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Construction of expression secretion vector pESV-nlp1(a-c) for use in lactobacillus reuteri
乳酸菌(lactic acid bacteria)長期被利用於食品工業上，其對於人類及動物健康的助益不斷地被發現，目前已被大量利用於表現外來基因，應用於醫療保健或是食品工業。其中洛德乳酸桿菌(Lactobacillus reuteri)代謝甘油後產生的中間產物洛德因（reuterin），具有廣效性的殺菌效果，使洛德乳酸桿菌在腸道中屬於優勢的菌種，故洛德乳酸桿菌廣泛被認定為優良的疫苗載體。本實驗利用洛德乳酸桿菌與大腸桿菌（E. coli）穿梭載體pSTE32(+)為基礎，嵌入一段洛德乳酸桿菌染色體來源的啟動子序列(promoter sequence)R18，並在R18基因下游接上質體pET-28a-c(+)的多重選殖區（multiple cloning site；MCS），完成3個表現分泌載體pLE-R18(a-c)。為測試載體pLE-R18(a-c)的功能，我們將Pasteurella multocida的毒素基因片段（tox2）嵌入pLE-R18c的MCS，成為質體pLER18-Tox2。將質體pLER18-Tox2電孔轉型（electroporation）至L. reuteri DSM 20016後，以豬抗Tox2的抗體作探針進行西方墨點法（Western blot），可以在L. reuteri的菌體內偵測到訊號，證明質體pLER18-Tox2確實能在L. reuteri 中表現tox2基因。為使表現載體具有分泌的功能，依據1998年Poquet等人對Lactococcus lactis訊號序列之研究報告（Poquet et al, 1998），找出在E. coli及L. reuteri中皆具有啟動子（promoter）及訊號序列（signal sequence）功能之Lactococcus lactis來源的基因nlp1序列，取代R18基因，使表現載體具有分泌外源性蛋白之功能。|
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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