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目的研究噬菌体Legendre的生物学特性并初步探索其用于抗耐药结核的潜力。方法双层平板法制备Legendre的噬菌斑,观察其特点,纯化噬菌体,电镜观察Legendre形态;提取Legendre基因组,限制性内切酶酶切分析确定其核酸类型;以不同感染复数扩增Legendre,找出最佳MOI和最小MOI;通过一步生长实验找出Legendre潜伏期、裂解期和裂解量;纯化Legendre颗粒,免疫家兔,获得抗血清,通过中和反应实验测定Legendre以及其他8种噬菌体和Legendre抗血清之间的吸附反应常数K值;采用单斑法测定Legendre的宿主谱;检测Legendre对紫外线、温度、氯仿、酒精、酸碱度的耐受性。结果 Legendre的噬菌斑圆形透明,边界清楚,Legendre头部呈多面体立体对称,直径平均为65 nm,尾长平均为215 nm;基因组核酸能被双链DNA内切酶EcoRⅠ,HindⅢ及BamHⅠ切开,大小约65 kb;Legendre最佳MOI为10-4,最小MOI为10-3,Legendre对耻垢分枝杆菌极度易感;Legendre感染宿主菌的潜伏期为180 min,裂解期为120 min,裂解量为13;Legendre K值为697,Legendre抗血清对非对应噬菌体的中和活性有差异,对DNAⅢ、Bo4、Clark、Sedge、Leo高,对TM4、D29中和活性较低;Legendre能裂解耻垢分枝杆菌、结核分枝杆菌标准株、多数临床耐药株;Legendre对紫外线、温度、氯仿、酒精、酸碱度均敏感。结论 Legendre属于长尾噬菌体科,双链DNA噬菌体,抗原性低,宿主谱广,具有抗耐药结核潜力。
Objective To study the biological characteristics of phage Legendre and to explore its potential for anti-drug resistant tuberculosis. Methods Legendre plaque was prepared by double-layer plate method. The phage were purified and the phage were purified. The Legendre morphology was observed under electron microscope. The Legendre genome was extracted and the restriction endonuclease digestion was used to determine its nucleic acid type. The best MOI and the lowest MOI were obtained. The Legendre latency, lysis and lysis were determined by a one-step growth experiment. The Legendre particles were purified and immunized rabbits to obtain antiserum. Legendre and eight other phage and Legendre antibodies were determined by neutralization reaction The K value of the adsorption reaction between serums was determined. The host spectrum of Legendre was determined by monochromatic method. The tolerance of Legendre to UV, temperature, chloroform, alcohol and pH was tested. Results Legendre’s plaque was round and transparent with clear boundary. The head of Legendre was polyhedron with an average diameter of 65 nm and tail length of 215 nm. Genomic DNA was digested with EcoRⅠ, HindⅢ and BamHⅠ Legendre is extremely susceptible to Mycobacterium smegmatis; Legendre host strain has an incubation period of 180 min and a lysis period of 120 min, with an optimal MOI of 10-4 and a minimum MOI of 10-3. Legendre antiserum has different neutralizing activity to non-corresponding bacteriophage, higher DNAⅢ, Bo4, Clark, Sedge and Leo, lower neutralizing activity to TM4 and D29; Legendre can cleave Mycobacterium smegmatis, Mycobacterium tuberculosis standard strain, the majority of clinical resistant strains; Legendre UV, temperature, chloroform, alcohol, pH are sensitive. Conclusion Legendre belongs to the family of phage phage and double-stranded DNA phage. It has low antigenicity and broad host spectrum and possesses the potential of resistance to drug-resistant tuberculosis.