分枝杆菌噬菌体DNAⅢ的遗传学信息及抗结核潜力初步研究

Genetic information of mycobacteriophages DNAⅢ and its anti-tuberculosis potential

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。

Objective To understand the genetic information of mycobacteriophage DNA 111, and explore its anti- tuberculosis potential. Methods Mycobacteriophage DNAHI 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 myeobaeteriophage DNA Ⅲ genome was further explored by colinearity analysis and phylogenetic tree construction. The ability of DNA Ⅲ to lyse M. tuberculosis was investigated in vitro, and the effects of temperature, alcohol, pH values on DNAHI survival was determined. Results The genome of DNA 111 possessed 39 520 bp in size, and was linear double- stranded DNA, with G ＋ C content of 66.83%. DNAm 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, ant there was also no gene encoding a repressor. DNA II1 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 DNA m group were significantly smaller than those in control group （P 〈 0.05）. DNA Ⅲ was unstable to temperature, alcohol and Ⅲ values. Conclusion DNA Ⅲ which belongs to cluster G, is a mycobacteriophage capable of lysing M. tuberculosis.