vector systems, pNlp1 and p203, developed for use in Lactobacillus reuteri
作者: 詹啟甫
Chan, Chi-Fu
關鍵字: lactic acid bacteria
乳酸菌
promoter
signal sequence
啟動子
訊號序列
出版社: 獸醫微生物學研究所
摘要: 乳酸菌 (lactic acid bacteria;LAB) 在食品工業及農業作物上有很大的功用,舉凡乳製品、發酵性飲料、肉製品的保存及蔬菜的醃製等都有相當的貢獻,對動物的健康及營養均亦具有不錯的效果,對於人類及動物是有助益的,因而常被作為生菌劑之用,目前已經有許多乳酸菌表現分泌載體被開發用於基因表現並使之分泌外來蛋白。本實驗分別以來自L. lactis 和 B. subtilis 的不同啟動子-訊號序列 (promoter-signal sequence) ,構築2個不同表現分泌載pESV-Amy-nlp和pESV-Amy-nisA,經利用澱粉酶分解試驗,評估其在L. reuteri標準株DSM 20016中的表現能力。此外,本實驗亦構築不同重組質體,pESV-Gfp-nlp、pESV-Nuc-nlp、pESV-Nuc-nisA,分別轉形至L. reuteri DSM 20016與L. reuteri 3-3中。在生長速率方面,L. reuteri 3-3轉形株生長速度明顯快於20016轉形株,質體穩定性試驗結果顯示: (1) 重組質體在3-3轉形株中較為穩定,明顯優於20016轉形株 (2) pESV-Nlp表現系統之穩定性明顯優於pESV-nisA表現系統。綜合實驗結果,我們認為pESV-Nlp表現分泌系統,不論在表現能力、分泌效率與質體穩定性上皆優於pESV-nisA系統,故pESV-Nlp表現分泌系統將更適合應用於載體疫苗上;此外,分離自老鼠腸道的洛德乳酸桿菌3-3菌株,具有宿主特異性、生長速度快、穩定保有質體等優點,適合用於將來小鼠模式之免疫試驗上,期能日後做為黏膜免疫疫苗開發的技術平台。
Lactic acid bacteria (LAB) has been widely used in food engineering, and agriculture. It has contributed enormously to the conservation of foods, including dairy products, flesh products, fermentative drinks, and pickled vegetables. Evidences, accumulated thus far, have proved that some stains of LAB are beneficial to the health of animals, strongly suggesting them as good proiotic candidates. The aim of this study was trying to utilize Lactobacillus reuteri as a vaccine carrier to express and secret products of foreign genes, such as partially deleted toxin genes, in gastrointestinal tract of animals to stimulate their membrane immune response. Consequently, two expression-secretion vectors (ESV), termed pESV-nisA and pESV-nlp, were constructed by using two different promoter-signal sequence form L. lactis and B. subtilis, respectively. Subsequent cloning of amylase gene, green fluorescent gene, and Nuclease gene into these two ESVs resulted in the generation of recombinant plasmids pESV-Amy-nisA, pESV-Amy-nlp, pESV-Gfp-nisA, pESV-Gfp-nlp, pESV-Nuc-nisA, and pESV-Nuc-nlp, respectively. L. reuteri DSM20016 and L. reuteri 3-3, transformed with each of these recombinant plasmids, were employed to analyze the growth efficiency and plasmid stability. Analysis of the growth condition revealed that transformants of L. reuteri 3-3 had better growth rate than those of L. reuteri DSM20016. In the plasmid stability test, it showed that: (1) recombination plasmids in L. reuteri 3-3 were more stable than those in L. reuteri DSM20016. (2) pESV-Nlp was more stable than pESV-nisA. In conclusions, pESV-Nlp was found to be more efficient than pESV-nisA with regard to gene expression, protein secretion and plasmid stability in L. reuteri, indicating pESV-Nlp as being more suitable for the development of vaccine vector. In addition, L. reuteri 3-3, an isolate from mice intestinal tract with properties of high host specificity, better growth rate and good maintenance of heterologous plasmids, should be a good host candidate for ESV in the future vaccine carrier study by employing mice as an animal model.
URI: http://hdl.handle.net/11455/66408
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