马耳他布鲁氏菌sRNA Bmsr10缺失株的构建及其对毒力的影响

Construction of sRNA Bmsr10 deletion strain of Brucella melitensis and the effect on its virulence

为评估布鲁氏菌sRNA Bmsr10对布鲁氏菌毒力的影响,本研究以布鲁氏菌M28为亲本株,利用同源重组方法构建了稳定遗传的sRNA Bmsr10缺失株M28ΔBmsr10和回补株M28ΔBmsr10-com。对其生长曲线进行测定,结果显示与亲本株M28相比,M28ΔBmsr10体外生长速率无明显变化;将M28ΔBmsr10按MOI 100感染RAW264.7细胞后,分别于感染后6 h、24 h和48 h裂解细胞进行细菌计数,结果显示:与亲本株M28相比,M28ΔBmsr10感染24h后,在巨噬细胞内的分菌数显著低于M28;以1×10^6cfu/100μL/只感染小鼠后于不同时间采集小鼠脾脏进行细菌计数和称重,检测M28ΔBmsr10在小鼠体内的增殖水平,结果显示感染M28ΔBmsr10后各时间点小鼠脾脏分菌数(p<0.01)和脾脏重量(p<0.01)与M28相比均显著降低,表明sRNA Bmsr10缺失显著降低了M28在小鼠体内的增殖能力和引起的炎症反应。结果证明:sRNA Bmsr10缺失可显著降低布鲁氏菌的毒力。本研究为探究Bmsr10影响布鲁氏菌毒力的分子机制及疫苗开发奠定了重要基础。

In order to evaluate the effect of Brucella sRNA Bmsr10 on the virulence of Brucella,a stable inherited sRNA Bmsr10 deletion strain was constructed by using homologous recombination from the parent strain Brucella M28 in this study.The growth curve of M28ΔBmsr10 was measured,and no significant change in the vitro growth rate was observed between M28ΔBmsr10 and the parent strain M28.100 MOI of M28ΔBmsr10 and M28 strains were inoculated in RAW264.7 cells,respectively,bacteria counting was performed by using cell lysis at 6 hours,24 hours and 48 hours post infection.The results showed that the number of M28ΔBmsr10 in macrophages had significantly reduced than the M28 strain in macrophages at24 hours post-infection.Mice were inoculated with 100μL of 1×10^6 cfu M28ΔBmsr10 or M28 strains to evaluate the proliferation property,spleens of infected mouse were collected/at different time point for bacteria counting and body weight measurement.The results showed that the number of bacteriain spleen(p<0.01)and spleen weight(p<0.01)were significantly reduced at each time point in M28ΔBmsr10 infection group compared with that in M28 strain infection group.It showed that the deletion of sRNA Bmsr10 significantly reduced the proliferation property of M28 and caused inflammation in mice.The results prove that the deletion of sRNA Bmsr10 can significantly reduce the virulence of Brucella.This study laid an important foundation for exploring the molecular mechanism of Bmsr10 affecting Brucella virulence and vaccine development.