铜绿假单胞菌噬菌体PaP4的分离与鉴定

Isolation and identification a novel Pseudomonas aeruginosa phage PaP4

摘要:

【目的】鉴定一株新分离的铜绿假单胞菌噬菌体PaP4的生物学特性。【方法】双层琼脂培养法制备PaP4的单个噬斑, 观察噬斑特点; 用聚乙二醇8000浓缩PaP4颗粒后, 再用氯化铯密度梯度离心纯化; 用透射电子显微镜观察磷钨酸负染色的PaP4颗粒; 提取PaP4基因组核酸, 通过限制性内切酶图谱分析其核酸类型; 按照感染复数(MOI)分别为0.000 1、0.001、0.01、0.1、1和10加入噬菌体纯培养液和宿主菌, 充分裂解细菌后, 测定噬菌体滴度; 以MOI=10的比例加入噬菌体及宿主菌, 进行一步生长实验, 绘制一步生长曲线。【结果】PaP4的噬斑直径约3 mm?5 mm, 圆形透明边缘清晰; PaP4噬菌体呈多面体立体对称的头部, 直径约50 nm, 有一个约30 nm的短尾; 限制性酶切实验表明PaP4基因组为双链DNA; 当MOI为0.001时PaP4感染其宿主菌产生的子代噬菌体滴度最高; 用一步生长曲线描绘了其生长特性。【结论】PaP4属dsDNA短尾科裂解性噬菌体; 最佳感染复数是0.001; 由一步生长曲线得出感染宿主菌的潜伏期是25 min, 裂解期是20 min, 平均裂解量是150。

Abstract:

[Objective] To isolate and identify a novel Pseudomonas aeruginosa phage named PaP4 via its biological properties. [Methods] We used the double-layer agar culture method to make a single plaque. Phage particles of PaP4 were purified with CsCl2 gradient centrifugation and the ultrastructure of PaP4 was examined by electron microscopy. We extracted the genome of PaP4 and identified the type of nucleic acid with restriction enzyme analysis. The host bacteria were infected with PaP4 at six different MOI. Three hours and thirty minutes later, the phage titers were determined, respectively. For one step growth experiments, phages and its host with an MOI of 10 were used with a 15 min adsorption. Following centrifugation, the pellet was washed twice and resuspended with LB broth and incubated. Samples were taken at 15 min-intervals (up to 120 min) and immediately tittered by the double layer agar plate method. [Results] The plaques of PaP4 were transparent and their sizes were about 3 mm?5 mm in diameter. PaP4 had an isometric head approximately 50 nm in diameter and a long tail with 30 nm in length. 0.001 MOI-infected host bacteria gave the highest phage offsprings. One-step growth curve was determined according to the results of one-step growth experiments. [Conclusion] PaP4 is a virulent phage and belongs to Podoviridae. The whole genome can be digested by the restriction enzyme, indicating that PaP4 is a dsDNA virus. The optimal MOI of PaP4 is 0.001 and one-step growth experiment shows latent and burst periods are 25 min and 20 min, respectively, burst size is 150.