摘要
目的筛选能有效裂解结核分枝杆菌的噬菌体,并了解其生物学和遗传学信息。方法双层平板法扩增噬菌体,观察噬菌斑形态;电镜观察噬菌体颗粒形态;单斑法检测噬菌体宿主谱。采用λ噬菌体提取试剂盒提取噬菌体DNA,鸟枪法测序,得到的DNA序列用Phred/PhraD/Consedh软件包组装,拼接重叠群,PCR扩增重叠群缺口。采用DNAStar软件包、Glimmer3.0基因预测软件、Promoter prediction/Prokaryotic程序进行基因成分分析,基因功能预测,启动子区预测,并同时对Leo作共线性分析及构建系统进化树。结果噬菌体Leo由多面体头部和尾部构成,头部直径(70.0±3.0)nm,尾长(211.0±31.7)nm。其噬菌斑清晰透明,能裂解不同的结核分枝杆菌临床分离株。Leo基因组全长39 981bp,GC含量66.86%,含3个可能的启动子区,不含串联重复序列和tRNA区。Leo含59个基因,gene32编码整合酶,不含编码阻遏蛋白的基因和不利基因,Leo与BPs和Angel相似性最高。结论噬菌体Leo是一株不含不利基因的裂解性噬菌体,能裂解结核分枝杆菌,可以作为鸡尾酒疗法的候选噬菌体。
Objective To screen the phage that can effectively lyse Mycobacterium tuberculosis,and examine its genetic in formation and biological characteristics.Methods The mycobacteriophage Leo was amplified by using the double-layer agar plate method and the phage plaques were observed.The morphological characteristics were observed by electron microscopy,and the host range was measured by single-spot examination.The phage DNA was extracted by lamda DNA extraction protocol and sequenced by shotgun method.Phred/PhraD/Consedh software packages were used to assemble the shotgun sequences,and then the gaps were filled with PCR fragments.DNAStar software packages,and Glimmer3.0 and Promoter prediction/Prokaryotic software packages were used to predict the general characteristic of the genome of Leo,the function of the coding gene and the promoter region.In addition,the colinearity analysis was performed and phylogenetic tree constructed.Results The mycobacteriophage Leo consisted of a polyhedron head and a tail.The head was （70.0±3.0） nm in diameter and the tail was （211.0± 31.7） nm in length.Leo could lyse various clinical isolates of Mycobacterium tuberculosis.It had a transparent and distinct phage plaque.The full length of Leo genome was 39 981 bp with a G＋C content of 66.86%.It contained 59 predicted genes and 3 possible promoter region.Neither tRNA nor tandem repeat sequences was found in Leo.Leo gene 32 could encode integrases.There were no genes encoding repressor protein and non-toxin genes.Leo was highly similar to BPs and Angel.Conclusion Leo is a lytic phage that contains non-toxin genes.It can lyse mycobacterium tuberculosis and is expected to become the candidate phage for phage cocktails.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2014年第5期506-511,共6页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家"十二五"重大专项课题资助项目(No.2012ZX10003009)
国家临床重点专科专项经费资助项目(No.卫办医政函【2012】649号)
