分枝杆菌噬菌体Chy2的分离及生物学特性

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。

Isolation and biological characteristics of mycobacteriophage Chy2

Objective To isolate and identify a new mycobacteriophage Chy2,and to explore its biological characteristics.Methods The new mycobacteriophage was isolated and purified from soil by single-plate method.The phage was induced by ultraviolet ray(UV) to identify its lytic or lysogenic capacity.Chy2,D29 and TM4 were compared by the plaque size,particle morphology and restriction enzyme analysis.Chy2 was amplified by double-layer agar plate method in different multiplicity of infection(MOI) to find the optimal MOI.One-step growth experiment was performed to find the latent period,burst period and burst size of Chy2.The host range of Chy2 was examined by single-spot examination.The ability of Chy2 to crack Mycobacterium smegmatis mc2155 at different pH values was examined.Results A new mycobacteriophage was successfully isolated and purified,which was named as Chy2.The plaques of Chy2 were round and transparent,and their sizes were about 3 mm in diameter.Plaques did not appear with the host bacteria induced by UV.Chy2 has an oval head and a long tail.The size of Chy2 genome was about 49 kb,which was different from that of D29 or TM4,and its genome can be digested by the restriction endonucleases of EcoRⅠ,HindⅢ,BamHⅠ and XbaⅠ.The optimal MOI and the highest titer of Chy2 were 9.58×10-5 and 7.2×1010 PFU/ml,respectively.The one-step growth cycle of Chy2 was 270 min,with the latent period 210 min,the burst period 60 min and the burst size 23.Chy2 could crack Mycobacterium smegmatis mc2155,Mycobacterium tuberculosis H37Rv and a majority of clinical drug-resistant bacterial strains.Chy2 could crack Mycobacterium smegmatis mc2155 in solid medium at pH 5.0 and pH 7.4.Conclusion Chy2 belongs to Siphoviridae family and has genome of double-stranded DNA.Chy2 has a broad host range and,therefore,has a potential role in anti-tuberculosis treatment.