上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

分枝杆菌噬菌体DNAⅢ的遗传学信息及抗结核潜力初步研究

甘易玲1，刘 平2，邬亭亭3，郭述良1   

  1. 1.重庆医科大学附属第一医院呼吸与危重症医学科， 重庆 400016； 2.重庆市长寿区人民医院呼吸内科， 重庆 401220； 3.成都市第三人民医院呼吸内科， 成都 610031
  • 出版日期:2013-10-28 发布日期:2013-10-31
  • 通讯作者: 郭述良, 电子信箱: guosl999@sina.com。
  • 作者简介:甘易玲（1986—）， 女， 硕士生; 电子信箱:307323601@qq.com。
  • 基金资助:

    国家“十二五”科技重大专项（2012ZX10003-009）；国家临床重点专科专项经费（卫办医政函【2012】949号）

Genetic information of mycobacteriophages DNAⅢ and its anti-tuberculosis potential

GAN Yi-ling1, LIU Ping2, WU Ting-ting3, GUO Shu-liang1   

  1. 1.Department of Respiratory and Critical Care Medicine, the First Affiliated Hospital of Chongqing Medical  University, Chongqing 400016, China; 2.Department of Respiratory Medicine, People's Hospital of Changshou District, Chongqing 401220, China; 3.Department of Respiratory Medicine, the Third People's Hospital of Chengdu, Chengdu 610031, China
  • Online:2013-10-28 Published:2013-10-31
  • Supported by:

    National Major Science and Technology Program in “Twelfth Five-year” Plan, 2012ZX10003-009; National Key Clinical Specialty Foundation, 2012-949

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摘要：

目的 了解分枝杆菌噬菌体DNAⅢ的遗传学信息，初步探索其在抗结核治疗中的应用潜力。方法 试剂盒提取噬菌体DNA，鸟枪法测序，Phred/Phrad/Consed 软件包组装、拼接重叠群，PCR扩增连接重叠群缺口。通过DNAStar软件包中的EditSeq软件、Glimmer 3.0基因预测软件、共线性分析及构建DNAⅢ的系统发育树等对DNAⅢ基因组的遗传学信息进行分析。检测DNAⅢ对理化因素的抵抗力并行体外抗结核菌实验。结果 DNAⅢ基因组是含39 520 bp的线性双链DNA，G+C 含量是66.83%，不含tRNA和串联重复序列；DNAⅢ有60个基因，gene 32编码整合酶，但未发现编码阻遏蛋白的基因；DNAⅢ与Angel和BPs核苷酸序列相似度高，且具有清晰的共线性；与DNAⅢ关系最近的是Liefie。DNAⅢ对温度、乙醇及酸碱度均敏感；抗结核菌实验第1、2、3天的DNAⅢ组结核菌数量与对照组比较差异均有统计学意义（P<0.05）。结论 DNAⅢ是一株能有效杀灭结核菌的G簇烈性分枝杆菌噬菌体。

关键词: 噬菌体DNAⅢ, 基因组, 结核菌, 抗结核菌

Abstract:

Objective To understand the genetic information of mycobacteriophage DNAⅢ, and explore its anti-tuberculosis potential. Methods Mycobacteriophage DNAⅢ DNA was isolated and purified using lambda DNA extraction protocol. The shotgun sequences were assembled with Phred and PhraD and edited with Consed, then gaps were filled by sequencing PCR fragments. The general characteristics of genome were analysed using EditSeq software of DNAStar software package. The genome was scanned for open reading frames using Glimmer 3.0.  The genetic information of mycobacteriophage DNAⅢ genome was further explored by colinearity analysis and phylogenetic tree construction. The ability of DNAⅢ to lyse M.tuberculosis was investigated in vitro, and the effects of temperature, alcohol, pH values on DNAⅢ survival was determined. Results The genome of DNAⅢ possessed 39 520 bp in size, and was linear double-stranded DNA, with G+C content of 66.83%. DNAⅢ contained 60 predicted protein-coding genes, and did not encode any tRNAs. There was no tandem repeat sequence in the genome of DNAⅢ. DNAⅢ gene 32 encoded integrase, and there was also no gene encoding a repressor. DNAⅢ was highly similar and colinear in nucleotide acid level to Angel and BPs. At three different time points (1 d, 2 d and 3 d), the numbers of M.tuberculosis in DNAⅢ group were significantly smaller than those in control group (P<0.05). DNAⅢ was unstable to temperature, alcohol and pH values. Conclusion DNAⅢ, which belongs to cluster G, is a mycobacteriophage capable of lysing M.tuberculosis.

Key words: mycobacteriophage DNAⅢ, genome, M.tuberculosis, anti-tuberculosis