炭疽芽胞杆菌sigB基因缺失株的构建与研究  

Construction of sigB deletion mutant of Bacillus anthracis vaccine strain A16R

作　　者：顾梦恩 陈蒙 王东澍 吕宇飞 王恒樑[1,2] 刘先凯 GUMeng-en;CHEN Meng;WANG Dong-shu;LU Yu-fei;WANGHeng-liang;LIU Xian-kai(College of Food Science and Technology,Shanghai Ocean University,Shanghai 201306,China;State Key Laboratory of Pathogen and Biosecurity,Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China)

机构地区：[1]上海海洋大学食品学院,上海201306 [2]军事科学院军事医学研究院生物工程研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《军事医学》2019年第1期57-62,共6页Military Medical Sciences

基　　金：艾滋病和病毒性肝炎等重大传染病防治专项(2018ZX10714002);国家自然科学基金(81571958)

摘　　要：目的构建炭疽芽胞杆菌A16R株sigB基因缺失突变株,通过分析其表型,为后续的炭疽杆菌生命过程展开系统性研究奠定基础。方法采用同源重组技术以sigB为目标缺失基因,利用Golden-gate法构建含同源臂的打靶载体,结合基于I-SceⅠ酶驱动的芽胞菌高效敲除体系,筛选出炭疽杆菌sigB缺失突变株。通过生长曲线绘制、芽胞形成率测定、糖代谢能力比较,以及抗逆性实验等进行表型分析研究。结果成功构建了炭疽杆菌sigB缺失突变株,发现SigB对细菌生长能力、芽胞形成率和糖代谢能力均无显著影响。在抗逆性实验中,经不同浓度H2O2作用下,与野生株相比,sigB缺失突变株抑菌环无明显差异。经过48与55℃处理后,与A16R野生株比较,sigB缺失突变株菌落数减少,说明sigB缺失突变株抗热性减弱。结论成功获得了炭疽杆菌sigB基因缺失突变株。该研究表明SigB不影响芽胞形成,对细菌的抗热性有显著影响。Objective To construct a strain sigB gene mutant of Bacillus anthracis A16R strain and analyze its phenotypein order to facilitate the systematic study of the life cycle of B.anthracis. Methods The homologous recombination methodwas used to delete the targeted gene, sigB. The targeting vector containing the homology arm was constructed by Golden-gate method, and the strain sigB mutation was selected based on the I-SceⅠ endonuclease efficient gene knockout system.The phenotypic analysis was performed by measuring the growth curve and the rate of sporulation, and comparing the glucose metabolism ability and the stress resistance test. Results The strain sigB mutant of B.anthracis was successfully constructed. Our result showed that SigB had no significant effect on bacterial growth, sporulation efficiency or glucose metabolism. In the stress resistance experiment, the strain △sigB was treated with different concentrations of H2O2. The antibacterial ring in the strains △sigB was slightly smaller than that of the wild-type strain.After 48 and 55℃ treatment, there werefewer colony-forming units(CFU)in the strain △sigB than in the A16R wild strain. The result indicated that the heat resistance of the strain △sigB was weakened. Conclusion The strain sigB mutant of B.anthracis is successfully obtained. Thisstudy shows that SigB does not affect spore formation,but has a significant effect on heat resistance.

关 键 词：芽胞杆菌 炭疽 SIGB 抗热性 基因缺失 

分 类 号：R378.72[医药卫生—病原生物学]