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

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℃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.%[Objective] The study in order to screen the strain with high yield ofautoinducer-2 (AI-2) of lactic acid bacteria strains which isolated from koumiss of Ximeng region in Inner Mongolia,and optimize the condition of recombinant protein to synthesize signal molecule AI-2 in vitro.[Methods] Using biological luminescence method to compare the contents of signal molecule AI-2 which produced by different lactic acid bacteria,with high production of signal molecule AI-2 lactic acid bacteria genomic DNA as a template,expanded its S-adenosine homocysteine nucleoside enzyme (Pfs) gene to build the prokaryotic expression vector.Isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce expression of recombinant proteins.The culture medium,induction temperature,the density of bacteria,IPTG concentration and inducing time were optimized to get the high expression of Pfs protein,and finally synthesized signal molecule AI-2 in vitro.[Results] Ten strains of lactic acid bacteria could produce AI-2,and the relative luminescence intensity of AI-2 secreted by Enterococcusfaecium 8-3 was obviously higher than other strains.The optimal inducing condition of the recombinant protein was as follows:when the OD600 was 0.5-0.7,using SOC medium as the induction medium,the induction were initiated with 0.1 mmol/L IPTG at 37 ℃ for 12 h;The optimal inducing condition was used to induce the target protein and obtained the purified Pfs protein with the concentration of 4.08 g/L,and successfully synthesized AI-2 in vitro.[Conclusion] The strains of ten lactic acid bacteria isolated from Koumiss could produce AI-2 and the signal molecule AI-2 of Enterococcusfaecium 8-3 could be synthesized by Pfs gene.