蜡样芽胞杆菌plcR基因功能及其无义突变株特征分析  

Biological characteristics of Bacillus cereus plcR gene nonsense mutant strain

作　　者：王晓景 王东澍 吕宇飞 冯尔玲 潘超 朱力 王恒樑 刘先凯 WANG Xiao-jing;WANG Dong-shu;LÜYu-fei;FENG Er-ling;PAN Chao;ZHU Li;WANG Heng-liang;LIU Xian-kai(Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China)

机构地区：[1]军事科学院军事医学研究院生物工程研究所,北京100071

出　　处：《军事医学》2020年第2期116-121,共6页Military Medical Sciences

基　　金：国家自然科学基金(81571958,81671979);“艾滋病和病毒性肝炎等重大传染病防治”国家科技重大专项(2018ZX10714002)

摘　　要：目的分析比较蜡样芽胞杆菌HN001与plcR基因无义突变株HN1M的生物特征,并研究蜡样杆菌中plcR基因的功能。方法绘制生长曲线比较蜡样杆菌HN001和HN1M的生长性能;鉴别培养基平板比较两菌株产胞外酶能力;结晶紫染色法测定两菌株生物膜形成;RT-PCR分析两菌株中溶血酶、磷脂酶和肠毒素基因的转录水平;以C57BL/6N小鼠为动物模型比较两菌株的毒力大小。结果与HN001相比,HN1M稳定期菌密度和生物膜形成能力显著升高,溶血能力、产卵磷脂酶能力和对小鼠致病性显著减弱;HN1M中毒力基因hlyD、clO、hly、cerA、cerB、plcA、cytK、nhe和hblCDB转录水平显著下调;tlyA和hblA转录水平无显著差异。结论与HN001相比,蜡样杆菌plcR基因无义突变株HN1M溶血酶和磷脂酶的活性明显降低,毒力明显减弱,该研究为开发蜡样杆菌工程菌和研究plcR的功能提供了新思路。Objective To analyze and compare the differences in biological characteristics of Bacillus cereus HN001 and its plcR gene nonsense mutant strain HN1 M,and study the function of plcR gene in B.cereus.Methods HN001 and HN1 M strains were cultured under the same condition to contrast their growth rate by drawing growth curves.The ability of these strains to produce extracellular enzymes was compared using the identification medium plate.The biofilm formation ability of these strains was determined by crystal violet staining.The transcription levels of hemolysin genes,phospholipase genes and enterotoxin genes were analyzed by RT-PCR.The virulence of these strains was evaluated in a C57 BL/6 N mouse model.Results Compared with B.cereus HN001,the optical density in the stationary phase and the biofilm-forming ability of HN1 M increased significantly,while the hemolytic and lecithinase capacity,and the pathogenicity to mice were significantly reduced.The transcription levels of genes hlyD,clO,hly,cerA,cerB,plcA,cytK,nhe and hblCDB in HN1 M were significantly down-regulated,and the transcription levels of genes tlyA and hblA did not change significantly.Conclusion Compared with HN001,the hemolytic and phospholipase activity of HN1 M is significantly reduced,and its virulence is significantly contained.This study provides a new idea for the development of engineered strains of B.cereus and the study of functions of plcR regulon.

关 键 词：蜡样芽胞杆菌 plcR调节子 溶血素蛋白质类 磷脂酶类 生物膜 肠毒素类 毒力 基因表达调控 

分 类 号：R378.7[医药卫生—病原生物学] Q754[医药卫生—基础医学]