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T4SS targeted bacteriophage vBEcoMX4 and application thereof

A phage and targeting technology, applied in the field of bioengineering, can solve the problem of killing intestinal flora and achieve efficient blocking effect

Active Publication Date: 2021-10-22
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, it has been found that the main adsorption sites of phages on the surface of bacteria are polysaccharides and proteins, and the above sites exist in both drug-resistant and non-resistant bacteria. Therefore, when traditional phages are applied to intestinal drug-resistant bacterial infections, they may affect normal Gut flora produces a killing effect

Method used

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  • T4SS targeted bacteriophage vBEcoMX4 and application thereof
  • T4SS targeted bacteriophage vBEcoMX4 and application thereof
  • T4SS targeted bacteriophage vBEcoMX4 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 The acquisition of T4SS plasmid-carrying bacteria

[0042]Select the tet (X4) positive bacterial strain preserved in our laboratory, which carried a typical T4SS plasmid (Genbank number: NZ_MT219822), and electrotransform this plasmid into Escherichia coli standard strains J53, DH5a, JM109, BL21 and K12 to obtain Various standard strains carrying only T4SS plasmids were used as host bacteria to isolate T4SS-targeted phages.

Embodiment 2

[0043] Example 2 Initial isolation of T4SS targeting phage

[0044] The sample of the present invention is collected from a broiler farm sewage in Nanjing City, Jiangsu Province. The supernatant is filtered through double-layer filter paper, centrifuged at 10,000 rpm for 20 minutes, and then 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 constructed above, and then add sterile CaCl 2 After mixing the mother liquor to a final concentration of 1.25mM, add 20mL of LB medium, react at room temperature for 30min, and then place it at 37°C. After culturing for 6-8h, take the above culture at 12000rpm, 4°C, centrifuge for 30min, and take the supernatant ; Then take 10 mL of the supernatant, add 0.5 mL of the overnight culture of the host bacteria, and add sterile CaCl 2 After mixing the mother liquor to a final concentration of 1.25mM, add 20mL LB medium, cultivate and centrifuge according to ...

Embodiment 3T4

[0047] Example 3 Purification and Confirmation of T4SS Targeting Phage

[0048] On the double-layer plate forming phage plaques, use the tip of a pipette to pick up a single phage plaque with a larger diameter, inoculate it in 3-5mL LB liquid medium, add 0.1mL of phage host bacterial solution, and mix well. , act at room temperature for 15 min, incubate at 37°C for 10-14h, centrifuge at 12000rpm at 4°C for 10min, take the supernatant, and add 0.3% chloroform. In this way, single phage plaques were picked repeatedly 4-5 times, and the phages were purified into phage plaques of the same size.

[0049] Spot the purified phages of different sizes: use Escherichia coli standard strains (J53, DH5a, JM109, BL21, and DH10B) carrying T4SS plasmids and their corresponding standard strains without T4SS plasmids as host bacteria at the same time, and pipette each Drop 0.1mL of the host bacterial solution on the center of different LB agar plates, and spread the bacterial solution evenly ...

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Abstract

The invention discloses a T4SS targeted bacteriophage vBEcoMX4 and application thereof, and belongs to the field of bioengineering. The T4SS targeted bacteriophage vBEcoMX4 is obtained from sewage through separation, purification and screening and can split multiple drug-resistant bacteria, and the number of drug-resistant bacteria is remarkably decreased after the bacteriophage vBEcoMX4 is applied to a drug-resistant bacteria environment for 1-3 h. The bacteriophage vBEcoMX4 is preserved in the China Center for Type Culture Collection, and the preservation number thereof is CCTCC NO: M2021519. The bacteriophage vBEcoMX4 can be independently used or compounded with other substances for use, multiple drug-resistant bacteria in an animal breeding environment, a food processing environment and a human medical environment can be killed, and propagation and diffusion of drug-resistant genes in the environment can be simply, conveniently, rapidly and efficiently blocked.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a T4SS targeting phage vB_EcoM_X4 and its application. Background technique [0002] Antibiotics are effective drugs for treating bacterial infections. However, the large-scale use of antibiotics in human medicine and agricultural production has led to the rapid spread and spread of bacterial resistance in the environment, especially the combined use of multiple antibiotics has promoted multiple infections. The emergence of drug resistance is a serious threat to human and animal health, food and environmental safety, and related problems have aroused people's vigilance. [0003] Finding alternatives to antibiotics is an important means of controlling the development of drug resistance. Common alternatives to antibiotics include immunomodulators, phages, antimicrobial peptides, prebiotics, and plant extracts. Among them, bacteriophages have many advantages compared with antibiotics...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/31A01N63/40A01P1/00A01P3/00C12R1/92
CPCC12N7/00C07K14/195A01N63/40C12N2795/10121C12N2795/10131Y02A50/30
Inventor 李俊何涛王冉魏瑞成龚兰吉星
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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