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A kind of Klebsiella pneumoniae phage and its application

A Klebsiella and bacteriophage technology, applied in the field of bioengineering, can solve the problems of limiting the application of bacteriophage, narrow lysis spectrum, etc., and achieve the effects of less toxic and side effects, strong killing activity and high safety

Active Publication Date: 2020-07-14
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Jilin University published a phage of NDM-1 Klebsiella pneumoniae in 2013 (Lu Rong, "Isolation and identification of lytic phage of NDM-1 Klebsiella pneumoniae and its treatment of bacteremia in mice ", master's thesis, 2013.6), the phage NKP-1 disclosed by Lu Rong was isolated from ATCC BAA-2146 (ATCC Collection Center in the United States) as the host. , which limits the further application of this phage

Method used

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  • A kind of Klebsiella pneumoniae phage and its application
  • A kind of Klebsiella pneumoniae phage and its application
  • A kind of Klebsiella pneumoniae phage and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Phage Isolation and Preparation

[0037] Streak-inoculate the host bacteria on 1.2% LB agar medium, culture overnight, pick a single clone and inoculate it in 1ml LB liquid medium, culture it with shaking at 37°C for 5-6 hours, and use it as the host bacteria culture for later use.

[0038] In the summer of 2015, sewage from a dairy farm in Nanjing City, Jiangsu Province was collected, and the supernatant was filtered through double-layer filter paper, centrifuged at 10,000rpm for 20min, and then filtered with a 0.22μm filter membrane; 10mL of the filtered supernatant was added to 0.5mL of the host bacteria Overnight culture, followed by sterile CaCl 2 After mixing the mother liquor to a final concentration of 1.25mM, add 20ml of LB liquid medium, react at room temperature for 30min, and place it at 37°C for 6-8h; then take the culture and centrifuge at 12000rpm, 4°C for 30min, and take the supernatant; 10mL of this supernatant was added to 0.5mL of the overn...

Embodiment 2

[0041] Example 2 Phage Amplification and Purification

[0042] On the double-layer plate forming plaques in Example 1, use the tip of a pipette to pick up a single plaque with a larger diameter, inoculate it in 3-5ml LB liquid 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, add 0.3% chloroform; repeat the double-layer experiment, and pick a single phage plaque repeatedly for 4-5 times. Phage were purified into plaques of equal 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 LB liquid medium, then add CaCl 2 The mother liquor was grown to a final concentration of 1.25mM, cultured with shaking at 37°C f...

Embodiment 3

[0048] Example 3 Transmission electron microscope observation of phage vB_KpnM_he1

[0049] Take the PEG-purified phage in Example 2 for electron microscope observation. The specific operation steps are: add 10 μL of sample and drop it on the copper grid, wait for it to settle for 15 minutes, absorb excess liquid with filter paper, and stain with 2% phosphotungstic acid (PTA) 1-2min, after drying, use a transmission electron microscope (Hitachi H-7650) to observe; the observation results are as follows figure 2 As shown, the head is in the shape of an icosahedron, the diameter of the head is about 80 nm, and the length of the tail is about 110 nm. According to "Classification of Viruses—The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2005, vB_KpnM_he1 belongs to Myoviridae.

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Abstract

The invention discloses a strong lysis bacteriophage for splitting NDM-1 positive Klebsiella pneumoniae clinical isolates, and an application of the strong lysis bacteriophage used as a biological preparation in the prevention and treatment of NDM-1 Klebsiella pneumoniae induced infection and pollution. The preservation number of the bacteriophage is CCTCC M 2015760, and the bacteriophage has a regularly icosahedral head and a telescopic tail, and belongs to a Myoviridae double-stranded DNA bacteriophage. The bacteriophage has strong kilign activity and wide bacteriophage lysis spectrum, can be used for preparing medicines for preventing and treating NDM-1 Klebsiella pneumoniae induced bacterium infection, and also can be used for killing NDM-1 positive Klebsiella pneumoniae in culture environment and medical environment.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a Klebsiella pneumoniae phage vB_KpnM_he1 (klebsiella pneumoniae phage vB_KpnM_he1) and its application. Background technique [0002] Klebsiella (Klebsiella) is a class of encapsulated Gram-negative bacilli in the Enterobacteriaceae, including Klebsiella pneumoniae (also known as Klebsiella pneumoniae), Klebsiella stinky nose and rhinosclerosis Klebsiella is closely related to humans, especially Klebsiella pneumoniae, which accounts for more than 95% of Klebsiella infections. It is an important pathogen of respiratory tract infection, often causing severe pneumonia, and can also It can cause serious diseases such as urinary tract infection, biliary tract infection, sepsis and suppurative meningitis. [0003] Klebsiella pneumoniae (Klebsiella pneumoniae) is widely distributed in soil, water, agricultural and forest products in nature, and is also common in the intestinal tract and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04
CPCA61K35/76C12N7/00C12N2795/10121
Inventor 何涛王冉葛展霞孙利厂庞茂达
Owner JIANGSU ACAD OF AGRI SCI
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