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Bacteriophage preparation and application thereof

A bacteriophage and preparation technology, applied in the field of bacteriophage preparations, can solve the problems of not providing the lysis ability of piglet diarrhea pathogenic bacteria isolates, not publishing phage transmission electron microscopy images, and unable to effectively control piglet diarrhea, etc. Scope, good killing effect

Inactive Publication Date: 2016-04-06
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in follow-up experiments, it was found that the infection of E. coli K88 in piglets was also very serious, and the diarrhea of ​​piglets could not be effectively controlled only by K99 phage
[0008] There have been literature reports about lytic phages of Escherichia coli K88 and K99, such as the PK88-4 phage of Escherichia coli K88 (Wang Ran et al., North China Agricultural Science Journal, 2012), and the phages were separated from EK88-4 in the present invention simultaneously Obtained, both of them belong to Myocygidae phages with stretchable tails, but EK88-4 has a wider phage spectrum and stronger bactericidal efficacy than PK88-4; He Mizhi published his master's thesis in 2012 In (http: / / www.doc88.com / p-9973640509846.html), one Escherichia coli K88 phage Bp8A0811 and two Escherichia coli K99 phages Bp9B1226 and Bp9C1213 were isolated from a pig breeding farm, but the author did not disclose the phages The transmission electron microscope images of the bacteriophages did not provide the bacteriophage lysis spectrum, but only the lysis effect on the host bacteria, and did not provide relevant data on the lysis ability of the isolates of pathogenic bacteria isolated from piglet diarrhea and the application of drugs for the prevention and treatment of piglet diarrhea.

Method used

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  • Bacteriophage preparation and application thereof
  • Bacteriophage preparation and application thereof
  • Bacteriophage preparation and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1 Isolation, amplification and purification of enterotoxigenic Escherichia coli K88 phage

[0024] Phage isolation: CVCC1502 (serum type O9:K88ac) purchased from China Veterinary Microbiology Culture Collection Center was used as host to isolate phage. The bought bacteria were revived and cultivated separately, and a single K88 colony on the LB plate was picked, inoculated in 5 mL of LB medium, shaken at 150 rpm overnight at 37°C, and an overnight bacterial solution was prepared. Fecal and urine sewage samples from pig farms (a pig farm in Jiangsu) were collected, filtered through double-layer filter paper, centrifuged at 12000×g for 20 min, and then the supernatant was filtered with 0.45 μm and 0.22 μm filter membranes respectively; 10 mL of the filtrate was added to 0.5mL overnight cultured host bacteria (the bacterial content is 10 9 cfu / mL), then add 20mL of 4×LB medium, place it at 37°C for 12-16h, and the next day, take the above culture and centrifuge it...

Embodiment 2

[0029] The preparation of embodiment 2 phage preparation JS-15

[0030] Take the Escherichia coli K88 phage EK88-4 obtained in Example 1 and the Escherichia coli K99 phage EK99-C disclosed in the patent ZL200910263295.0, the titers of the two phages are all ≥ 10 8 Pfu / mL, the two phages are fully mixed according to the titer ratio, and an appropriate amount of SM buffer is added to prepare the compound phages of enterotoxigenic Escherichia coli K88 and K99. The preparation is self-named JS-15, and the total content of bacteriophage should be ≥10 6 pfu / g or 10 6 pfu / mL.

[0031] In the actual operation process, one, two or more mixtures of sodium alginate, sucrose, maltodextrin, and glucose can also be added to the phage preparation JS-15, as long as the total content of phage in the obtained preparation is guaranteed ≥10 6 pfu / g or 10 6 pfu / mL can achieve the purpose of the present invention.

Embodiment 3

[0032] Phage spectrum analysis of embodiment 3 phage preparation JS-15

[0033] Take 55 strains of Escherichia coli (see Table 1 for strain sources), recover the strains, cultivate them, and evenly spread 100 μl of the overnight cultured bacterial solution on the LB plate. After drying, take 10 μl of the phage JS-15 prepared in Example 2, Escherichia coli K88 phage EK88-4 and Escherichia coli K99 phage EK99-C were spotted on the surface of the bacterial lawn respectively, and normal saline was used as a control. Each sample was repeated three times, and the droplets were dried and then placed upside down at 37°C for incubation box, cultivated for 12-16 hours and observed the effect the next day. The results are shown in Table 1:

[0034] Table 1 Bactericidal spectrum of phage preparation JS-15 for preventing and treating piglet diarrhea

[0035]

[0036]

[0037]

[0038]

[0039] It can be seen from Table 1 that for the 55 Escherichia coli epidemic strains, the ...

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Abstract

The invention discloses a bacteriophage preparation and an application thereof. The bacteriophage preparation is prepared from enterotoxigenic escherichia coil K88 bacteriophage with a preservation number of CCTCC No: M209243 and a bacteriophage EK99-C by adding an adjuvant material, wherein the total bacteriophage effective quantity is more than or equal to 10<6>PFU / mL. The bacteriophage preparation can be used for preparing a medicine for preventing and treating piglet scours or killing escherichia coils K88 and K99. The bacteriophage preparation provided by the invention has broad-spectrum bactericidal activity, and the active ingredients of the preparation are separated from a livestock farm environment and belong to substances in the environment, so that the bacteriophage preparation cannot generate poisoning to the environment and animals, and bacteriophages are colorless, tasteless and nonirritant after purification, and are safe and environment-friendly.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a phage preparation JS-15 and its application. Background technique [0002] Piglet diarrhea is a common problem in the pig industry and has become a bottleneck restricting the healthy development of the pig industry, especially for piglets aged 1-3 months. The incidence of diarrhea in piglets after weaning is as high as 30%, and the mortality rate is as high as 10% %-15%. After the onset of piglets, the growth rate is slow, and the feed remuneration is reduced. At the same time, due to diarrhea, physical decline, decreased immune function, and weakened resistance to epidemic diseases, it is easy to cause other infectious diseases. If the treatment is not timely, it will cause a large number of deaths. cause huge economic losses. [0003] In order to pursue high yield of sows and increase the parity of sows, the early weaning technology of piglets is promoted internationally, that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/76A61P1/12A61P31/04A01N63/00A01P1/00
CPCA61K35/76A01N63/00A61K2300/00A01N2300/00
Inventor 王冉张辉包红朵周艳
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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