霍乱弧菌分型噬菌体VP2部分生物学特性及基因组分析

孙惠惠; 范宇峰; 李臻鹏; 赵硕; 王晓勋; 逄波; 阚飙

doi:10.3784/jbjc.202105190282

霍乱弧菌分型噬菌体VP2部分生物学特性及基因组分析

doi: 10.3784/jbjc.202105190282

孙惠惠^{1, 2,},
范宇峰¹,
李臻鹏¹,
赵硕¹,
王晓勋³,
逄波¹,
阚飙^1, ,

1.
中国疾病预防控制中心传染病预防控制所，北京 102206
2.
中国疾病预防控制中心环境与健康相关产品安全所，北京 100021
3.
国药中生生物技术研究院有限公司，北京 102600

基金项目: 国家重点基础研究发展计划（No. 2015CB554201）

详细信息

作者简介:
孙惠惠，女，山东省诸城市人，博士研究生在读，主要从事霍乱弧菌分型噬菌体相关研究工作，Email：sunhuihui@nieh.chinacdc.cn

通讯作者:
阚飙，Tel：010–58900742，Email：kanbiao@icdc.cn

中图分类号: R211; R378.3
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出版历程
- 收稿日期: 2021-05-19
- 网络出版日期: 2021-09-16

Partial biological characteristics and genomic analysis of Vibrio cholerae typing phage VP2

Sun Huihui^{1, 2
,},
Fan Yufeng¹,
Li Zhenpeng¹,
Zhao Shuo¹,
Wang Xiaoxun³,
Pang Bo¹,
Kan Biao^{1
, ,}

1.
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100026, China
2.
National Institute of Environment Health, Chinese Center for Disease Control and Prevention, Beijing 100026, China
3.
Sinopharm Biotechnology Research Institute Co. Ltd, Beijing 102600, China

Funds: This study was supported by the National Basic Science and Technology Fund (No. 2015CB554201)

More Information

Corresponding author: Kan Biao, Email: kanbiao@icdc.cn

摘要

摘要: 目的对霍乱弧菌分型噬菌体VP2基因组、蛋白质组和增殖特征等开展分析，获取VP2的生物学特征。方法电镜观察噬菌体VP2的形态，测定最佳感染复数和一步生长曲线；通过在线RAST（https://rast.nmpdr.org/）对噬菌体VP2基因组进行编码基因预测及功能注释；提取VP2成熟颗粒中的蛋白质进行质谱分析；使用pyani（https://github.com/widdowquinn/pyani）进行噬菌体全基因组平均核苷酸一致性（ANI）聚类分析。结果噬菌体VP2为典型的20面体短尾噬菌体，最佳感染复数为1∶100，一步生长曲线结果显示其潜伏期约为60 min，60～120 min为爆发期，120 min后为稳定期。预测存在49个开放阅读框，VP2成熟颗粒蛋白质谱分析显示有34个蛋白与预测的基因相对应，ANI聚类分析结果显示VP2与弧菌噬菌体CJY的ANI最高。结论明确了分型噬菌体VP2的形态和基因组特征，预测的功能蛋白及结构蛋白的鉴定为后续研究同源噬菌体及其与霍乱弧菌的相互作用奠定了基础。
- 分型噬菌体VP2 /
- 霍乱弧菌 /
- 生物学特性 /
- 基因组分析
Abstract: Objective This study aimed to further understand the genetic information of VP2 by analyzing the whole genome of Vibrio cholerae typing phage VP2, and analyzing the protein of mature particles in combination with biological characteristics. Methods The morphology of phage VP2 was observed under electron microscope, and some of its biological characteristics were determined. Online RAST (https://rast.nmpdr.org/) was used to predict and annotate the encoding genes of the phage VP2 genome, and the proteins in the mature VP2 particles were analyzed by mass spectrometry. Phage genome average nucleotide consistency (ANI, business, nucleotide identity) was analysis by pyani (https://github.com/widdowquinn/pyani). Results Phage VP2 was a typical 20-hedron short-tailed phage, the optimal MOI is 0.01. One step growth curve showed that incubation period is about 60 minutes, 60 minutes to 120 minutes for the outbreak period, after 120 minutes for the stable period. Forty-nine open reading frames (ORFs) were predicted. Protein spectrum analysis of mature VP2 particles showed that 34 proteins corresponded to the predicted genes. ANI cluster analysis showed that ANI was the highest in VP2 and vibro phage CJY. Conclusion The morphological and genomic characteristics of typing phage VP2 were identified, which laid a foundation for the further study of homologous phage and its interaction with Vibrio cholerae.
- Typing phage VP2 /
- Vibrio cholerae /
- Biological characteristics /
- Genomic analysis

HTML全文

图 1 VP2噬菌体在电镜下形态（×97 000）

注：红色箭头示短尾

Figure 1. Morphology of VP2 phage under transmission electron microscopy (the short tail of the phage is shown at the red arrow) (×97 000)

下载: 全尺寸图片幻灯片

图 2 噬菌体VP2的一步生长曲线

Figure 2. One-step growth curve of phage VP2

下载: 全尺寸图片幻灯片

图 3 VP2全基因组模式图

注：最外层表示噬菌体基因组全长，第二圈代表正向阅读框；第三圈代表负向阅读框，其中蓝色为功能基因，橙色为结构基因，绿色为假定基因；第四圈黄色与绿色代表GC含量，其中黄色表示低于全基因组平均GC含量，绿色则相反；最内圈玫红色表示GC偏嗜性G－C/G＋C＜0，蓝色表示＞0

Figure 3. Genome map of phage VP2

下载: 全尺寸图片幻灯片

图 4 VP2全基因组核苷酸一致性分析

Figure 4. Average nucleotide identity of VP2

下载: 全尺寸图片幻灯片

表 1 VP2噬菌体与宿主菌16017的最佳感染复数

Table 1. The optimal MOI between VP2 phage and bacteria 16017

噬菌体滴度	宿主菌浓度	MOI（噬菌体∶菌）	结果
10⁶	10⁶	1∶1	2.00×10⁶
10⁵	10⁶	1∶10	5.20×10⁷
10⁴	10⁶	1∶100	6.80×10⁷

下载: 导出CSV

表 2 VP2颗粒全蛋白质谱与VP2全基因组测序预测蛋白^［14］比对结果

Table 2. Comparison between the results of VP2 granule protein spectrum and the predicted proteins of VP2 whole genome sequencing^［14］

VP2ORF	起始基因位点	终止基因位点	氨基酸长度（aa）	功　　能	蛋白覆盖度（%）
1	409	630	222	hypothetical protein	−
2	647	1147	501	terminase small subunit	36
3	1134	2840	1707	terminase	15
4	2851	4494	1644	Head-tail connecting protein	78
5	4494	4721	228	hypothetical protein	96
6	4734	4991	258	hypothetical protein	−
7	4996	5838	843	hypothetical protein	58
8	6006	6974	969	structural protein	84
9	7041	7310	270	hypothetical protein	97
10	7325	8173	849	hypothetical protein	77
11	8170	8535	366	hypothetical protein	59
12	8537	9010	474	LysM peptidoglycan-binding domain-containing protein	27
13	9003	9305	303	Phage protein	93
14	9329	11554	2226	PE family protein/tail protein	76
15	11563	13347	1785	structural protein	76
16	13353	13757	405	Phage protein	98
17	13754	15838	2085	Phage protein	93
18	15838	17010	1173	hypothetical protein	78
19	17012	19360	2349	structural protein	79
20	19364	20437	1074	Phage tail fibers	54
21	20439	21821	366	Outer capsid protein	53
22	22220	21846	474	hypothetical protein	−
23	22677	22240	303	hypothetical protein	45
24	22828	22667	2226	hypothetical protein	−
25	23022	22825	1785	metal dependent phosphohydrolase	65
26	23336	23019	405	Hydrolase	91
27	23598	23338	2085	hypothetical protein	−
28	24631	23600	1032	Adenylosuccinate synthetase	71
29	26961	24673	2289	integrase	54
30	28819	26951	1869	DNA polymerase I	17
31	29337	28861	477	Single stranded DNA-binding protein，phage-associated	15
32	30212	29403	810	hypothetical protein	29
33	30800	30333	468	hypothetical protein	17
34	32332	30863	1470	superfamily II DNA/RNA helicase	29
35	32750	32397	354	hypothetical protein	17
36	33442	32750	693	hypothetical protein	20
37	33832	33575	258	hypothetical protein	18
38	34446	34036	411	structural protein	−
39	34774	34448	327	hypothetical protein	−
40	35624	34998	627	hypothetical protein	14
41	35828	35667	162	hypothetical protein	−
42	36418	35933	486	hypothetical protein	−
43	36859	36428	432	Phage protein	38
44	37407	37036	372	hypothetical protein	−
45	37631	37407	225	hypothetical protein	−
46	37999	37709	291	hypothetical protein	−
47	38345	38106	240	hypothetical protein	−
48	38946	38356	591	hypothetical protein	−
49	39817	39548	270	hypothetical protein	−

下载: 导出CSV

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