| Saccharomyces boulardii is used in clinical application for prophylaxis and the treatment of a variety of diseases caused by bacterial infection, such as Escheria coli, Salmonella typhimurium, Vibrio cholera, Clostridium difficile, Helicobacter pylori, Candida albicans and protozoa. Besides, the AP of S. boulardii with detoxification of LPS via dephosphorylated the lipid A of LPS. In this research, we partly revealed the prevention mechanism of S. boulardii against Salmonella enteritidis infection, and heterologously expressed the alkaline phosphatase of S. boulardii in P. pastoris. The details of the research are as follows:1To reveal the mechanism of S. boulardii against S. enteritidis infection, we used a mouse model of S. enteritidis infection, which pre-treatment with S. boulardii. After S. enteritidis challenged, we evaluated the amounts of S. enteritidis in cecum and in the liver. Besides, cecum were collected to analyse the adsorption between S. boulardii and intestinal microflora. Hepatic tissues damage were also analysed using HE staining. The results showed that S. boulardii remitted weight loss of mice caused by S. enteritidis infection (25.9g to22.3g)(p<0.05) at the11th day, the colonisation of S. enteritidis in cecum contents was decreased from107cfu/g to105cfu/g and the translocation bacteria in liver was decreased from104cfu/g to4×102cfu/g at the10th day, and remitted the damage of the liver caused by the infiltration of neutrophile granulocyte, lymphocyte and plasmocyte, via decreasing the amounts of S. enteritidis in the liver.2In this research, in order to get the recombination alkaline phosphatase (rAP). We cloning of the SBAP gene, and construction of the recombinant plasmids pPICSAP, which were linearized by digestion with Pmel and transformed into P. pastoris X-33by electroporation. After screening for transformants, the positive transformants were selected for enlarge expression in200ml shake flasks. The fermentation supernatant was used for purification of rAP by HisTrap HP column. The results showed that a novel SBAP gene (without introns) was cloned from5. boulardii ATCC MYA-796with a GenBank accession number KF471017. After screening for the enzyme activity of transformants, the rAP was expressed in P. pastoris X-33with a yield of43.66mg/1at the end of120h of induction in a shaker flask with200ml fermentation broth. After purification by affinity-column chromatography, optimum concentration of imidazole in elution buffer was250mM, the peak fractions were separated by SDS-PAGE and the purity of rAP was over90%.3In this study, the enzymatic properties of rAP including effects of Mg2+, EDTA, temperature and pH were evaluated, as were the thermal stability and specific activity of rAP in optimum conditions. Additionally, the ability of rAP to reduce inflammation caused by LPS was investigated. The results showed that the optimal reaction conditions of rAP were pH9.6, temperature at60℃and2mM Mg2+ in diethanolamine buffer. And EDTA was a potent inhibitor of rAP activity, the enzyme activity of rAP was inhibited to18.37%by1mM EDTA. The specific activity of rAP was9912U/mg under the optimal conditions, which was3times than preciously espressed calf intestinal AP. The rAP maintained more than80% of its enzyme activity after incubation at37℃for1h, as the test temperature increased, the activity gradually decreased, and it was almost nonexistent after incubation at100℃for1h. Furthermore, rAP showed a broad dephosphorylation activity to LPS over a broad pH range (pH2-10) in Tris-HCl buffer. After LPS dephosphorylated by rAP was injected intraperitoneally into mice, the serum level of tumor necrosis factor (TNF)-a was significantly reduced compared to that of the wild LPS group (p<0.01).In a word, S. boulardii prevention of the hepatic tissue damage caused by S. enteritidis infection in this mouse model. Besides, rapid cloning of a novel SBAP gene from S. boulardii. Expression of high-activity rAP in P. pastoris, and rAP with detoxication of LPS. These findings suggest that rAP has great potential to cure diseases caused by LPS. |
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