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【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。
【Objective】 To identify the biological characteristics of a newly isolated Pseudomonas aeruginosa phage PaP4. 【Method】 A single plaque of PaP4 was prepared by double agar culture. The plaque characteristics were observed. PaP4 particles were concentrated by polyethylene glycol 8000 and then purified by cesium chloride density gradient centrifugation. Transmission electron microscopy was used to observe the negative Stained PaP4 particles; extraction of PaP4 genomic nucleic acid, by restriction enzyme analysis of the nucleic acid type; according to the multiplicity of infection (MOI) were 0.000 1,0.001,0.01,0.1,1 and 10 added phage pure culture medium and host bacteria After sufficient bacterial lysis, phage titers were measured; phage and host bacteria were added at a ratio of MOI = 10, and one-step growth experiments were performed to draw a one-step growth curve. 【Result】 The PaP4 plaque diameter was about 3 mm 5 mm and the circular transparent edge was clear. The PaP4 phage showed a polyhedron with a symmetrical head and a diameter of about 50 nm with a short tail of about 30 nm. The PaP4 genome was double-stranded DNA. When the MOI was 0.001, PaP4-infected host bacteria produced the highest titer of offspring phage. The growth characteristics were characterized by a one-step growth curve. 【Conclusion】 PaP4 is a dsDNA short tail family lysate bacteriophage. The optimal multiplicity of infection is 0.001. The incubation period of the infected host strain is 25 min and the lysis period is 20 min. The average lysis amount is 150.