高产信号分子AI-2乳酸菌的筛选及其Pfs基因的克隆和表达

发布时间：2018-03-19 05:04

本文选题：乳酸菌　切入点：信号分子AI-　出处：《微生物学通报》2017年05期 　论文类型：期刊论文

【摘要】：【目的】从锡盟地区酸马奶酒分离的乳酸菌中筛选出高产信号分子自体诱导物2(Autoinducer-2,AI-2)的乳酸菌,通过优化其重组蛋白Pfs的诱导条件体外合成信号分子AI-2。【方法】利用生物学发光法对不同乳酸菌产信号分子AI-2的产量进行比较,以高产信号分子AI-2乳酸菌基因组DNA为模板,扩增其S-腺苷高半胱氨酸核苷酶(S-adenosylhomocysteine nucleosidase,Pfs)基因,构建原核表达载体。利用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)进行重组蛋白的诱导表达,通过优化培养基、诱导温度、诱导前菌体密度、IPTG浓度以及诱导时间得到高表达的Pfs蛋白,使其与底物作用最终体外合成信号分子AI-2。【结果】10株乳酸菌均可产信号分子AI-2,其中屎肠球菌8-3分泌信号分子AI-2的产量明显高于其他菌株;重组蛋白的最佳诱导条件为:选取SOC(Super optimal broth with catabolite repression)作为诱导表达培养基,菌液OD600为0.5 0.7时加入终浓度为0.1 mmol/L的IPTG,37°C诱导12 h;利用最优诱导条件获得了浓度为4.08 g/L的纯化Pfs蛋白,体外合成了信号分子AI-2。【结论】酸马奶酒中分离出的10株乳酸菌均可产生信号分子AI-2,且屎肠球菌8-3可通过Pfs基因的作用生成信号分子AI-2。
[Abstract]:[objective] to screen the high-yield signal molecule autoinducer 2AI-2 from the lactic acid bacteria isolated from the sour horse milk wine in Ximeng area. The signal molecule AI-2was synthesized by optimizing the induction conditions of its recombinant protein Pfs. [methods] the production of signal molecule AI-2 from different lactic acid bacteria was compared by using biological luminescence method. The genomic DNA of high yield signal molecule AI-2 was used as template. The Sadenosylhomocysteine nucleosidase gene was amplified and the prokaryotic expression vector was constructed. The recombinant protein was induced and expressed by isopropyl- 尾 -Dthiopyranoside galactoside (IPTG). The high expression of Pfs protein was obtained by the concentration of IPTG and the induction time before induction. The results showed that 10 strains of lactic acid bacteria could produce signal molecule AI-2, in which Enterococcus faecium 8-3 secreted signal molecule AI-2 was significantly higher than other strains. The optimal induction conditions of recombinant protein were as follows: SOC(Super optimal broth with catabolite expression was used as induction medium, and the final concentration of 0.1 mmol/L was added at 37 掳C for 12 h, and the purified Pfs protein was obtained with the concentration of 4.08 g / L, when OD600 was 0.5-0. 7, and the final concentration was 0.1 mmol/L at 37 掳C for 12 h. The signal molecule AI-2 was synthesized in vitro. [conclusion] all 10 strains of lactic acid bacteria isolated from yoghurt wine can produce signal molecule AI-2, and Enterococcus faecium 8-3 can produce signal molecule AI-2 through the action of Pfs gene.
【作者单位】：内蒙古农业大学食品科学与工程学院;
【基金】：国家自然科学基金项目(No.31360396) 内蒙古自治区研究生科研创新项目(No.B20151012908Z)~~
【分类号】：Q78;Q933

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