迟缓爱德华菌luxS基因在不同生长时期表达水平的差异

Expression of Edwardsiella tarda lux S gene at different growth stage

【目的】探索迟缓爱德华菌Lux S/AI-2型密度感应系统关键基因lux S的分布及在不同生长时期的表达特征和生物功能。【方法】克隆迟缓爱德华菌lux S基因,利用生物信息学工具和网络数据库分析该基因序列的特征及编码蛋白的基本特征和保守结构;原核表达Lux S蛋白,纯化后制备抗Lux S的抗体,运用Western-blot技术分析Lux S在不同毒力、不同来源迟缓爱德华菌中的分布情况及在不同生长时期的表达水平;利用抗体中和方法,分析抗Lux S抗体对迟缓爱德华菌生长的影响,探索Lux S是否为信号分子AI-2的特异依赖模式。【结果】克隆到迟缓爱德华菌lux S基因,长度为516 bp,序列分析结果表明,该基因在迟缓爱德华菌属中高度保守;对多株迟缓爱德华菌Lux S蛋白的检测结果表明,该基因在该菌属中普遍存在;对不同生长时期Lux S蛋白的检测结果表明,Lux S蛋白的表达量在迟缓期较低,进入对数生长期逐渐增加,在对数生长后期最大,稳定后期逐渐减少;抗体中和生长试验结果表明,1%抗血清(效价1:40000)能延长迟缓爱德华菌生长的平台期,但对细菌生长无显著影响。【结论】Lux S/AI-2介导的密度感应系统在迟缓爱德华菌中普遍存在;关键基因lux S的序列高度保守,在不同生长时期表达量不一致,在对数生长后期达到峰值。

[ Objective] Purpose of this work was to explore the distribution of LuxS/AI-2 quorum sensing system in Edwardsiella, and analyze expression characteristics and biological function of the key gene luxS accompanying the growth of Edwardsiella. [ Methods ] The full-length of AI-2/LuxS of Edwardsiella tarda was cloned by PCR based on the sequence on NCBI, then characteristics and conservative structure of this protein-coding gene were analyzed using web database and bioinformatics tools. The anti-rabbits serum was prepared after this protein was purified through prokaryotic expression. The expression level of luxS gene was analyzed during different growth stages using Western blot and further the distribution of luxS gene in Edwardsiella tarda was studied by this technique. To explore whether the specific LuxS is AI-2 dependent we used the method of antibody neutralization to analyze the effect of the anti-rabbits serum on the growth of Edwardsiella tarda. [ Results] The luxS gene was obtained by PCR, its length was 516 bp, and the sequence was highly conserved in Edwardsiella tarda. Results of Western blot analysis showed that LuxS expression level was the lowest in the lag phase and began increasing when entered index phase. It reached the peak in the late index phase and decreased in decline phase. Moreover, Antibody neutralization results showed that, it can elongate the growth plateau phase, but it has no significant effect on bacterial growth. [ Conclusion] The key gene of luxS was highly conserved, and LuxS/AI-2 was widely distributed among Edwardsiella tarda. The expression level of luxS gene was different during every growth period, expression of LuxS protein reached the highest level in the late index phase.