CN117965459A - Coliphage strain 04922C and expression product depolymerase Depo thereof and application - Google Patents
Coliphage strain 04922C and expression product depolymerase Depo thereof and application Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The application relates to the technical field of phages, and particularly discloses an escherichia coli phage strain 04922C and an expression product depolymerase Depo and application thereof. E.coli phage strain 04922C (ESCHERICHIA COLI PHAGE 04922C) is preserved in China Center for Type Culture Collection (CCTCC) NO: M2020467, and the preservation time is 9 months and 4 days in 2020; coli phage 04922C can be metabolized to produce phage depolymerase Depo54, and phage depolymerase Depo54 has excellent effects of degrading bacterial polysaccharide and assisting antibiotics in killing bacteria.
Description
Technical Field
The application relates to the technical field of phages, in particular to an escherichia coli phage strain 04922C and an expression product depolymerase Depo and application thereof.
Background
Bacterial biofilms are mainly composed of mucus secreted by bacteria, and are wrapped around bacteria, so that the bacteria are not only protected from being attacked by host immune cells, but also are difficult to kill by antibiotics or common disinfectants, and great difficulty is added to clinical treatment of infectious diseases. In addition, the presence of biofilm formation by bacteria on the surfaces of medical devices is also a major cause of problems in disinfecting devices. It has been shown that bacteria encapsulated in biofilms have a 10-1000 fold improvement in their resistance to antibiotics compared to the planktonic form. But humans are not completely incapable of biofilm. At present, a great deal of researches show that phage and the encoded depolymerase thereof have good cleaning effect on a biological envelope.
Phage depolymerase (Depolymerase) is an EPS hydrolase that recognizes and degrades bacterial polysaccharides, and is generally synthesized from phage tail structural proteins (tail tube, tail thorn, tail fiber). When the phage specifically binds bacteria, tail protein synthesizes and secretes depolymerizing enzyme to degrade biofilm, so that the phage can kill bacteria specifically. The depolymerase has the ability to diffuse in agar, which is faster than phage and can break down bacterially produced polysaccharides. Therefore, in order to determine whether phage can produce depolymerase, it is necessary to observe its transparent plaques (plaque). Around this area there is a translucent halo (halo) and the halo expands with increasing latency. This is the phenomenon in which phages produce depolymerases, ranging in size from 197 to 2417 amino acids, to cleave bacterial EPS.
Phage depolymerases may be classified as hydrolases or lyases, depending on the mechanism of action of the phage depolymerase. Hydrolytic enzymes include, for example, sialidases that degrade capsular polysialic acid, and rhamnosidases that hydrolyze LPS O-antigen; depolymerases having phosphatase activity towards Wall Teichoic Acid (WTA) are also hydrolases. The lyase includes hyaluronidases capable of cleaving hyaluronic acid-based capsules and EPS, pectin/pectin lyase responsible for degrading biofilm EPS, alginate lyase encoded by certain azotobacter and pseudomonas phages capable of degrading host alginic acid EPS. Depolymerase may refer to any general purpose protein capable of degrading a polymer. From this point of view, phage-encoded endolysins are also depolymerases, as they cleave peptidoglycans. Since these enzymes target and disrupt structures important for cell survival and virulence, they may find use in controlling bacterial pathogens.
There have been studies to date that have found a depolymerase derived from a different phage, such as phage depolymerase Depo58 derived, for example, from Klebsiella pneumoniae strain P719, such as phage depolymerase Depo43 derived from Klebsiella pneumoniae strain P560, and the like. But there are fewer reports about the depolymerase enzyme against coliphage and its metabolites.
Disclosure of Invention
The application provides an escherichia coli phage strain 04922C and an expression product depolymerase Depo thereof and application, wherein the expression product depolymerase Depo of the escherichia coli phage 04922C can obviously degrade exomembrane polysaccharide of bacteria and inhibit formation of bacterial biofilm; the depolymerizing enzyme Depo and Depo can be used for preparing phage depolymerizing enzyme medicaments, can be applied to the sterilization of living environments and instruments, and has wide application prospects.
In a first aspect, the present application provides an escherichia coli bacteriophage strain 04922C, which adopts the following technical scheme: an escherichia coli phage strain 04922C, wherein the escherichia coli phage strain 04922C (ESCHERICHIA COLI PHAGE 04922C) is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2020467 and the preservation time of 2020, 9 and 4 days.
By adopting the technical scheme, the coliphage strain 04922C can express the depolymerase Depo54, and the depolymerase Depo54 can degrade the exomembrane polysaccharide of bacteria, so that the bacteria can be efficiently cracked, and an excellent bactericidal effect is achieved.
Alternatively, the open reading frame at position 54 of the genome of the E.coli phage strain 04922C encodes a depolymerase.
In a second aspect, the present application provides a phage depolymerizing enzyme Depo, comprising the following steps:
A phage depolymerase Depo, said phage depolymerase Depo being prepared from the above-described escherichia coli phage strain 04922C.
The 54 th open reading frame of the coliphage strain 04922C is expressed to obtain phage depolymerase Depo54, and the depolymerase Depo54 can obviously degrade bacterial exomembrane polysaccharide and inhibit bacterial biofilm formation. Furthermore, the depolymerase Depo can be used for preparing phage depolymerase medicaments, the obtained phage depolymerase medicaments are used for disinfecting living environments and instruments, and the phage depolymerase Depo has wide application prospect.
Alternatively, the amino acid sequence of phage depolymerase Depo is shown in SEQ ID NO. 1.
Alternatively, the gene sequence of phage depolymerizing enzyme Depo is shown in SEQ ID NO. 2.
Optionally, the preparation method of the phage depolymerase Depo comprises the following steps:
Obtaining Depo gene fragment of an open reading frame at a 04922C 54 th position of an escherichia coli phage strain through PCR, double enzyme digestion and connecting molecule cloning, and connecting the Depo gene fragment to a plasmid vector to obtain a recombinant plasmid;
Screening the recombinant plasmid host cells to obtain recombinant host cells containing the recombinant plasmid;
Culturing the recombinant host cell to obtain a culture solution, and collecting the liquid after solid-liquid separation of the culture solution to obtain the phage depolymerizing enzyme Depo.
Optionally, the plasmid vector is a plasmid vector pGAPZalpha A; the host cell is pichia pastoris;
Preferably, the pichia pastoris is selected from any one of pichia pastoris SMD1168, pichia pastoris GS115 and pichia pastoris X-33.
Optionally, when culturing the recombinant host cell, the culturing method comprises the steps of:
The recombinant host cells were inoculated onto YPD liquid medium, cultured at 28-32℃and 120-180rpm to the logarithmic growth phase, and then inoculated onto new YPD liquid medium for 40-52h.
In a third aspect, the present application provides the use of a bacteriophage depolymerizing enzyme Depo as described above to degrade bacterial polysaccharides.
In a fourth aspect, the present application provides the use of an auxiliary antibiotic of the phage depolymerase Depo as described above to kill bacteria.
Optionally, the antibiotic is selected from any one or more of enrofloxacin, florfenicol, doxycycline and compound neotame.
In summary, the application has the following beneficial effects:
1. the application firstly provides an escherichia coli phage strain 04922C, wherein the 54 th open reading frame of the genome codes phage depolymerase Depo; the phage strain is capable of expressing a depolymerase Depo, 54.
2. The phage depolymerase Depo54 obtained from the coliphage 04922C of the application can destroy the exomembrane polysaccharide of bacteria, reduce bacterial virulence and inhibit the formation of biofilm with high efficiency.
3. The phage depolymerase Depo54 obtained from the coliphage 04922C can be synergistic with various antibiotics, and the sterilization effect is obviously improved.
Drawings
FIG. 1 is a plaque plot of E.coli phage 04922C in example 1 of the present application;
FIG. 2 is an electron microscope image of E.coli phage 04922C in example 1 of the present application;
FIG. 3 is an agarose gel electrophoresis chart of PCR amplified products of the phage depolymerizing enzyme Depo gene of example 2 of the present application;
FIG. 4 is a diagram showing the degradation of E.coli polysaccharide by phage depolymerizing enzyme Depo54 of application example 1 of the present application;
FIG. 5 is a graph showing the effect of phage depolymerizing enzyme Depo54 of application example 2 of the present application on the auxiliary antibiotic to kill E.coli.
Detailed Description
The application is described in further detail below with reference to the drawings and examples, in which: the following examples, in which no specific conditions are noted, are conducted under conventional conditions or conditions recommended by the manufacturer, and the raw materials used in the following examples are commercially available from ordinary sources except for the specific descriptions.
Example 1
An coliphage strain 04922C, wherein the coliphage strain 04922C (ESCHERICHIA COLI PHAGE 04922C) is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2020467 and the preservation time of 2020, 9 and 4 days. The 54 th open reading frame of the genome of the E.coli phage strain 04922C encodes a depolymerase.
The method for separating and purifying the coliphage 04922C comprises the following specific steps:
(1) The original sample is from chicken farm in Fujian province, excrement collected by chicken farm and water sample on ground are taken, mixed evenly and then put into 2L enriching barrels respectively, and the total sample amount is not more than 1/4 of the barrel volume.
(2) Pathogenic E.coli (ATCC 25922) was grown in an expanded culture using 100mL of LB liquid medium, and 100mL of the pathogenic E.coli culture broth obtained by the culture was put in an enrichment vessel containing the sample. 300mL of fresh LB liquid culture was added to the enrichment vessel, which was then incubated overnight at 37 ℃. The next day 10mL of the enriched solution was placed in a centrifuge tube, centrifuged at 10000rpm for 10min, 5mL of the supernatant was filtered twice through a 0.22 μm filter, and the filtrate was stored at 4 ℃. Phage isolation was performed using spot method: 10. Mu.L of the prepared filtrate was pipetted onto agar plates coated with host bacteria, each agar plate was spotted twice, labeled, and incubated upside down at 37℃overnight to observe plaques.
(3) Purifying: the isolated phage were purified using a double-layer agar method. Single plaques were picked in a 2mL centrifuge tube, 500. Mu.L of SM buffer was added and the plaques were triturated and incubated overnight at 4 ℃. Centrifuging at 12000rpm for 5min after vortex shaking for 5min on the next day, filtering the supernatant with 0.22 μm filter membrane, collecting phage filtrate and equal volume of host bacteria liquid, and culturing by double-layer flat plate method. Purifying for 3 times to obtain phage with uniform plaque morphology, namely named as coliphage 04922C, adding glycerol with a final concentration of 20Vol.% into coliphage 04922C bacterial liquid, and storing in an ultralow temperature refrigerator at-80 ℃ for later use.
The preparation method of the purified coliphage 04922C proliferation liquid comprises the following steps: respectively taking 500 mu L of coliphage 04922C bacterial liquid and host bacterial liquid (colibacillus liquid), adding into 7mL of LB upper layer agar, mixing uniformly, then pouring uniformly on an LB solid plate, inverting the solid plate into a biochemical incubator after the upper layer is solidified, and culturing overnight at 37 ℃. The next day the plate was observed, as in FIG. 1, the appropriate plate was selected, the upper agar was scraped, triturated and placed in SM buffer for extraction overnight at 4℃and then vortexed, centrifuged and filtered with a 0.22 μm filter to give E.coli phage 04922C proliferation.
And (3) observing a morphological structure by using an electron microscope: and (3) dripping 20 mu L of purified coliphage 04922C proliferation liquid on a copper mesh, standing for 10min, sucking the residual liquid on the side surface of the copper mesh by using filter paper, dripping 10 mu L of 2% phosphotungstic acid on the copper mesh, sucking the residual liquid, standing, drying, and then placing under 100kv of a Philips CM100 type transmission electron microscope to observe the shape of coliphage 04922C. The results are shown in fig. 2: coli phage 04922C consisted of a hexagonal head with a diameter of 75.6.+ -. 2.2nm, a collapsible tail sheath and a tail tube with a length of 151.2.+ -. 3.7nm.
Example 2
A phage depolymerizing enzyme Depo and its preparation method specifically comprises:
Step1, extraction of coliphage genome
The genome of E.coli phage 04922C was extracted using a viral DNA extraction kit (purchased from North View organism, nanjing).
Step 2, constructing pGAPZ alpha A-Depo recombinant plasmid
Designing primers for amplifying phage depolymerase genes:
an upstream primer: 5'-GAATTCTTGGCTATCGAGACTAATGC-3', SEQ ID NO 3;
a downstream primer: 5'-GCGGCCGCTTAGCTAATACGTCTCCAAA-3', SEQ ID NO 4;
The PCR amplification reaction system is as follows: 2.0. Mu.L of upstream primer, 2.0. Mu.L of downstream primer, 2. Mu.L of phage DNA template, 25. Mu.L of 2×Taq mix enzyme, and 19. Mu.L of ddH 2 O;
The reaction conditions are as follows: pre-denaturation at 95℃for 3min, denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 45s,28 cycles, 72℃for 10min, and 4℃for 5min.
And (3) connecting the phage depolymerizing enzyme gene product amplified by PCR to pGAPZ alpha A plasmid after enzyme digestion and purification, wherein enzyme digestion sites are EcoRI and NotI, and obtaining pGAPZ alpha A-Depo54 recombinant plasmid.
Step 3, preparing recombinant Pichia pastoris GS115-Depo54
Sucking 5 mu L of pGAPZalpha A-Depo recombinant plasmid and 80 mu L of pichia pastoris GS115 competent cells, mixing well, and adding into a precooled electric shock cup. The electric shock conditions are as follows: the voltage is 1.5KV, the capacitance is 25 muF, the resistance is 200-400 omega, and the time is 10sec.
After the electric shock is finished, 1mL of precooled 1M sorbitol is sucked into an electric shock cup to uniformly mix bacterial liquid, and the bacterial liquid is transferred into a precooled 2mL sterile centrifuge tube; the bacterial solution was plated on 50. Mu.g/mL bleomycin-resistant YPD plates and incubated at 30℃until single colonies appeared.
Verification by amplification primers of phage depolymerase gene, the result is shown in FIG. 3, and the correct positive clone strain was selected and named recombinant Pichia pastoris GS115-Depo54.
And 4, inoculating recombinant Pichia pastoris GS115-Depo to YPD liquid culture medium, culturing for 8 hours to logarithmic phase at 150rpm/min in a constant temperature shaking box at 30 ℃, and then inoculating to fresh YPD liquid culture medium for 48 hours according to an inoculum size of 1:100. After the culture is finished, the culture solution is centrifuged and filtered by a 0.22 mu m filter membrane, and the filtrate is collected to obtain the phage depolymerizing enzyme Depo.
The amino acid sequence of the phage depolymerizing enzyme Depo is shown as SEQ ID NO. 1, and the gene sequence is shown as SEQ ID NO. 2.
The amino acid sequence of phage depolymerizing enzyme Depo is shown as SEQ ID NO. 1, specifically:
LAIETNAVVVTDLNPLYPRDRDYIYEGAAQIRLIKQTLQNTFPNVTEPVDIDSDTFKIMSEKL
KFTGDSMDVGGLMIKNVTPGTGDKDVVTKGQMEAFMKNWMENKVFRIGSYYITEEDINP
GDSISLGFGSWAKVTGVIMGTGVVNPDGSVPNAQRVEFQAGGTGGRVFNTIRTENVPLMT
VNGSSFSLSSNTHSHNMVFGRGDASGHNSSPNWYSPGGGYSQRTENDTHSHTISGSVSLGR DDISRQPINTLPPFRSAHIWRRIS；
the gene sequence of phage depolymerizing enzyme Depo is shown as SEQ ID NO. 2, specifically:
>ORF54
TTGGCTATCGAGACTAATGCGGTAGTTGTTACCGACTTAAACCCGCTCTATCCTAGAGAC
AGGGATTACATCTACGAAGGGGCGGCTCAAATCCGCCTCATTAAACAAACACTACAGAA
CACATTCCCTAATGTCACAGAACCAGTTGATATTGATTCTGACACTTTTAAGATTATGTCA
GAGAAGCTCAAGTTTACTGGTGATTCAATGGATGTTGGCGGTCTAATGATTAAGAACGTC
ACTCCGGGGACTGGTGACAAAGATGTTGTTACTAAAGGTCAGATGGAAGCGTTCATGAA
AAACTGGATGGAGAATAAAGTATTCCGTATCGGCTCTTACTATATCACTGAAGAAGATATT
AACCCCGGTGATTCTATCTCCTTAGGGTTTGGCTCTTGGGCTAAAGTGACTGGTGTTATC
ATGGGTACTGGTGTCGTAAACCCTGATGGCTCTGTTCCTAACGCTCAGAGGGTTGAGTTC
CAAGCAGGTGGCACAGGTGGCCGTGTCTTCAATACCATTCGTACAGAGAACGTACCTCT
GATGACAGTTAATGGCTCCAGCTTCTCCTTATCTAGTAACACCCACAGTCACAACATGGT
ATTTGGTCGTGGAGATGCTAGTGGTCACAACAGCTCGCCTAACTGGTACAGTCCGGGTG
GTGGTTATAGTCAGAGAACAGAGAATGACACACACTCTCATACAATCTCTGGTAGCGTG
AGTCTTGGTCGTGATGATATTTCTCGTCAACCTATTAACACTTTGCCACCATTCAGATCGGCTCACATTTGGAGACGTATTAGCTAA。
Application example
Application example 1
Use of phage depolymerase Depo to degrade bacterial polysaccharides.
E.coli was cultured with fresh LB medium to log phase and bacterial extracellular polysaccharide was extracted by Soxhibao bacterial polysaccharide extraction kit.
4 Test groups were set up, respectively: 1) 200. Mu.L of depolymerase Depo; 2) 200 μl of polysaccharide extract; 3) 200. Mu.L of polysaccharide extract+200. Mu.L of depolymerase Depo54; 4) 200. Mu.L of depolymerase Depo54 +200. Mu.L of PBS buffer. And simultaneously incubating the mixed solution of each experimental group in a water bath at 37 ℃ for 1h, and taking the mixture of each group to carry out SDS-PAGE gel electrophoresis after the incubation is finished, and carrying out silver staining to observe the degradation effect of the depolymerizing enzyme Depo on the polysaccharide on the bacterial surface. The results are shown in FIG. 4: the polysaccharide bands in group 3 treated with phage depolymerase Depo disappeared and the polysaccharide bands in group 4 with control added PBS buffer were identical to those in group 2, indicating that the recombinantly expressed phage depolymerase Depo was active and able to degrade the extracted polysaccharide.
Application example 2
Use of a secondary antibiotic of phage depolymerase Depo to kill escherichia coli.
And (3) designing a test for killing escherichia coli by using phage depolymerase to assist antibiotics, selecting escherichia coli bacterial liquid OD 600 =0.8 in the test, preparing each group of test samples (3 parallel samples are designed for each test group), and then placing the test samples in a biochemical incubator at 37 ℃ for incubation for 24 hours to determine the OD value of the samples. The specific test groups and results are shown in table 1:
TABLE 1 grouping of experiments for different test groups
Referring to table 1 and fig. 5, the results indicate that: the killing effect of antibiotics alone (e.g., group 2 and group 4) or phage depolymerase Depo alone (e.g., group 6) on bacteria is not very strong; however, the two antibiotics were better at killing bacteria when combined with phage depolymerase Depo and 54, respectively, than when one antibiotic alone, indicating better killing when the added phage depolymerase Depo assisted the antibiotic.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Claims (10)
1. The coliphage strain 04922C is characterized in that the coliphage strain 04922C (ESCHERICHIA COLI PHAGE 04922C) is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2020467 and the preservation time of 2020, 9 and 4 days.
2. An escherichia coli phage strain 04922C as claimed in claim 1, wherein the genomic open reading frame at position 54 of escherichia coli phage strain 04922C encodes a depolymerase.
3. A phage depolymerase Depo and Depo, wherein phage depolymerase Depo and Depo are prepared from the escherichia coli phage strain 04922C of any one of claims 1-2.
4. The phage depolymerizing enzyme Depo of claim 3, wherein said phage depolymerizing enzyme Depo has an amino acid sequence as set forth in SEQ ID No. 1;
alternatively, the gene sequence of phage depolymerizing enzyme Depo is shown in SEQ ID NO. 2.
5. The phage depolymerizing enzyme Depo of claim 3, wherein the process of preparing phage depolymerizing enzyme Depo comprises the steps of:
Obtaining Depo gene fragment of an open reading frame at a 04922C 54 th position of an escherichia coli phage strain through PCR, double enzyme digestion and connecting molecule cloning, and connecting the Depo gene fragment to a plasmid vector to obtain a recombinant plasmid;
Screening the recombinant plasmid host cells to obtain recombinant host cells containing the recombinant plasmid;
Culturing the recombinant host cell to obtain a culture solution, and collecting the liquid after solid-liquid separation of the culture solution to obtain the phage depolymerizing enzyme Depo.
6. The phage depolymerizing enzyme Depo of claim 5, wherein said plasmid vector is plasmid vector pgapzαa; the host cell is pichia pastoris;
Preferably, the pichia pastoris is selected from any one of pichia pastoris SMD1168, pichia pastoris GS115 and pichia pastoris X-33.
7. The phage depolymerizing enzyme Depo of claim 5, wherein when said recombinant host cell is cultured, the culturing method comprises the steps of:
The recombinant host cells were inoculated onto YPD liquid medium, cultured at 28-32℃and 120-180rpm to the logarithmic growth phase, and then inoculated onto new YPD liquid medium for 40-52h.
8. Use of a bacteriophage depolymerizing enzyme Depo, according to any one of claims 3-7, to degrade bacterial polysaccharides.
9. Use of a supplementary antibiotic of phage depolymerase Depo according to any one of claims 3 to 7 to kill bacteria.
10. The use of the phage depolymerizing enzyme Depo to assist in killing bacteria according to claim 9, wherein the antibiotic is selected from any one or more of enrofloxacin, florfenicol, doxycycline and compound neotame.
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