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Novel bacteriophage, bacteriophage mixed formulation and application of novel bacteriophage and application of bacteriophage mixed formulation to prevention and treatment of pseudomonas pneumonia of minks

A phage and preparation technology, applied in the field of microorganisms, can solve the problems of no safe and effective phage preparations, and achieve the effects of broadening the scope of sterilization, increasing bactericidal activity, and improving the cure rate

Active Publication Date: 2020-07-03
QINGDAO PHAGEPHARM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, no safe and effective phage preparation with a wide cleavage spectrum has been found so far for the prevention and treatment of mink hemorrhagic pneumonia. Therefore, further screening and research work on new phages is needed

Method used

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  • Novel bacteriophage, bacteriophage mixed formulation and application of novel bacteriophage and application of bacteriophage mixed formulation to prevention and treatment of pseudomonas pneumonia of minks
  • Novel bacteriophage, bacteriophage mixed formulation and application of novel bacteriophage and application of bacteriophage mixed formulation to prevention and treatment of pseudomonas pneumonia of minks
  • Novel bacteriophage, bacteriophage mixed formulation and application of novel bacteriophage and application of bacteriophage mixed formulation to prevention and treatment of pseudomonas pneumonia of minks

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The isolation and purification culture of embodiment 1 phage

[0038] 1. Isolation and culture of host bacteria

[0039] The host bacterium of the Klebsiella phage in the embodiment is a clinical strain of Klebsiella pneumoniae, which is obtained from a mink infected with Klebsiella pneumoniae and died from a mink farm in Weifang, Shandong Province.

[0040] The host bacterium of the Pseudomonas aeruginosa phage in the embodiment is a clinical strain of Pseudomonas aeruginosa, which is obtained from a dead mink infected with Pseudomonas aeruginosa in a mink farm in Qingdao, Shandong Province.

[0041] Take the host bacteria and inoculate them on 2% nutrient agar. After culturing overnight, pick a single colony and inoculate it in 5ml of nutrient broth medium, shake and culture it in a shaking table at 37°C and 170rpm for 12h as the host bacteria culture for later use.

[0042] 2. Isolation and culture of phage

[0043] Put the sewage obtained from the mink farm into t...

Embodiment 2

[0049] Morphological observation of embodiment 2 phage

[0050] Experimental method: take 1×10 10 20 μl of PFU / ml phage sample was dropped on the microporous copper grid, settled for 15 minutes, and the excess liquid was absorbed with filter paper. Add 15 μl of 2% phosphotungstic acid (PTA) dropwise on the copper grid, stain for 5 minutes, absorb excess dye solution with filter paper, observe and take pictures with transmission electron microscope after drying.

[0051] Such as figure 1 As shown in the electron microscope picture of Klebsiella phage, the head length of Klebsiella phage is 90-100nm, the head width is 110-120nm, and the tail length is 120-130nm. According to the definition of the International Committee on Taxonomy of Viruses (ICTV), the morphology of the phage PH35 conforms to the characteristics of the myotail phage family, and it is named vB_KpnM_PH35.

[0052] Such as figure 2 As shown in the electron microscope picture, the head of Pseudomonas aerugino...

Embodiment 3

[0053] Whole Genome Analysis of Example 3 Phage

[0054] Extract the genome of Pseudomonas aeruginosa phage vB_PaeS_PA69, and perform whole genome sequencing to obtain:

[0055] The length of the entire genome of phage vB_PaeS_PA69 is: 45414bp. In its genome, the tail protein gene sequence related to phage host recognition is shown in sequence 1 in the sequence table, and the highly conserved terminase large subunit gene sequence See Sequence 2 in the Sequence Listing, the sequences of two DNA polymerase genes are shown in Sequences 3-4 in the Sequence Listing, and the lysozyme gene sequence related to cleavage ability is shown in Sequence 5 in the Sequence Listing. The specific information of the above genes is shown in Table 1 below.

[0056] Table 1 Gene sequence information table of bacteriophage vB_PaeS_PA69

[0057]

[0058]

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Abstract

The invention discloses a novel bacteriophage, a bacteriophage mixed formulation and an application of the novel bacteriophage and an application of the bacteriophage mixed formulation to prevention and treatment of pseudomonas pneumonia of minks. The novel bacteriophage is klebsiella pneumoniae bacteriophage vB_KpnM_PH35 of which the preservation No. is CGMCC NO.18858, the bacteriophage mixed formulation comprises a klebsiella pneumoniae bacteriophage vB_KpnM_PH35 of which the preservation No. is CGMCC NO.18858 and a pseudomonas aeruginosa bacteriophage vB_PaeS_PA69 of which the preservationNo. is CGMCC NO.18855. The novel bacteriophage and the bacteriophage mixed formulation have broad spectrum sterilization effects, the sterilizing scope is greatly widened, the bactericidal activity isimproved, and the cure rate of the minks can be greatly increased. Through mouse test, the bacteriophage mixed formulation is free from toxic and side effects, high in safety and convenient for large-scale production. The bacteriophage mixed formulation has good hydrophilic phase, and is easy to make into spraying liquid or injections, and klebsiella pneumoniae and pseudomonas aeruginosa in environment can be effectively killed.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a phage mixed preparation and its application in medicines for preventing and treating mink hemorrhagic pneumonia. Background technique [0002] Pseudomonas aeruginosa and Klebsiella are important zoonotic opportunistic pathogens causing hemorrhagic pneumonia in fur animals. Early mink hemorrhagic pneumonia was mainly caused by Pseudomonas aeruginosa infection. Its onset was rapid and difficult to control. It was endemic in many countries and regions, resulting in the death of a large number of minks and serious economic losses. In recent years, there have been more and more cases of Pseudomonas aeruginosa and Klebsiella being mixed to cause disease, leading to the main clinical manifestations of the disease, such as mental depression, loss of appetite, sepsis and sudden death. Hemorrhage, lymph node abscess. Mink are infected by ingesting contaminated feed, and can also...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K35/76A61P11/00A23K10/18A01N63/40A01P1/00C02F1/50C02F3/34C12R1/92C02F103/20
CPCA01N63/00A61K35/76A23K10/18A61P11/00C02F1/50C02F3/34C02F2101/20C12N7/00C12N2795/10321C12N2795/10331C12N2795/10332A61K2300/00
Inventor 潘强任慧英孙虎芝闫艳新袁嘉婧
Owner QINGDAO PHAGEPHARM BIO TECH CO LTD
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