| Summary: | 碩士 === 國立中興大學 === 獸醫學系 === 90 === Abstract
Lactic acid bacteria(LAB) has been applied in food industry for a long time. Their benefits for animals and human health were shown and proven in much of recent research. Among Lactobacillus species, Lactobacillus reuteri was found to be unique in producing an intermediate metabolite during anaerobic fermentation of glycerol, called “ reuterin ”, which was demonstrated as a broad-spectrum antimicrobial substance against protozoa, yeast, gram-positive and gram-negative bacteria. This activity let L. reuteri to be capable of colonizing in gastrointestinal tract quickly. Consequently, L. reuteri was regarded as an attractive candidate for vaccine design. In this work, three L. reuteri expression secretion vectors (ESV), called pLE-R18a-c, were constructed by integrating promoter sequence R18 from L. reuteri chromosome DNA and a multiple cloning site (MCS) from pET-28a-c(+) into the L. reuteri-E. coli shuttle vector-pSTE32(+). Further evaluation of the expression of these ESVs by cloning the Tox2-DNA fragment from Pasteurella multocida toxin gene as a reporter gene into pLE-R18c, generating a recombinant plasmid pLE-R18/Tox2, was able to detect the Tox2 product in cell lysate of L. reuteri DSM 20016 transformed with pLE-R18/Tox2. With an aim of constructing an efficient ESV capable of expression and secretion of foreign gene products, a DNA fragment termed nlp1, 570 bp in length with a standard promoter-signal sequence from Lactococcus lactis subsp. lactis, was found to be functional in both E. coli and L. reuteri and was used to replace the original R18 fragment in pLE-R18a-c. Preliminary analysis of the recombinant plasmid with R18 being replaced by nlp1-DNA fragment was able to detect protein products of the gene, downstreamed of nlp1, to be present in the supernatant as well as lysate of its L. reuteri transforment indicating the success of constructing a L. reuteri ESV
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