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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。
Objective To investigate the role of BSR1526 in the intracellular survival of Brucella, to construct BSR1526 mutant and overexpression strains and analyze their viability in mimicking intracellular environment and in mice. METHODS: Northern blot and RT-PCR were used to verify the transcription of BSR1526 in Brucella 16M stimulated in vitro. Then, BSR1526 deletion was constructed by fusion PCR. BSR1526 was inserted into plasmid pBBR1-MCS4 and electrotransformed into 16M Expressing strain 16M-BSR1526. Finally, the survival and viability of BSR1526-deficient and overexpressing strains in a simulated intracellular environment and in mice were analyzed. Results BSR1526 was transcribed in Brucella 16M and had different transcriptional levels under different stimulation conditions. The deletion or overexpression of BSR1526 affected the growth of Brucella 16M in vitro. After deletion, the viability of Brucella 16M in heat stress, oxidative stress and acidic environment decreased significantly, and the number of bacteria in spleen of mice significantly decreased, indicating that BSR1526 affected The virulence of Brucella. Conclusion The non-coding small RNA BSR1526 affects the viability of Brucella 16M in vitro. The ability of Brucella 16M to resist the adverse environment is decreased after BSR1526 is absent, while its virulence is reduced in mice.