分枝杆菌噬菌体的分离纯化及生物学特性研究.docVIP

噬菌体Legendre的生物学特性及抗耐药结核潜力刘平 邬亭亭 彭丽郭述良* 罗永艾 （重庆医科大学附属第一医院呼吸科，重庆 400016）[摘要] 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。 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以及其它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。 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215nm；基因组核酸能被双链DNA内切酶EcoRⅠ，Hind Ⅲ及BamH 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DNA、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。 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。 [关键词] Legendre；耻垢分枝杆菌；结核分枝杆菌；生物学特性；耐药结核病 [中图分类号] R52 he Biological Characteristics of Mycobacteriophage Legendre and Its Potentials in Drug-resistant Tuberculosis Control Liu Ping, Wu Tingting, Peng Li, Guo Shuliang*, Luo Yongai (Department of Respiratory Medicine, the First Hospital of Chongqing Medical University, Chongqing, 400016, China) [Abstract] Objective This paper aimed to research on the biological characteristics of mycobacteriophage Legendre to explore its anti-drug-resistant tuberculosis potentials. Methods The plaques morphological property of Legendre was observed by double-layer plating and the ultrastructuer of Legendre was observed by electron microscopy. The genome of Legendre was extracted and the type of nucleic acid was identified with restriction enzyme analysis. Legendre was amplified by double-layer agar plate method in different multiplicity of infection (MOI) to find the optimal MOI and the lowest MOI. One step growth experiment was done to find the latent period, burst period and burst size of Legendre. The rabbits were immuned with purified Legendre particles to prepare anti-Legendre serum and cross neutralization test was done to examine the reaction constant k valu