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目的分离鉴定新分枝杆菌噬菌体并研究其生物学特性。方法以平板法从泥土中分离纯化噬菌体,紫外线诱导法鉴定溶原性,从噬菌斑大小、颗粒形态和限制性内切酶分析三方面比较Chy2、D29、TM4异同;以不同感染复数(MOI)扩增Chy2,测定最佳MOI;通过一步生长实验测出Chy2潜伏期、裂解期和裂解量;采用单斑法测定MOI的裂解谱;检测MOI在不同pH值培养基中裂解耻垢分枝杆菌mc2155的能力。结果成功分离纯化1株分枝杆菌噬菌体,命名为Chy2,其噬菌斑圆形透明,紫外线诱导法处理宿主菌后不能形成噬菌斑,头部呈椭圆形,长尾,基因组核酸能被双链DNA内切酶EcoRⅠ、HindⅢ、BamHⅠ及XbaⅠ切开,大小约49 kb,与D29、TM4差异大;Chy2最佳MOI为9.58×10-5;Chy2一步生长周期为270 min,潜伏期210 min,裂解期60 min,裂解量为23;Chy2能裂解耻垢分枝杆菌mc2155、结核菌标准株H37Rv、多数临床耐药株;在7H9固体培养基pH值为5.0和7.4时,Chy2均能裂解耻垢分枝杆菌mc2155。结论 Chy2属长尾噬菌体科,基因组为双链DNA,宽噬谱广,是一种潜在的抗结核药物资源。
Objective To isolate and identify new Mycobacterium phage and study its biological characteristics. Methods The phage were isolated and purified from soil by plate method and the lysogenicity was identified by UV induction. The similarities and differences of Chy2, D29 and TM4 were compared in terms of plaque size, particle morphology and restriction enzyme analysis. ) To amplify Chy2 and determine the optimal MOI. The Chy2 latency, lysis and lysis were measured by one-step growth assay. The lysis pattern of MOI was determined by monolayer assay. MOI was detected in different pH mediums for cleavage of M. smegmatis mc2155’s ability. RESULTS: One Mycobacterium phage was successfully isolated and purified, named Chy2. The phage plaque was circular and transparent. The plastids were not formed after the host strain was treated by ultraviolet light. The head was oval and the tail was long. The genomic nucleic acid could be double Chy2 had the best MOI of 9.58 × 10-5; Chy2 had a one-step growth cycle of 270 min and an incubation period of 210 min, with the size of about 49 kb, which was different from that of D29 and TM4; Chy2 can cleave Mycobacterium smegmatis mc2155, Mycobacterium tuberculosis standard strain H37Rv, most of the clinical drug-resistant strains in the pyrolysis period of 60 min, the amount of cleavage was 23; Chy2 was able to be cleaved by shading at pH 7.0 and 7.4 of 7H9 solid medium Mycobacterium smegmatis mc2155. Conclusion Chy2 is a spermatophore of phage phage. The genome of Chy2 is double-stranded DNA with broad-spectrum and wide-spectrum. It is a potential anti-tuberculosis drug resource.