羊种布鲁菌lpsA基因缺失株的构建及生物学特性研究

Construction of Brucella lpsA Gene Deletion Strain and ITS Biological Characteristics

为构建羊种布鲁菌lpsA基因缺失株并分析其对胚胎滋养层细胞(HPT-8)的损伤作用及引起炎症反应的因子变化。以羊种布鲁菌M5-90株为模板,同源重组和抗性替换法构建自杀载体,电穿孔法转入M5-90感受态细胞构建lpsA基因缺失株M5-90ΔlpsA,将lpsA基因亚克隆至pBBR1MCS4载体电转至M5-90ΔlpsA感受态细胞构建回补株M5-90ΔlpsA-C,对缺失株及回补株的体外生长、胞内生存、粘附力等生长特性和感染HPT-8产生的细胞毒性及细胞因子变化进行分析。结果显示,成功构建缺失株和回补株,体外生长趋势与亲本株相比,缺失株M5-90ΔlpsA粘附力较低且对环境的应激能力降低;在侵染HPT-8细胞12 h后缺失株生存率显著低于亲本株,细胞毒性降低,且诱导的细胞因子IL-1β和IL-18均比亲本株高,差异显著;回补株M5-90ΔlpsA-C恢复了这些表型变化。本研究表明lpsA基因在羊种布鲁菌M5-90株的致病能力中发挥了重要作用,为进一步揭示布鲁菌生物特性及感染宿主细胞分子机制奠定基础。

To construct the lpsA gene deletion strain of Brucella melitensis and analyze its damage to embryonic trophoblast cells(HPT-8)and the changes of factors that cause infl ammation.Using the M5-90 strain of Brucella melitensis as the template,the suicide vector was constructed by homologous recombination and resistance replacement method,and the M5-90 competent cells were transformed into M5-90 competent cells by electroporation to construct the lpsA gene deletion strain M5-90ΔlpsA.The lpsA gene was subcloned into pBBR1MCS4 vector and electrotrans formed into M5-90ΔlpsA competent cells to construct a complement strain M5-90ΔlpsA-C.The in vitro growth,intracellular survival,adhesion and aggregation and other growth characteristics of the deletion strains and the complement strains,as well as the cytotoxicity,cytokine changes produced by HPT-8 infection were analyzed.The results showed that the deletion strain and the complement strain were successfully constructed.Compared with the parent strain M5-90,the in vitro growth trend of the deletion strain M5-90ΔlpsA has lower adhesion and aggregation ability and lower stress ability to the environment.The survival rate of the deletion strain was significantly lower than that of the parent strain 12 h after infection of HPT-8 cells,the cytotoxicity was reduced,and the induced cytokines IL-1βand IL-18 were both higher than the parent strain,with significant differences.However,the complement strain M5-90ΔlpsA-C restored these phenotypic changes.This study showed that the lpsA gene played an important role in the pathogenicity of Brucella melitensis M5-90 strain,and laid the foundation for further revealing the biological characteristics of Brucella and the molecular mechanism of host cell infection.