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
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摘要
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 ofdsbG gene significantly reduces the adhesion invasiveness and intracellular viability of Brucella species 2308.
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 ofdsbG gene significantly reduces the adhesion invasiveness and intracellular viability of Brucella species 2308.
引文
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[2] 彭英波.家畜布鲁氏菌病的疫情监测与启示[J].中国畜牧兽医文摘,2017,33(9):107.
[3] 徐晓阳.布鲁氏菌胞内生存相关sRNA的筛选及非编码小RNA BSR0441功能的初步研究[D].成都:四川农业大学,2016.
[4] 陆承平.兽医微生物学[M].4版.北京:中国农业出版社,2007:151.
[5] Eskra L,Mathison A,Splitter G.Microarray analysis of mRNA levels from RAW264.7 macrophages infected with Brucella abortus[J].Infect & Immunity,2003,71(3):1125-1133.DOI:10.1128/IAI.71.3.1125-1133.2003
[6] 李志强.布鲁氏菌二元调控系统TceSR和TcfSR的生物学功能研究[D].石河子:石河子大学,2015.
[7] Shao F,Bader MW,Jakob U,et al.DsbG,a protein disulfide isomerase with chaperone activity[J].J Biol Chem,2000,275(18):13349-13352.DOI:10.1074/jbc.275.18.13349.
[8] Moreno E,Cloeckaert A,Moriyón I.Brucella evolution and taxonomy[J].Vet Microbiol,2002,90(1):209-227.
[9] Hui Z,Dou X,Li Z,et al.Expression and regulation of the ery operon of Brucella melitensis in human trophoblast cells[J].Exp Ther Med,2016,12(4):2723.
[10] 张众,黄华樑.大肠杆菌二硫键形成相关蛋白的结构、功能及其在基因工程表达外源蛋白上的应用[J].生物工程学报,2002,18(3):261-266.
[11] Heras B,Edeling M A,Schirra H J,et al.Crystal structures of the DsbG disulfide isomerase reveal an unstable disulfide[J].Pantl Acad Sci USA,2004,101(24):8876.