北京和非北京基因型结核分枝杆菌生长曲线及mazEF系统的研究

Growth curves and expression of Mycobacterium tuberculosis Beijing and non-Beijing genotype and the mazEF system

目的研究北京与非北京基因型结核分枝杆菌的生长曲线、mazEF3,6,9系统表达有无差异。方法测绘上述菌株及其mazEF3,6,9基因缺失和过表达菌株在标准、低氧和营养饥饿培养条件下的生长曲线。使用qRT-PCR技术检测各菌株的mazEF3, 6, 9、mazF3, 6,9和mazE3, 6,9基因mRNA表达水平;利用Western Blot技术检测MazF9蛋白表达水平。结果在标准和低氧条件下培养的第4、6、8、10 d,营养饥饿条件下培养的第2、4、6、8 d,北京基因型菌株的OD值(细菌数量)均高于非北京基因型菌株,差异有统计学意义(P<0.05);在上述3种培养条件下,北京和非北京基因型mazEF3,6,9缺失突变株和过表达株菌数均低于其亲本株。在mRNA、蛋白水平上,与非北京基因型菌株相比,北京基因型菌株mazEF3高表达9.55倍、mazF3高表达4.86倍、mazF6高表达2.64倍、mazF9高表达5.27倍和mazE9低表达0.16倍;mazF9蛋白高表达1.62倍,差异均具有统计学意义(P<0.05)。结论北京与非北京基因型结核分枝杆菌的生长曲线、mazEF3,6,9系统表达存在差异,mazEF系统与北京基因型菌株流行相关。

To study the difference in the growth curves and expressionlevels of mazEF3,6,9 systems between Beijing and non-Beijing genotype Mycobacterium tuberculosis(MTB)strains,growth curves of Beijing and non-Beijing genotype strains as well as their mazEF3,mazEF6,mazEF9 genes deletion and overexpressing were surveyed and drawn in standard,hypoxia and nutrient starvation culture conditions.The mRNA expression levels of mazEF 3,6,9,the mazF 3,6,9 toxin genes,and the mazE 3,6 and 9 antitoxin genes in all strains were detected using Quantitative real time-PCR(qRT-PCR).Expression level of the MazF9 was determined by Western Blot.Results showed that OD values(numbers of the strains)of Beijing genotype strains were higher than that of non-Beijing genotype strains on the 4th,6th and 10th days under standard and hypoxia culture conditions,and on the 2nd,4th and 8th day under nutritional hunger culture condition,differences were significant(P<0.05).The numbers of Beijing and non-Beijing genotype strains with mazEF3,mazEF6,mazEF9 genes deletion and overexpressing were fewer than that of their parents under the above three culture conditions.At the mRNA and protein levels,compared with non-Beijing genotype strains,the mazEF3 has a higher expression of 9.55 times,a mazF3 higher expression of 4.86 times,a mazF6 higher expression of 2.64 times,a mazF9 higher expression of 5.27 times,a mazE9 lower expression of 0.16 times and mazF9 protein is higher expression 1.62 times in Beijing genotype strains,and the differences were statistically significant.There are significant differences in the growth curves and mazEF system expression levels between Beijing and non-Beijing genotype strains.MazEF system is related to the prevalence of Beijing genotype strains.