CN117187126A - Isolation of coliphage JNUW3 and development of anti-JNUW 3 phage colibacillus - Google Patents
Isolation of coliphage JNUW3 and development of anti-JNUW 3 phage colibacillus Download PDFInfo
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Abstract
The invention discloses separation of an escherichia coli bacteriophage JNUW3 and development of an anti-JNUW 3 bacteriophage escherichia coli, and belongs to the technical field of microorganisms. The invention screens and separates the bacteriophage JNUW3 from the pollution fermenter, and the bacteriophage can inhibit various escherichia coli. The invention successfully screens out a chassis cell JNK4 with phage resistance, and compared with the escherichia coli in the prior art, the invention can resist infection of phage JNUW3; and can also resist T7 phage, and has certain broad-spectrum anti-phage property. The problem that fermentation products are easy to be infected by phage in production to cause fermentation failure is fundamentally solved, and the economic benefit of fermentation production enterprises is improved.
Description
Technical Field
The invention relates to separation of an escherichia coli bacteriophage JNUW3 and development of an anti-JNUW 3 bacteriophage escherichia coli, and belongs to the technical field of microorganisms.
Background
Phages offer a special perspective for the diversity, origin and evolution of viruses, not only because they are extremely abundant with more than 1031 phage particles in the biosphere, but also their distant origin, possibly more than 30 billion years ago. In addition, virus on-state scientists calculated about 1023 phage infections per second worldwide, indicating that the population is not only huge but also old, with a high degree of dynamics. Phages have some characteristics of viruses: individuals are tiny, only a single nucleic acid, and have no intact cellular structure. The use of ribosomes, proteins, etc. in microbial cells synthesizes the various factors, amino acids, and energy required for self-growth and proliferation, and once exiting the host cell, the phage is neither able to grow nor to replicate. The parasitism of phage is highly specific, which is determined by the surface architecture of the host cell and the architecture of the phage. The infection process of virulent phages is generally divided into 5 stages of adsorption, injection, replication, assembly and release.
The modern fermentation industry tends to a large-volume fermenter and a continuous fermentation process, which provides favorable conditions for the growth, propagation and propagation of phage, and in the microbial fermentation production process, once phage is infected, the fermentation process is stopped quickly, and the thalli disappear quickly. Causing serious economic loss. Moreover, once phage infection occurs, continuous failure of fermentation production is often caused if effective measures are not taken in time. Bringing huge losses to enterprises.
After colibacillus infects phage, measures such as formaldehyde fumigation, pipeline sterilization or strain rotation are generally adopted in the past to ensure the production sustainability, but the corresponding strategies are long in time consumption, strict in requirements, high in cost and poor in effect, and cannot fundamentally solve the problem of phage infection. Therefore, the breeding of the efficient bacteriophage-resistant industrial escherichia coli chassis cells has important significance for solving the problem of bacteriophage infection in the fermentation industry.
Disclosure of Invention
The invention provides a bacterial strain of Escherichia coli (Escherichia coli) JNK4 with phage resistance, which is preserved in China Center for Type Culture Collection (CCTCC) in 2023, month 07 and 05, and the preservation number is CCTCC NO: m20231188. According to the invention, a strain which can resist the coliphage JNUW3 is screened out by taking the colibacillus BL21 as an initial strain through natural evolution, so that the method is applied to industrial production.
The invention also provides an evolution method of the Escherichia coli JNK4 resistant to the Escherichia coli phage, which comprises the following steps:
(1) The separated JNIW 3 phage and 5mL LB liquid medium are inoculated, 100uL of corresponding host escherichia coli bacterial liquid is added and mixed evenly, and the culture is carried out overnight at 37 ℃ in an incubator.
(2) The cultures were streaked onto LB plates and incubated in an incubator at 37℃for 12h in an inverted manner. Single colonies were picked and cultured overnight at 37℃at 180rpm in LB liquid medium.
Repeating the steps (1) and (2) 5 times to obtain the escherichia coli K4 which can resist JNIW 3 phage.
The invention provides a phage JNUW3 which is preserved in the XX preservation center in the 10 th month 26 of 2022, and the preservation number is CCTCC NO: m20221670.
The phage JNUW3 is obtained by taking escherichia coli W3110 as a host, is a long-tail phage, has a full length of 43624bp and a G+C content of 51.00%, and has 58 ORFs in total.
The invention also provides a separation and purification method of the coliphage JNUW3, which comprises the following steps:
(1) Collecting a sample in a fermentation tank which is failed to ferment due to phage infection from a certain company, simply preprocessing the sample, centrifuging at 6000rpm for 10min, taking supernatant, filtering and sterilizing, uniformly mixing filtrate with the same volume of LB liquid culture medium and 1mL of coliform bacteria liquid in logarithmic phase, culturing overnight at 37 ℃ in an incubator, and enriching phage;
(2) Centrifuging the sample enrichment solution at 5000rpm for 10min, collecting supernatant, and sterilizing with microporous membrane of 0.22 μm to obtain filtrate containing phage;
(3) Mixing 100 μl of filtrate with 500 μl of host Escherichia coli bacterial liquid, standing for 15min to allow the mixture to bind with the receptor on the surface of bacteria;
(4) Adding 5mL of LB semisolid agar medium cooled to 50 ℃, uniformly mixing, immediately spreading on a solidified LB plate, culturing for 12h in an incubator at 37 ℃ in an inverted mode after agar is solidified, and observing plaque growth;
(5) On a double-layer flat plate for forming plaque, a large transparent plaque is picked up by a sterile gun head, and is subjected to shaking desorption in 1mL of LB liquid, then is subjected to sterilization by a microporous filter membrane with the thickness of 0.22 mu m to obtain phage filtrate, and is inoculated into 5mL of LB liquid culture medium, 100 mu L of corresponding host escherichia coli bacterial liquid is added and mixed uniformly, and is cultured overnight at 37 ℃ in an incubator, the supernatant is centrifuged at 5000rpm for 10min, and is filtered by a bacterial filter membrane, the plaque morphology is observed by a double-layer flat plate method, and the plaque with the consistent shape and size can be obtained after repeated operation 35 times.
The invention provides a composition, which contains coliphage JNUW3.
The invention provides a bactericide which contains coliphage JNUW3.
The invention provides a bactericidal medicament, which contains coliphage JNUW3 as a main component.
The invention provides a disinfectant, which contains coliphage JNUW3.
The invention provides a cleaning agent, which contains coliphage JNUW3.
The invention provides a feed additive composition, which contains coliphage JNUW3.
The invention also provides application of the coliphage JNUW3 in preparing bactericidal medicines or medicines for preventing and treating infectious diseases caused by colibacillus.
The invention also provides application of the coliphage JNUW3 in preparing bactericides, disinfectants, cleaners and feed additive compositions.
The invention provides a genetic engineering bacterium with phage resistance, which is used for preparing a fermentation product by taking escherichia coli K4 as a chassis cell.
In one embodiment of the invention, the fermentation product includes, but is not limited to, enzymes, organic acids, amino acids, polysaccharides, pigments.
The invention also provides application of the escherichia coli JNK4 or the genetically engineered bacterium in preparation of fermentation products and resistance to phage infection.
In one embodiment of the present invention, the phage is phage JNUW3, T4 phage, lambda phage, and the phage JNUW3 has been preserved in the chinese collection at 10 months and 26 days 2022 with a preservation number of cctcrno: m20221670.
In one embodiment of the invention, the fermentation product includes, but is not limited to, enzymes, organic acids, amino acids, polysaccharides, pigments.
The invention also provides application of the escherichia coli JNK4 or the genetically engineered bacterium in preparation of a product capable of producing a fermentation product and resisting phage infection.
In one embodiment of the present invention, the phage is phage JNUW3, T4 phage, lambda phage, and the phage JNUW3 has been preserved in the chinese collection at 10 months and 26 days 2022 with a preservation number of cctcrno: m20221670.
In one embodiment of the invention, the fermentation product includes, but is not limited to, enzymes, organic acids, amino acids, polysaccharides, pigments.
Advantageous effects
(1) The invention screens and separates bacteriophage JNUW3 from a pollution fermentation tank, and the bacteriophage can inhibit various escherichia coli, and the bacteriophage is respectively: e.coli O78:H21 (ATCC 35401), E.coli BL21, E.coli W3110, E.coli MG1655, E.coli JM109.
(2) The invention successfully screens out a chassis cell JNK4 with phage resistance, and compared with the escherichia coli in the prior art, the invention can resist infection of phage JNUW3; and the T7 phage can be resisted, the broad-spectrum phage resistance characteristic is provided, the difficult problem that fermentation failure is caused by phage infection in the production of fermentation products is fundamentally solved, and the economic benefit of fermentation production enterprises is improved.
Preservation of biological materials
Coli (Escherichia coli) JNK4, taxonomic designation Escherichia coli JNK, was deposited at the chinese collection center at month 05 of 2023 under the accession number cctccc NO: m20231188, the preservation address is Wuhan, university of Wuhan, china.
Coli phage (colibacillus) JNUW3, taxonomic designation Colibacteriophage JNUW, was deposited at the chinese collection center for typical culture at 2022, 10 months, 26, with a deposit number of cctccc NO: m20221670, the preservation address is Wuhan, university of Wuhan, china.
Drawings
Fig. 1: e.coli phage (Colibacteriophage) JNUW3 Transmission Electron Microscope (TEM) photographs.
Fig. 2: e.coli phage (Colibacteriophage) JNUW3 Transmission Electron Microscope (TEM) photographs.
Fig. 3: resistance of E.coli (Escherichia coli) JNK4 to phages; wherein 4 on the plate is phage JNUW3.
Fig. 4: resistance of E.coli (Escherichia coli) BL21 to phage; wherein 4 on the plate is phage JNUW3.
Detailed Description
Staphylococcus aureus Staphylococcus aureus, bacillus subtilis Bacillus subtilis, salmonella enterica subspecies Salmonella enterica subsp.enterica, bacillus licheniformis Bacillus licheniformis, vitis vinifera Blastobotrys adeninivorans referred to in the examples below were deposited by the laboratory.
T4 phage, lambda phage referred to in the examples below were purchased from: china Center for Type Culture Collection (CCTCC).
The following examples relate to the following media:
(1) LB liquid medium: 10g/L peptone, 5g/L, naCl g/L yeast extract.
(2) LB solid medium: 10g/L peptone, 5g/L, naCl g/L yeast extract and 15g/L agar.
(3) LB semisolid culture medium: 10g/L peptone, 5g/L, naCl g/L yeast extract and 5g/L agar.
The detection method involved in the following examples is as follows:
example 1: isolation, purification, identification and preservation of phage JNUW3
The method comprises the following specific steps:
1. isolation and purification of phages
(1) Samples collected from fermenters from a company that failed to ferment due to phage infection were dispensed into sterile centrifuge tubes and centrifuged for 5min.
(2) Sucking the supernatant after centrifugation by a disposable syringe, and filtering the supernatant by a sterilizing filter head with the diameter of 0.22 mu m after autoclaving to obtain phage filtrate; diluting the filtrate with sterilized ultrapure water to a concentration of 10 -6 The method comprises the steps of carrying out a first treatment on the surface of the Vortex for more than 10s by a vortex instrument before each dilution; the phage dilutions were stored in a refrigerator at 4℃for further use.
(3) Isolation, purification, identification and preservation of coliphage JNUW3
Preparing high-pressure sterilized LB culture solution, a sterile 10mL centrifuge tube and other necessary materials, culturing escherichia coli BL21, and putting into a shaking table; the shaking table is set at 37 ℃ and 180r/min, and the bacteria are cultured overnight.
Mixing 100 μl of filtrate with 500 μl of host Escherichia coli bacterial liquid, standing for 15min to allow the mixture to bind with the receptor on the surface of bacteria; adding 5mL of LB semisolid agar medium cooled to 50 ℃, uniformly mixing, immediately spreading on a solidified LB plate, and after agar solidification, inversely culturing for 12h at 37 ℃ in an incubator.
(4) Observing plaque growth;
(5) On a double-layer flat plate for forming plaque, a large transparent plaque is picked up by a sterile gun head, and is subjected to shaking desorption in 1mL of LB liquid, then is subjected to sterilization by a microporous filter membrane with the thickness of 0.22 mu m to obtain phage filtrate, and is inoculated into 5mL of LB liquid culture medium, 100 mu L of corresponding host escherichia coli bacterial liquid is added and mixed uniformly, and is cultured overnight at 37 ℃ in an incubator, the supernatant is centrifuged at 5000rpm for 10min, and is filtered by a bacterial filter membrane, the plaque morphology is observed by a double-layer flat plate method, and the plaque with the consistent shape and size can be obtained after repeated operation 35 times.
According to the plaque morphological characteristics of JNIW 3 and the identification result of a transmission electron microscope (figures 1-2), the JNIW 3 phage is long-tail double-stranded DNA mild phage; the rice is preserved in China Center for Type Culture Collection (CCTCC) at the 10 th month of 2022 and the 26 th day, and the preservation number is CCTCC NO: m20221670.
Example 2: phage (Colibacteriophage) JNUW3 whole genome sequencing
The whole genome sequencing of E.coli phage (Colibacteriophage) JNUW3 was performed using the I-pillar Miseq sequencing platform and the results are shown in Table 1.
Table 1: phage JNUW3 genomic genes
The results show that: the whole genome of phage JNUW3 is double-stranded DNA, the length is 43624bp, and the GC content is 51.00%. A total of 58 coding sequences were identified. However, more than half of CDSs are predicted to be hypothetical or phage proteins with unknown function.
Example 3: determination of phage JNUW3 host profile
The test strains studied included:
11 strains of common pathogenic escherichia coli such as escherichia coli O157:H7, escherichia coli O55:H7, escherichia coli O111:K58:H2, escherichia coli O103:H2 8, escherichia coli O78:K80, escherichia coli O78:H2 11, escherichia coli O128:H2, escherichia coli O114:H2, escherichia coli O145:H2, escherichia coli O26:H2 11, escherichia coli O111:H2 8 and the like with different numbers; staphylococcus aureus Staphylococcus aureus, bacillus subtilis Bacillus subtilis, salmonella enterica subspecies Salmonella enterica subsp. Whether this is the host for the bacterium is determined by a spot test on potential host plates.
The specific method comprises the following steps:
(1) Preparation of phage (colibacillus) JNUW3 suspension;
1) Preparing a culture medium: liquid LB medium was selected.
2) Proliferation of host bacteria: coli e.coli W3110 was inoculated into LB medium and cultured at 37 degrees celsius for 8h for proliferation.
3) Phage addition: phage solution was added to the above-described medium containing E.coli W3110 to infect host bacteria.
4) Incubation and culture: and 3) incubating the system added with the phage in the step 3) at 37 ℃ for 6 hours to obtain a culture solution.
5) Mixing and centrifuging: after incubation, the culture broth is removed and the bacteria and phage are separated by mixing and centrifugation.
6) And (3) filtering and clarifying: residual host bacteria and cell debris are removed by a filter or centrifugation operation to render the suspension purer.
7) Adjusting the concentration: centrifugation or an appropriate amount of solution can be used to adjust the concentration of phage suspension, as desired.
(2) mu.L of phage suspension (1X 10) obtained in step (1) 10 PFU·mL -1 ) Dropping on a plate covered with potential host bacteria, and after the potential host bacteria are dried, inversely placing the potential host bacteria in an incubator, wherein the culture conditions are as follows: 37℃for 4h.
The plates covering the potential host bacteria are respectively: 11 strains of common pathogenic escherichia coli such as escherichia coli O157, escherichia coli O55, escherichia coli O7, escherichia coli O111, K58, escherichia coli O21, escherichia coli O103, escherichia coli O8, escherichia coli O78, K80, escherichia coli O78, escherichia coli O128, escherichia coli O114, escherichia coli O2, escherichia coli O145, escherichia coli O26, escherichia coli O11, escherichia coli O111, and escherichia coli O8 are respectively added; staphylococcus aureus Staphylococcus aureus, bacillus subtilis Bacillus subtilis, salmonella enterica subspecies Salmonella enterica subsp.enterica, lb+1.8% agar powder medium of bacillus licheniformis Bacillus licheniformis;
judging whether the phage is the host of the phage to be tested according to the presence or absence of plaques on the plate.
For the E.coli epidemic strains tested, phage JNUW3 had lytic activity against E.coli O78:H11 (ATCC 35401), E.coli BL21, but did not show lytic activity against non-E.coli strains.
Example 4: screening and separation of escherichia coli JNK4
The method comprises the following specific steps:
(1) Inoculating the separated JNUW3 phage into 5mL of LB liquid medium, adding 100 mu L of corresponding host escherichia coli BL21 bacterial liquid, uniformly mixing, and culturing overnight at 37 ℃ in an incubator for the following period of time: 12h; a culture was obtained.
(2) Streaking the culture obtained in the step (1) on an LB plate, and inversely culturing for 12 hours at 37 ℃ in an incubator. Single colonies were picked and inoculated into LB liquid medium and cultured overnight at 37℃at 180 rpm. Repeating the steps until the escherichia coli JNK4 which can resist JNUW3 phage is obtained; the rice is preserved in China Center for Type Culture Collection (CCTCC) in the year 2023, namely, the month 07 and the day 05, and the preservation number is CCTCC NO: m20231188.
Example 5: experiment of resistance of E.coli JNK4 Strain to different phages
The resistance experiment of the JNK4 strain to different phages is verified through a double-layer plate spot plate experiment, and the broad-spectrum phage resistance capability of the JNK4 is verified through different phage infection JNK4 strains.
The method comprises the following specific steps:
(1) Preparation of phage suspensions
Preparation of phage (Colibacteriophage) JNUW3 (4 in FIGS. 3-4, namely the phage), T4 phage suspension, lambda phage suspension, T1 phage suspension, T7 phage suspension, and phage (named 5, 6, JNUWH, respectively) suspensions for subject group contemporaneous screening.
1) Preparing a culture medium: liquid LB medium was selected.
2) Proliferation of host bacteria: coli e.coli W3110 was inoculated into LB medium and cultured at 37 degrees celsius for 8h for proliferation.
3) Phage addition: phage solution was added to the above-described medium containing E.coli W3110 to infect host bacteria.
4) Incubation and culture: and 3) incubating the system added with the phage in the step 3) at 37 ℃ for 6 hours to obtain a culture solution.
5) Mixing and centrifuging: after incubation, the culture broth is removed and the bacteria and phage are separated by mixing and centrifugation.
6) And (3) filtering and clarifying: residual host bacteria and cell debris are removed by a filter or centrifugation operation to render the suspension purer.
7) Adjusting the concentration: centrifugation or an appropriate amount of solution can be used to adjust the concentration of phage suspension, as desired.
(2) Resistance of JNK4 strains to different phages
Whether this is the host for the bacterium is determined by a spot test on potential host plates.
10 mu L of each of the above-mentioned solutions was 1X 10 10 PFU·mL -1 The phage suspensions (JNUW 3 phage (4 in fig. 3 to 4, namely the phage) suspension, T4 phage suspension, lambda phage suspension, T1 phage, T7 phage suspension, and subject group contemporaneously screened phage (designated 5, 6, JNUWH, respectively) were dropped onto a plate covering potential host bacteria, and after air-dried, placed upside down in an incubator for overnight culture.
The plate covered with the potential host bacteria is added with the bacterial concentration: LB+1.8% agar powder culture medium of E.coli JNK4 strain with OD600 of 0.6-0.8.
Judging whether the phage is the host of the phage to be tested according to the presence or absence of plaques on the plate.
The verification result shows that (in figures 3-4, 4 on the plate is phage JNUW 3), the JNK4 escherichia coli strain can resist JNUW3 and T7 phage, and has a certain broad-spectrum anti-phage property.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. Coli (Escherichia coli) JNK4 with phage resistance was deposited in the chinese collection at month 05 of 2023 under the accession number cctccc NO: m20231188.
2. Coli phage (colibacillus) JNUW3 was deposited at the chinese collection at 10 months and 26 days 2022 under the accession number cctccc NO: m20221670.
3. A product, characterized in that the product contains the escherichia coli bacteriophage JNUW3 according to claim 2; the product comprises a composition, a bactericide, a bactericidal medicament, a disinfectant, a cleaning agent and a feed additive; the main active ingredient in the composition is coliphage JNUW3.
4. A genetically engineered bacterium with phage resistance is characterized in that the genetically engineered bacterium takes the escherichia coli JNK4 as a chassis cell in claim 1 to prepare a fermentation product.
5. The use according to claim 4, wherein the fermentation products include, but are not limited to, enzymes, organic acids, amino acids, polysaccharides, pigments.
6. Use of the escherichia coli JNK4 of claim 1 or the genetically engineered bacterium of claim 4 or 5 for the preparation of a fermentation product or for the preparation of a product capable of producing a fermentation product.
7. The use according to claim 6, wherein the phage is phage JNUW3, T4 phage, lambda phage, and the phage JNUW3 has been deposited at the chinese collection at 10.10.26 of 2022 with a deposit number of CCTCC NO: m20221670.
8. The use according to claim 7, wherein the fermentation products include, but are not limited to, enzymes, organic acids, amino acids, polysaccharides, pigments.
9. The use of the coliphage JNUW3 of claim 2 for preparing a bactericidal medicament or a medicament for preventing and treating infectious diseases caused by escherichia coli.
10. Use of the coliphage JNUW3 of claim 2 in the preparation of bactericides, disinfectants, cleaners, feed additives.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311130419.4A CN117187126A (en) | 2023-09-04 | 2023-09-04 | Isolation of coliphage JNUW3 and development of anti-JNUW 3 phage colibacillus |
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| CN202311130419.4A CN117187126A (en) | 2023-09-04 | 2023-09-04 | Isolation of coliphage JNUW3 and development of anti-JNUW 3 phage colibacillus |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114828868A (en) * | 2019-11-27 | 2022-07-29 | 艾发可持续能源创新发展股份公司 | Bacteriophage resistant microorganisms |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114828868A (en) * | 2019-11-27 | 2022-07-29 | 艾发可持续能源创新发展股份公司 | Bacteriophage resistant microorganisms |
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