| Summary: | 博士 === 國立中興大學 === 獸醫微生物學研究所 === 95 === Lactobacillus reuteri (Lb. reuteri, LR), frequently found in the gastrointestinal (GI) tracts of humans and animals, has been discovered to bearing advantages suitable for use as a probiotic. Further development of such a microbe with probiotic effects as a live vehicle carrying immunological molecules, if feasible, would certainly provide the GI tract with extra medical benefits. To accomplish this goal, a transferable cloning vector capable of expressing and secreting heterologous proteins of medical interest in LR is crucially needed.
In this study, the nisin-controlled gene expression (NICE) system discovered in Lactococcus lactis was, for the first time, adapted to LR by ligating the nisA promoter (PnisA) and nisRK DNA fragments into a E. coli-LR shuttle vector pSTE32, generating a chimerical plasmid (pNICE) capable of expressing heterologous amylase gene (amyL) and green fluorescent protein gene (gfp) under nisin induction. Optimization of induction-factors for this LR/pNICE system, including nisin concentration (i.e., 50 ng/ml), growth phase of culture at which nisin be added (i.e., at the early exponential phase), and the best time for analyzing the gene product after inoculation (i.e., at the 3rd h), allowed the induced protein to be produced at a prominent amount. However, a low level of constitutive gene expression was also observed in our LR/pNICE system. To explore this unusual constitutive expression, DNA sequence-deletion analysis was conducted and found the PnisA activity in LR, in contrast to be dependent of the NisRK products, was highly regulated by a TCT direct repeat [TCT-N8-TCT] upstream (-107 to -94) of the transcription initiation site. Disruption of this repeat was found to completely terminate the protein expression of PnisA, suggesting a potential pivotal role of this repeat in the transcription activity of NICE in LR.
For identifying excellent signal peptides (SP) functional in LR which may be used in the future construction of expression-secretion vector, a SP-probe vector pNICE-GFP:SP, employing a green fluorescent protein (GFP) as the SP-selection marker, was constructed in this work. The optimal conditions for inducing, activating, and enhancing the expression and secretion of the GFP in LR had also been determined. Subsequently, four putative SPs (i.e., SPamyL, SPmub, SPusp45 and a lipoprotein SPnlp1) were cloned into the pNICE-GFP:SP, demonstrating the capability of this SP-probe vector in identifying SP-DNA fragments by direct visualization of the expressed fluorescence activity around cells. Further assay of fluorescent intensity in the culture supernatants with spectrofluorometry clearly quantified the SPamyL as the best SP functional in LR. Consequently, the SPamyL was ligated to the downstream of PnisA in pNICE, successfully generating the first LR-specific expression-secretion vector (pNIES) in this study.
To further improve our pNIES, a gfp gene was included to construct a pNIES-GFP vector, which was able to secret the cloned gene as a GFP-fusion protein with fluorescent activity. Moreover, a recombinant ST-LTB gene, encoding a fusion protein of the heat-stable enterotoxin (ST) and heat-labile enterotoxin B (LTB) of the enterotoxigenic E. coli (ETEC), was inserted into pNIES-GFP to test the possible vaccine carrier role of this system. Following confirmation of possessing capabilities of adhering to mice gut and secreting GFP:STLTB product under induced and non-induced conditions, the resulting LR/pNIES-GFP:STLTB system was then verified for its expressed GFP:STLTB product to own ganglioside-binding ability, LTB antigenicity and relative freedom from the ST-associated toxicity, which are important properties for an oral live vaccine in mice.
Subsequent inoculation of the LR /pNIES-GFP:STLTB culture orally to mice was able to elicit significant serum IgG and mucosal IgA antibodies against the STLTB antigen, which was latterly found to provide full protection against the challenge of viable ETEC. This is the first report of using LR as a vaccine carrier to induce complete immunologic protection against ETEC.
Finally, a nisin-controlled cell-surface display vector pNIEA, which employed a cwaM6 gene encoding the cell-wall anchor region of Streptococcus pyogenes M6 protein, was also constructed in this study. This LR/pNIEA system was capable of displaying the alpha-amylase and STLTB antigen at the cell surface of LR and, more importantly, evoked a stronger systemic immune response, as compared with that in the pNIES system. The fixation of antigenic molecules on the surface of the colonized LR/pNIEA, avoiding the dilution of intestinal fluid, may provide a better antigen presentation to the mucosal immune system. We therefore suggest the Lb. reuteri/pNIEA system to be a highly potential candidate for the future development of live oral vaccine.
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