摘要
目的本研究构建了羊布鲁菌16M DK 63_426基因缺失株,分析其在细胞内的生存繁殖和致炎能力。方法以羊布鲁菌16M株基因组为模板,利用融合PCR技术将DK 63_426基因的上下游同源臂和卡那抗性基因融合,连接到克隆载体上,通过基因重组构建布鲁菌16M DK 63_426基因缺失株16MΔDK 63_426,观察布鲁菌亲本株16M和16MΔDK 63_426的生长变化趋势,建立侵染小鼠巨噬细胞RAW264.7模型,检测亲本株和缺失株16MΔDK 63_426在胞内的生存能力,检测IL-6、TNF-α细胞因子释放量。结果成功构建了16MΔDK 63_426;在体外相同培养条件下该缺失株与亲本株生长趋势相似,培养12 h即进入对数生长期,30 h进入平台期;16MΔDK 63_426在浸染RAW264.7细胞12 h胞内存活率极显著低于亲本株(P<0.01),在浸染8 h时IL-6的分泌量缺失株组均显著低于亲本株(P<0.05),TNF-α的分泌量缺失株组均显著高于亲本株(P<0.05),在侵染12 h时IL-6的分泌量缺失株组均极显著低于亲本株(P<0.01),TNF-α的分泌量缺失株组均极显著高于亲本株(P<0.01)。结论DK 63_426基因的缺失可影响布鲁菌的胞内存活,影响相关炎症因子的表达,为布鲁菌感染机制的研究奠定理论基础。
Objective In this study,16M DK 63_426 gene deletion strain of Brucella melitensis was constructed,and the survival,reproduction and inflammation ability of the deleted strains in cells were analyzed.Methods The genome of 16M strain of Brucella melitensis was used as a template,fusion of upstream and downstream homologous arms of DK 63_426 Gene with kanamycin resistance gene by fusion PCR and connected to the cloning vector.Construction of 16M DK 63_426 gene deletion strain of Brucella melitensis by gene recombination,Observed the Growth Trend of Brucella melitensis parent strains 16M and 16M DK 63_426,Established the infection model of mouse macrophages RAW264.7,tested the viability of parents and deleted strain 16M DK 63_426 in cells,and measured the amount of release of IL-6 and TNF-αcytokines.Results Show that 16M DK 63_426 gene deletion strain has been successfully constructed;Under the same culture conditions in vitro,the growth trend of the deleted strain was similar to that of the parent strain.After 12 hours of culture,both of them entered the logarithmic growth phase and after 30 hours into the plateau phase.The survival rate of 16M DK 63_426 gene deletion strain in RAW264.7 cells was extremely significantly lower than the parent strain(P<0.01)at 12 h.The amount of release of IL-6 about deletion strain was significantly lower than the parent strain(P<0.05)and the amount of release of TNF-αabout deletion strain was significantly higher than parent strain(P<0.05)at 8 h of infection.The amount of release of IL-6 about deletion strain was extremely significant lower(P<0.01)than the parent strain and the amount of release of TNF-αabout deletion strain was extremely significant higher than parent strain(P<0.01)at 12 h of infection.Conclusion Showed that the deletion of DK 63_426 gene could affect the survival and the expression of related inflammatory factors of Brucella melitensis,which laid a theoretical foundation for the study of the mechanism of Brucella melitensis infection.
作者
李明奇
王小凤
郭嘉
赵天艺
刘航
张樊
吴长新
张辉
LI Mingqi;WANG Xiaofeng;GUO Jia;ZHAO Tianyi;LIU Hang;ZHANG Fan;WU Changxin;ZHANG Hui(College of Animal Science&Technology/Key Laboratory of Xinjiang Endemic and Ethnic Disease,Shihezi University,Shihezi,Xinjiang 832003,China)
出处
《石河子大学学报（自然科学版）》
CAS
北大核心
2020年第1期31-36,共6页
Journal of Shihezi University(Natural Science)
基金
国际科技合作项目(GJHZ201709)
新疆生产建设兵团人才项目(CZ027202,2017CB002)
