变异链球菌自诱导物2信号分子的体外合成与活性检测

目的：利用分子生物学的相关技术体外合成具有良好生物学活性的自诱导物2（AI-2）信号分子，为进一步研究S-核糖高半胱氨酸裂解酶（LuxS）/AI-2信号系统对于变异链球菌和口腔生物膜的致病性、耐药性的调节机制奠定基础。方法???构建稳定高效的LuxS和S-腺苷高半胱氨酸核苷酶（Pfs）原核表达载体，异丙基β-D-1-硫代吡喃半乳糖苷（IPTG）诱导表达目的蛋白，并进行镍柱层析分离纯化与鉴定。利用纯化表达的LuxS蛋白和Pfs蛋白体外合成AI-2信号分子，以哈维氏弧菌菌株BB170作为报告菌株，通过生物发光效应检测体外合成的变异链球菌AI-2信号分子的生物活性，并以此间接检测LuxS蛋白和Pfs蛋白的生物学活性。结果???体外合成的AI-2可诱导哈维氏弧菌菌株BB170强烈发光。以变异链球菌UA159标准株菌液中自然生成的AI-2为对照，体外合成AI-2诱导发光效果强于菌液。结论??体外合成的AI-2具有良好的生物学活性，获得了高效表达、可溶性的LuxS和Pfs重组蛋白。

Biosynthesis of autoinducer-2 and determination of its bioactivity in vitro

Objective This study aimed to produce bioactive autoinducer-2(AI-2) in vitro via molecular biological techniques, as well as provide a basis for further study on the cariogenic virulence regulation mechanism of AI-2 in Streptococcus mutans. Methods Effective prokaryotic recombinant systems for the expression and purification of S-ribosylhomocysteinase( LuxS) and S-adenosylhomocysteine nucleosidase(Pfs) proteins were constructed. After induction by isopropyl β-D-1-thiogalactopyranoside, expressing products were purified with the Ni-NTA spin column. AI-2, which is an informational molecule in quorum sensing, was produced in vitro using purified LuxS and Pfs. The bioactivity of AI-2 was tested by Vibrio harveyi BB170 luminescence bioassay, which indirectly proved the bioactivities of purified LuxS and Pfs. Results AI-2 produced in vitro could significantly induce the luminescence of Vibrio harveyi. Furthermore, its luminous intensity was much higher than that induced by the AI-2 produced naturally by a bacterial colony of Streptococcus mutans UA159. Conclusion An efficient expression system for soluble recombinant proteins of LuxS and Pfs was established. The highly active AI-2 was manufactured, which could provide essential materials for further research.