分支杆菌噬菌体Leo生物学特性及抗结核作用

目的 明确分支杆菌噬菌体Leo的生物学特性及其抗结核作用,为“鸡尾酒疗法”筛选候选噬菌体。方法 采用双层平板法制备噬菌体Leo,观察噬菌斑形态;电镜观察噬菌体颗粒形态;提取噬菌体DNA,限制性内切酶酶切分析确定其核酸类型;以不同MOI扩增噬菌体Leo,确定最佳MOI及最低MOI;通过一步生长曲线,确定潜伏期及裂解量;采用终点滴定反浊法测定耻垢分支杆菌对噬菌体Leo的耐受突变率;检测噬菌体Leo对酒精、温度的耐受性及在不同pH下的裂解能力;通过体外杀结核分枝杆菌实验,明确噬菌体Leo对结核分枝杆菌的杀灭作用。结果 Leo噬菌斑圆形透明,边缘清晰,含对称的多面体头部,直径(70±3.0)nm, 可弯曲的尾部,尾长(211±31.7)nm;其基因组能被限制性内切酶HindⅢ、BglⅠ切开;最佳MOI为0.0 0001,最低MOI为0.0 001,潜伏期为150 min,裂解量74,耐受突变率为10^-7。Leo对温度、酒精敏感,在pH 7.4和pH 5.0时均能裂解宿主菌。Leo作用于结核分支杆菌72 h后,Leo组菌量明显低于空白组(P <0.05)。结论 分支杆菌噬菌体Leo属长尾噬菌体科,双链DNA噬菌体,能够杀灭结核分支杆菌,可作为“鸡尾酒疗法”的候选噬菌体。

Biological characteristics of mycobacteriophage Leo and its bactericidal effect on Mycobacterium tuberculosis

To investigate the biological characteristics of mycobacteriophage Leo and its bactericidal effect on Mycobacterium tuberculosis for cocktail therapy, we observed mycobacteriophage plaques after amplifying phage by double-layer agar plate method. The morphological characteristics were observed by electron microscopy. Mycobacteriophage Leo DNA was extracted and then digested by restriction enzyme to identify the nucleic acid type. Leo was amplified in different multiplicity of infection (MOI) to find the optimal MOI and the minimum MOI. One step growth experiment was carried out to find the latent period and burst size of Leo. The frequency of Mycobacterium smegmatis mutation treated with mycobacteriophage Leo was inspected by the endpoint titration test. The effect of temperature and alcohol on Leo survival was surveyed. The ability of Leo to crack host bacteria at different pH values was examined. The effect of Leo on Mycobacterium tuberculosis was determined by bactericidal assay. Results showed that the Leo plaque was round and transparent with a diameter of 1.5 nm. Leo has an isometric head (70±3.0 nm in diameter) and a flexible tail (211±31.7 nm in length). Its genome could be digested by restriction enzyme of Hind Ⅲ and Bgl Ⅰ. The optimal MOI and the minimum MOI of Leo were 0.000 01 and 0.000 1, respectively. The mutation frequency was 10^-7. The latent period was 150 min, and the burst size was 74. Leo could not only crack host bacteria in solid medium at pH 7.4 but also at pH 5.0. After 72 hours, the amount of Mycobacterium tuberculosis in Leo group was less than that in control group (P<0.05). In conclusion, Leo has a bactericidal effect on Mycobacterium tuberculosis and could be a candidate of phage cocktails.