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標題: 源自Bacillus subtilis之訊息序列供作構築洛德乳酸桿菌表現分泌載體之研究
Studies on the construction of Lactobacillus reuteri expression secretion vector by use of signal sequences from Bacillus subtilis
作者: 許清爭友
Hsu, Ching-Yu
關鍵字: lactobacillus reuteri
signal sequence
Bacillus subtilis
expression secretion vector
E2 gene
出版社: 獸醫微生物學研究所
摘要: 中文摘要 乳酸菌(lactic acid bacteria)在食品工業中對於一些農產品的發酵以及其他食品的應用上扮演著重要的角色,最近更發現乳酸桿菌屬(Lactobacillus)中特定的幾個菌種能促進人及動物的健康,其中Lactobacillus reuteri不但具有此性質,且可以分泌一種抗菌物質,稱為洛德因(reuterin),可抑制革蘭氏陽性菌、革蘭氏陰性菌、酵母菌、黴菌及原蟲等。目前認為L. reuteri是益生菌(probotics),且對人及動物皆無病原性。發展洛德乳酸桿菌為疫苗攜帶者(vaccine carrier), 使其攜帶病原菌之部分缺損腸毒素基因(partially deleted enterotoxin gene)或表面構造蛋白基因(surface protein gene),藉由分泌該些蛋白之刺激腸道免疫機制,應可達到保護動物的效果。 利用來自L. reuteri chromosome之promoter-signal sequence A3-2’及 Bacillus subtilis 之promoter-signal sequences,使分別嵌入E. coli-L. reuteri shuttle vector,完成ESV-A3(a~c)及ESV-Cwld(a~c)表現分泌載體的構築。更進一步利用轉殖來自Bacillus subtilis 的α-amylase 基因及豬瘟病毒的E2基因完成ESV-A3-amylase及ESV-Cwld-E2重組質體之構築利用SDS-PAGE及Western blot 來檢測表現分泌載體表現分泌外來基因能力,這是E2基因在L.reuteri的第一個表現報告。
Abstract Lactic acid bacteria (LAB) have long been playing an important role in the field of food fermentation industry. Recently , some species of LAB in the gastrointestinal tract were even proved to be beneficial to the health of human and animal. Among LAB, Lactobacillus reuteri was found to be unigue in its ability of producing a broad-spectrum antimicrobial substrance, termed reuterin, which could inhibit the growth of gram positive and gram negative bacteria, yeast, fungi and protozoas. In an attempt to develop L. reuteri as a vaccine carrier expressing surface antigen or partically deleted enterotoxin from pathogenic microorganism to stimulate the mucous membrane response of animal, expression-secretion vectors (ESV) were constructed in this study by employing promoter-signal sequence from L. reuteri and Bacillus subtilis. The promoter signal sequence A3-2' from L. reuteri and Cwld from B. subtilis , respectively , cloned into an E. coli-L. reuteri shuttle vector to generate ESV-A3(a~c) and ESV-Cwld(a~c). Further cloning of a-amylase gene from B. subtilis and E2 gene from Hog Cholera virus into ESV-A3-(b) and ESV-Cwld-(b) were able to detect the expression and secretion of the products of foreign gene in the SDS-PAGE and Western blot analysis. This is the frist expression of E2 gene in L. reuteri.
Appears in Collections:微生物暨公共衛生學研究所



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