布鲁氏菌非编码小RNA BSR1526突变株与过表达株的构建及毒力研究  

Construction of mutant and overexpression strains with small noncoding RNA BSR1526 and study of its virulence in Brucella

作　　者：徐晓阳[1] 欧红萍[2] 彭广能[1] 雷霜霜 田一男 曹雪峰[1] 王玉飞[3] 严光文[4] 钟志军[1] XU Xiao-yang OU Hong-ping PENG Guang-neng LEI Shuang-shuang TIAN Yi-nan CAO Xue-feng WANG Yu-fei YAN Guang-wen ZHONG Zhi-jun(College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China Chengdu Vocational College of Agricultural Science and Technology, Chengdu 611130, China Department of Laboratory Medicine, the General Hospital of Chinese People＇s Armed Police Forces, Beijing 100039, China College of Veterinary Medicine, Najing Agricaltural University, Najing 210095,China)

机构地区：[1]四川农业大学动物医学院,动物疫病与人类健康四川省重点实验室,成都611130 [2]成都农业科技职业学院,成都611130 [3]武警总医院检验科,北京100039 [4]南京农业大学动物医学院,南京216095

出　　处：《中国人兽共患病学报》2016年第12期1083-1090,共8页Chinese Journal of Zoonoses

基　　金：国家自然科学基金资助项目(No.31000548);成都大熊猫繁育研究基金会项目(No.CPF2015-4);北京市自然基金(No.6122030);北京市科技新星资助项目(No.Z131102000413062)联合资助

摘　　要：目的为探讨非编码小RNA BSR1526在布鲁氏菌胞内生存中的作用,构建BSR1526突变株与过表达株并分析它们在模拟胞内环境和小鼠体内的存活能力。方法首先采用Northern blot和RT-PCR验证布鲁氏菌16M在体外刺激条件中BSR1526的转录;然后采用融合PCR构建BSR1526缺失株;将BSR1526插入到质粒pBBR1-MCS4中,电转入16M中构建过表达株16M-BSR1526;最后分析BSR1526缺失株和过表达株在模拟胞内环境以及小鼠体内的存活力。结果 BSR1526在布鲁氏菌16M中存在转录,且在不同刺激条件下转录水平不同。BSR1526缺失或过表达影响了布鲁氏菌16M在体外的生长能力,缺失后在热应激、氧化压力及酸性环境下的生存能力下降,在小鼠脾脏内的细菌数量显著下降,表明BSR1526影响了布鲁氏菌的毒力。结论非编码小RNA BSR1526影响着布鲁氏菌16M在胞内生存能力,缺失BSR1526后布鲁氏菌16M抵抗外界不良环境的能力下降,同时在小鼠体内的毒力下降。To evaluate the roles of small noncoding RNA BSR1526 in Brucellaintracellular survival,the mutant and overexpression strains of BSR1526 were constructed and their survival capabilities under stresses that simulate intracellular environment and that of in mice were analyzed.Firstly,BSR1526 was verified by Northern blot and its transcription under stress conditions was verified by RT-PCR.The mutant strains of BSR1526 were constructed by fusion PCR.BSR1526 was inserted into the plasmid pBBR1-MCS4,which transformed into Brucella melitensis 16 M to generate the overexpression strain16MBSR1526.At last,the survival capabilities of 16M-△BSR1526and 16M-BSR1526 in different stimuli resembling that Brucella probably encounters during infection and mice were analyzed.Our results showed that BSR1526 was transcribed in Brucella,and with different transcription levels under the stress conditions.Deletion or overexpression of BSR1526 affected the growth of Brucella in vitro. The 16M-△BSR1526showed reduced survival under heat shock,oxidative stress and acidic conditions,and significant reduction in mice,indicating that BSR1526 influenced the virulence of Brucella.In summary,small noncoding RNA BSR1526 affects intracellular survival ability of Brucella melitensis 16 M.The ability adapting to hostile environments and virulence in mice with mutant BSR1526 strain was decreased.

关 键 词：布鲁氏菌 非编码小RNA BSR1526 胞内生存 

分 类 号：R378[医药卫生—病原生物学]