牛种布鲁氏菌2308强毒株dsbG基因缺失突变株的构建与初步评价

Construction and preliminary evaluation of dsbG gene deficient Brucella abortus 2308 virulent strain

目的研究dsbG基因对布鲁氏菌2308毒力的影响。方法以布鲁氏菌2308亲本株为模板,通过同源重组和抗性替代的方法,构建布鲁氏菌dsbG基因缺失株(2308ΔdsbG)。将毒力株2308、疫苗株RB51、2308ΔdsbG缺失株在相同起始浓度下恒温振荡培养,观察其生长变化趋势;将各菌株按200∶1的感染复数侵染人胚胎滋养层细胞(HPT-8),比较其在胞内的生存能力和粘附侵袭力。结果成功构建并获得了布鲁氏菌dsbG缺失株,且10代内未发现回复现象,遗传性较稳定。2308ΔdsbG缺失株与2308亲本株相比在体外具有相似的生长趋势,但其对宿主细胞的粘附侵袭和胞内的繁殖能力显著低于亲本株。结论 dsbG基因在布鲁氏菌的致病能力中发挥重要作用,dsbG基因的缺失显著降低了牛种布鲁氏菌2308的粘附侵袭和胞内生存能力。

To explore the role of dsbG genes on the virulence of Brucella abortus 2308.We regarded 2308 as a template then applied homologous recombination and resistant alternative method to construct Brucella abortus 2308(referred to 2308)dsbG gene mutant strain(2308ΔdsbG).In order to observe the growth status of Brucella,the B.abortus 2308 and 2308ΔdsbG were cultured under the same condition;the embryonic trophoblast cells(HPT-8)were infected with each strain at a multiplicity of infection(MOI)of 200∶1 to compare their viability and adhesion invasiveness.The results showed that the 2308ΔdsbG mutant strains were successfully constructed and obtained.The 2308ΔdsbG did not occur mutation and keep genetic stability after transfer 10 generations on Tryptic soy agar(TSA).The growth status and intracellular viability of 2308ΔdsbG were similar to parental strain,but the adhesion to the host cells and the number of cells in the cells were significantly lower than the parental strains.It is indicated that the dsbG gene plays an important role in influencing the pathogenicity of Brucella.The deletion of dsbG gene significantly reduces the adhesion invasiveness and intracellular viability of Brucella species 2308.