A phage capable of lysing bovine Streptococcus agalactiae and its application

A technology for Streptococcus lactis and Streptococcus, applied in the field of bioengineering, can solve the problems of narrow lysis spectrum and unclear sequence type of Streptococcus agalactiae, and achieve good killing effect, no toxic side effects, and high safety

Active Publication Date: 2020-11-10
JIANGSU ACAD OF AGRI SCI
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Problems solved by technology

The research on phages of Streptococcus agalactiae has also made some progress. Bai Qinqin et al. isolated and identified 4 strains of phages of Streptococcus agalactiae from bovine (Bai Qinqin, Yang Yongchun, Lu Chengping. Bovine Streptococcus agalactiae long Characteristics of tailed phages[J]. Acta Microbiology, 2016,56(2):317-326), and the genome characteristics of one of them were analyzed (Qinqin Bai, Wei Zhang, Yongchun Yang, FangTang, Xuanhoa Nguyen, Guangjin Liu, Chengping Lu.Characterization and genomesequencing of a novel bacteriophage infecting Streptococcus agalactiae with high similarity to a phage from Streptococcus pyogenes[J].Archives of Virology,2013,158:1733-1741), but the phage isolated by it was against the agalactiae chain The lysis spectrum of the coccus is too narrow, it does not have a lysis effect on the ST67 strain, and only has a lysis effect on some ST568 and ST103 strains, and its highest lysis rate is only 54.8% (23 / 42)
Bao Hongduo and others have also isolated a streptococcal phage (Bao Hongduo, Wang Ran, Zhou Yan, Zhang Hui, Zhang Lili. A streptococcal phage and its application [P]. Chinese patent: CN104845943A, 2015-08- 19), the characteristic of this phage is that it has a wider lytic spectrum, and it has a lytic effect on Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus viridans, etc. The spectrum is also narrow, the lysis rate is 52.4% (11 / 21), and the sequence type of the Streptococcus agalactiae strain used is not clear
[0005] At present, no bacteriophages capable of lysing all bovine Streptococcus agalactiae epidemic strains such as ST67, ST103 and ST568 have been reported.

Method used

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  • A phage capable of lysing bovine Streptococcus agalactiae and its application
  • A phage capable of lysing bovine Streptococcus agalactiae and its application
  • A phage capable of lysing bovine Streptococcus agalactiae and its application

Examples

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Effect test

Embodiment 1

[0036] Example 1 Isolation and preparation of bacteriophage

[0037] The host bacteria of the test phage of the present invention is the clinical isolate HZJG1201 of Streptococcus agalactiae.

[0038] The samples of the present invention were collected from mastitis milk samples from experimental farms in Yangzhou City, Jiangsu Province. The milk samples were centrifuged at 12,000 rpm for 20 minutes, and the supernatant was filtered through a 0.22 μm filter membrane. Take 10 mL of filtered supernatant, add 0.5 mL of host bacteria overnight culture, and then add sterile CaCl 2 The mother liquor was mixed to a final concentration of 1.25mM, and 20mL of THB medium was added. After culturing in a 37°C incubator for 6-8 hours, the above culture was taken at 12000rpm, centrifuged for 30min, and the supernatant was taken; then 10mL of the supernatant was taken. , add 0.5 mL of the host bacteria overnight culture, add sterile CaCl 2 The mother liquor was mixed to a final concentrati...

Embodiment 2

[0041] Example 2 Amplification and purification of phage

[0042] On the double-layered plate with plaque formation in Example 1, use the pipette tip to pick a single plaque with a larger diameter, inoculate it into 3-5 mL of THB medium, and add 0.1 mL of phage host bacterial solution. Mix well, act at room temperature for 15 minutes, incubate at 37° C. for 10-14 hours, centrifuge at 12,000 rpm for 10 minutes at 4° C., take the supernatant, and add 0.3% chloroform. Repeatedly picking individual plaques 4-5 times until the phages were purified into plaques of the same size.

[0043] Take 1 mL of freshly cultured host bacteria and add 0.3 mL of phage lysate (in a ratio of 1:1, 1:10 and 1:100 for a single phage culture and host bacteria, respectively). Incubate at 37°C for 20 min to make the phage particles adsorb to the host bacteria; add 100 mL of THB liquid medium, and then add CaCl 2 The mother solution was brought to a final concentration of 1.25 mM, shaken at 37 °C for 12...

Embodiment 3

[0047] Example 3 Transmission electron microscope detection of bacteriophage

[0048] Take the purified phage particles (provided in Example 2) for electron microscope observation, add 10 μL of the sample and drop it on the copper grid, wait for it to settle for 15 minutes, absorb the excess liquid with filter paper, and stain with 2% phosphotungstic acid (PTA) for 1-2 minutes , and observed by transmission electron microscope (Hitachi H-7650) after drying.

[0049] observe as figure 2 As shown, the phage head is a regular polyhedron with a head diameter of about 50 nm and a tail length of about 200 nm. According to "The Taxonomy of Viruses - The Ninth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2011, vB_SagS_FSN1 belongs to the family Siphoviridae.

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Abstract

The invention discloses a streptococcus phage which can split cattle streptococcus agalactiae. The applicant names the phage as vB_SagS_FSN1; the phage is preserved in China Centre for Type Culture Collection; the preservation number is CCTCC NO:M 2017670. The streptococcus phage belongs to siphoviridae, can survive stably at the temperature of 40 to 60 DEG C and the pH of 5 to 10, has high hydrophilcity, and has very strong splitting capacity to streptococcus agalactiae, particularly cattle streptococcus agalactiae epidemic strain, in vivo or in vitro, so that the feasibility of the phage inpreventing and controlling cow mastitis caused by the streptococcus agalactiae is verified, and a new way is provided for treatment of infection of the cattle streptococcus agalactiae.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a streptococcus phage strain and its application, in particular to a streptococcus phage capable of lysing the epidemic strain of Streptococcus agalactiae in Chinese dairy farms and its application in preventing and treating dairy cow mastitis . Background technique [0002] Dairy cow mastitis is one of the most serious and complex diseases in the dairy farming industry, which not only seriously threatens the health of dairy cows, but also affects milk production and quality, posing a serious threat to the healthy development of the dairy industry. At present, there are about 220 million dairy cows in the world, and the annual loss caused by dairy cow mastitis is as high as 35 billion US dollars. The annual loss caused by dairy cow mastitis in my country is also a huge 13.5 billion yuan. Streptococcus agalactiae is one of the main pathogens leading to mastitis in dairy co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K35/76A61K9/00A61P15/14A61P31/04C12R1/94
CPCA61K9/0017A61K9/0041A61K2035/11C12N7/00C12N2795/10321C12N2795/10332
Inventor 庞茂达王冉孙利厂包红朵葛展霞何涛张辉
Owner JIANGSU ACAD OF AGRI SCI
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