变形链球菌UA159合成自诱导分子2及其影响因素的实验研究

In vitro biosynthesis of autoinducer 2 of Streptococcus mutans UA159 and observation of the influence factors

目的：原核表达纯化变形链球菌（S．mutans）UA159 LuxS 蛋白，体外合成有活性的自诱导分子2（AI-2）并观察其合成的影响因素。方法：用基因重组的方法构建表达载体 pET21 a（＋）-luxS，在大肠杆菌（E．coli）BL21（DE3）中诱导表达 S-核糖基高半胱氨酸酶（LuxS）融合蛋白，镍柱亲和层析纯化、蛋白质印迹法分析鉴定 LuxS 蛋白，透析复性。纯化的 LuxS 蛋白和 S-腺苷高半胱氨酸核苷酶（Pfs）蛋白催化 S-腺苷 L 高半胱氨酸[SAH]合成 AI-2，哈氏弧菌 BB170检测 AI-2活性，并观察 LuxS 蛋白浓度、pH、氟化钠对 AI-2合成的影响。结果：与对照组相比，随着 LuxS 蛋白浓度的升高，体外合成的 AI-2活性增大（P ＜0．001）；当 pH 介于6～10之间时，AI-2活性最强，超出此 pH 范围，AI-2活性明显降低（P ＜0．001）；当氟化钠浓度≥0．3％时，AI-2活性显著降低（P ＜0．05）。结论：利用基因工程技术可以合成具有生物活性的 S．mutans UA159 AI-2；AI-2合成的最适 pH 在6～10之间；氟化钠浓度≥0．3％对 AI-2的合成有抑制作用。

Objective：To synthesize autoinducer-2 by the clone and prokaryotic expression of Streptococcus mutans（S.mutans）UAl59 luxS gene and to observe the influence factors.Methods：The expression vector pET21 a（＋）-luxS of S.mutans UAl59 was transformed into Escheriehia coli BL2l（DE3）.The S-ribosylhomocysteinase（Luxs）expression was induced by IPTG.The His tag fusion protein was isolated by Ni-chelating column and identified by Western blotting.Finally the protein was renatured by dialysis method.S-ribosylhomo-cysteine （SAH）was catalyzed by s-adenosylhomocysteine nucleosidas （Pfs）and LuxS,and then AI-2 was syntheszed.The AI-2 activi-ty was examined by luminescence of Vibrio harveyi BB1 70 when the concentration of LuxS protein or pH（4 -1 2）or the concentration of sodium fluoride was changed in reaction mixes of AI-2 synthesis.Results：Compared with the control group,with the increase of LuxS protein concentration,the relative activity of in vitro synthesized AI-2 increased gradually（P 〈0.001 ）.When pH was between 6 -1 0, the relative activity of AI-2 were the highest,beyond the range of pH,the relative activity of AI-2 decreased（P 〈0.001 ）.When a final concentration of sodium fluoride was more than 0.3%,the luminescence values decreased（P 〈0.05）.Conclusion：LuxS fusion protein can promote the production of AI-2.Optimum pH for AI-2 biosynthesis in vitro must be between 6-1 0.Biosynthesis of AI-2 is inhibited by sodium fluoride with final concentration of more than 0.3%.