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Phage capable of splitting cattle streptococcus agalactiae and application thereof

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

Active Publication Date: 2018-06-01
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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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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  • Phage capable of splitting cattle streptococcus agalactiae and application thereof
  • Phage capable of splitting cattle streptococcus agalactiae and application thereof
  • Phage capable of splitting cattle streptococcus agalactiae and application thereof

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Experimental program
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Embodiment 1

[0036] The isolation and preparation of embodiment 1 phage

[0037] The host bacterium of the phage used in the test of the present invention is the clinical isolate strain HZJG1201 of Streptococcus agalactiae.

[0038] The sample of the present invention is collected from a mastitis milk sample from an experimental farm in Yangzhou City, Jiangsu Province. The milk sample is centrifuged at 12,000 rpm for 20 minutes, and then the supernatant is filtered with a 0.22 μm filter membrane. Take 10 mL of the filtered supernatant, add 0.5 mL of the overnight culture of the host bacteria, and then add sterile CaCl 2 Mix the mother liquor to a final concentration of 1.25mM, add 20mL of THB medium, place it in a 37°C incubator and incubate for 6-8h, take the above culture at 12000rpm, centrifuge for 30min, and take the supernatant; then take 10mL of the supernatant , add 0.5mL overnight culture of host bacteria, add sterile CaCl 2 Mix the mother liquor to a final concentration of 1.25m...

Embodiment 2

[0041] Amplification and purification of embodiment 2 phage

[0042] On the double-layer plate that forms phage plaques in Example 1, pick a single phage plaque with a larger diameter with the tip of a pipette, inoculate it in 3-5mL THB medium, add 0.1mL of phage host bacterial solution, Mix well, act at room temperature for 15 minutes, incubate at 37°C for 10-14h, centrifuge at 12000rpm at 4°C for 10min, take the supernatant, and add 0.3% chloroform. Repeatedly pick 4-5 single phage plaques in this way until the phages are purified into phage plaques of the same size.

[0043] Take 1 mL of freshly cultured host bacteria and add 0.3 mL of phage lysate (the ratio of a single phage culture to host bacteria is 1:1, 1:10 and 1:100, respectively). Incubate at 37°C for 20 minutes to make the phage particles adsorb to the host bacteria; add 100mL THB liquid medium, then add CaCl 2 The final concentration of the mother liquor was 1.25mM, cultured with shaking at 37°C for 12-16h, 120...

Embodiment 3

[0047] The transmission electron microscope detection of embodiment 3 bacteriophage

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

[0049] observe as figure 2 As shown, the head of the phage is a regular polyhedron, the diameter of the head is about 50nm, and the length of the tail is about 200nm. According to the "Classification 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 long-tailed bacteriophage 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, and specifically relates to a streptococcal phage strain and its application, in particular to a streptococcal phage capable of cracking the endemic strain of Streptococcus agalactiae in Chinese dairy farms and its application in preventing and treating dairy cow mastitis . Background technique [0002] Cow mastitis is one of the most serious and complicated diseases in the dairy farming industry. It 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 due to cow mastitis is as high as 35 billion U.S. dollars. In my country, the annual loss caused by cow mastitis is also as huge as 13.5 billion yuan. Streptococcus agalactiae is one of the main pathogens that cause mastitis in dairy cow...

Claims

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Application Information

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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 ACADEMY OF AGRICULTURAL SCIENCES
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