食品科学 ›› 2023, Vol. 44 ›› Issue (18): 214-222.doi: 10.7506/spkx1002-6630-20221003-018

• 生物工程 • 上一篇    

1 株新的变形杆菌噬菌体的分离、鉴定与生物学特性分析

苏雅航，侯忠余，寇肖迪，刘爽，朱成林，陈娟，刀筱芳，唐俊妮   

  1. （1.西南民族大学食品科学与技术学院，四川 成都 610041；2.西南民族大学青藏高原研究院，青藏高原动物遗传资源保护与利用教育部重点实验室，四川 成都 610041）
  • 发布日期:2023-09-29
  • 基金资助:
    西南民族大学中央高校基本科研业务费专项（2020NTD04）；“十三五”国家重点研发计划重点专项（2018YFD0500500）

Isolation, Identification and Biological Characteristics Analysis of a New Proteus Phage

SU Yahang, HOU Zhongyu, KOU Xiaodi, LIU Shuang, ZHU Chenglin, CHEN Juan, DAO Xiaofang, TANG Junni   

  1. (1. College of Food Sciences and Technology, Southwest Minzu University, Chengdu 610041, China; 2. Key Laboratory of Qinghai-Tibetan Plateau, Animal Genetic Resource Reservation and Utilization, Ministry of Education, Institute of Qinghai-Tibetan Plateau, Southwest Minzu University, Chengdu 610041, China)
  • Published:2023-09-29

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摘要： 目的：对1 株新的变形杆菌（Proteus）噬菌体进行分离、鉴定与生物学特性及基因组分析。方法：将实验室从牦牛屠宰环节中分离到的菌株纯培养后，对其DNA提取进行16S rRNA测序，鉴定获得宿主菌，采集四川成都牦牛屠宰场环境污水，以获得的宿主菌进行噬菌体分离；运用双层琼脂平板法进行噬菌体的分离纯化，测定噬菌体的最佳感染复数、一步生长曲线和噬菌体耐受性等；通过磷酸负染、透射电镜观察噬菌体的形态；采用试剂盒提取噬菌体全基因组DNA，使用Illumina MiSeq测序平台进行噬菌体全基因组测序，对噬菌体全基因组序列进行组装、注释和基因组学分析。结果：从67 株分离菌株中鉴定出10 株Proteus，并从污水中分离得到1 株可裂解变形杆菌和粪肠球菌（Enterococcus faecalis）并且裂解性较强的噬菌体，命名为Proteus phage PV66；其最佳感染复数为0.1，一步生长曲线结果显示，感染宿主菌的潜伏期约为10 min，裂解期约为80 min，平均裂解量为321 PFU/cell；耐受pH值范围为3～12，耐受温度为65 ℃。电镜观察该噬菌体为C3形态噬菌体，其头部长（140±1）nm、宽（54±2）nm，尾部长为（36±1）nm。基因组测序结果表明PV66全序列长度为90 492 bp，系统发育树分析发现该噬菌体与奇异变形杆菌（P. mirabilis）噬菌体和克诺罗杆菌（Cronobacter）噬菌体同源性较高，与non-Kuravirus属处于同一分支。结论：本研究分离到1 株新的变形杆菌噬菌体并具有良好的应用潜力，为进一步开发噬菌体类新型抗菌剂提供基础，同时也丰富了变形杆菌噬菌体的数据库。

关键词: 变形杆菌；噬菌体；生物学特性；基因组分析

Abstract: Objective: To isolate, identify and characterize a new Proteus phage and analyze its genome. Methods: The strains isolated from yak slaughtering in the laboratory were cultured, their DNA was extracted and subjected to 16S rRNA sequencing, and the host bacteria were identified and used for bacteriophage separation from the environmental sewage of a yak slaughterhouse in Chengdu, Sichuan. The bacteriophage was separated and purified by the double-layer agar plate method, and its optimal multiplicity of infection (MOI), one-step growth curve and tolerance were determined. Its morphology was observed by negative staining with phosphoric acid and transmission electron microscopy (TEM). The whole genomic DNA of the phage was extracted using a commercial kit, sequenced on the Illumina MiSeq sequencing platform, assembled, annotated, and genomically analyzed. Results: Totally 10 Proteus strains were identified from the 67 isolates, and one phage with strong lytic activity, named Proteus phage PV66, was isolated from the sewage. PV66 could lyse Proteus and Enterococcus faecalis. The optimal MOI was 0.1. The results of one-step growth curve showed that the incubation period of bacteria infected with PV66 was about 10 min, the lytic period was about 80 min, and the average lytic amount was 321 PFU/cell. The tolerance pH range was 3–12, and the tolerance temperature was 65 ℃. The bacteriophage had a C3 morphotype under electron microscopy, with a head length of (140 ± 1) nm, a width of (54 ± 2) nm, and a tail length of (36 ± 1) nm. The genome sequencing results showed that the whole sequence length of PV66 was 90 492 bp. The phylogenetic tree analysis showed that the phage had high homology with Proteus mirabilis phage and Cronobacter phage and was in the same branch as non-Kuravirus. Conclusion: The Proteus phage has good application potential. This study provides a basis for future development of new bacteriophages, and enriches the database of Proteus phages.

Key words: Proteus; phage; biological characteristics; genome analysis

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