贝氏柯克斯体类脂A修饰突变株的转化与鉴定

Transformation and identification of lipid A modified Coxiella burnetii mutant

目的基于遗传克隆后的电转化技术,探索构建贝氏柯克斯体脂多糖修饰突变的实验室研究模型。方法设计并分子克隆引入衣原体KDO基因构建穿梭质粒,电转化贝氏柯克斯体后以半固体无生命培养克隆纯化并扩大培养,进一步用相差显微镜观察抑制剂LPC-011对贝氏柯克斯体的影响,利用qPCR定量对比测定转化突变与野生株在细胞或无细胞条件下的生长曲线并在Vero细胞中测得感染性集落形成单位。结果载体pCBGkdtA可稳定转化贝氏柯克斯体,LPC-011对贝氏柯克斯体最小抑菌浓度可能大于10μg/mL,类脂A修饰后的贝氏柯克斯体生长繁殖特性较野生型无显著改变。结论成功构建了贝氏柯克斯体类脂A修饰株CBkdtA并对其生长繁殖与感染特性进行了鉴定,本研究为进一步在动物模型中探究脂多糖的致病机理提供了新方法。

Objective To explore an experimental laboratory model of lipopolysaccharides modified Coxiella mutant,based on the electroporation technique of post genetic cloning.Methods The shuttle vector was constructed by designing and introducing the chlamydial KDO gene through molecular cloning.After electroporating Coxiella burnetii,clonal purification and expansion were performed using a semi-solid axenic medium.Additionally,the effects of LPC-011 inhibitors on Coxiella were observed using phase-contrast microscopy.Comparative growth curves of the transformed mutant and wild strain were measured under cellular and axenic conditions using qPCR quantitative analysis,and the infectivity of CFUs was detected in Vero cells.Results The shuttle vector pCBGkdtA can stably transform Coxiella.The minimal inhibitory concentration(MIC)of LPC-011 on Coxiella may be greater than 10μg/mL.Compared with the wild strain,the growth and propagation character of lipid A-modified Coxiella did not chang apparently.Conclusion CBkdtA,lipid A modified Coxiella transformant was successfully constructed,moreover,both its property of reproducing and infecting were identified.The study is provides a new method of further exploring the pathogenic mechanisms of Coxiella lipopolysaccharides in animal models.