CN116903710B - Phage targeting protein molecule and application thereof in identifying shiga toxin-producing escherichia coli of O103 antigen serotype - Google Patents
Phage targeting protein molecule and application thereof in identifying shiga toxin-producing escherichia coli of O103 antigen serotype Download PDFInfo
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Abstract
The invention provides a phage targeting protein molecule and application thereof in identifying shiga toxin-producing escherichia coli of O103 antigen serotype, wherein the amino acid sequence of the phage targeting protein molecule is shown as SEQ ID NO: 1. The phage targeting protein molecule can specifically identify the shiga toxin-producing escherichia coli of the O103 antigen serotype, can be used for identifying the shiga toxin-producing escherichia coli of the O103 antigen serotype, has the advantages of simplicity, rapidness, accuracy and the like, and has high application value.
Description
Technical Field
The present invention relates to the field of biology. In particular, the invention relates to the use of phage targeting protein molecules in shiga toxin-producing escherichia coli for the identification of O103 antigen serotypes.
Background
Shiga toxin-producing escherichia coli (Shiga toxin-producing)Escherichia coliSTEC) is a pathogen which is co-affected by humans and animals and can cause diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and other symptoms of human beings, and even threaten life. O157:H27 STEC is the serotype responsible for the disease, and cases other than O157 STEC have been increasing in recent years. The U.S. department of agriculture food safety inspection, 1994 announced O1036, O45, O103, O111, O121, and O145 as the first six non-O157 serotypes. In China, three serotypes of STEC O103 have high popularity in human beings and animals, and seriously threaten the life health of the human beings.
STEC releases shiga toxin Stx, which not only exacerbates tissue damage by locally produced pro-inflammatory cytokines, but also causes damage to kidney organs. Although the use of antibiotics can cope with infections with bacterial diseases, the disease caused by STEC is aggravated. Currently, identification of a specific serotype of STEC relies mainly on single serum factors, whole genome sequencing, and the like. These methods have the disadvantages of long time-consuming period, high price and the like. Therefore, there is a need to find a method for rapidly identifying STEC serum with a short identification period and low cost.
At present, no research report on the identification of STEC O103 by using phage targeting protein molecules exists, and the research is still yet to be performed.
Disclosure of Invention
The present invention aims to solve, at least to some extent, the technical problems existing in the prior art. Therefore, the invention provides a phage targeting protein molecule which can specifically identify the shiga toxin-producing escherichia coli of the O103 antigen serotype, can be used for identifying and enriching the shiga toxin-producing escherichia coli of the O103 antigen serotype, has the advantages of simplicity, rapidness, accuracy and the like, and has high application value.
In one aspect of the invention, the invention provides a phage targeting protein molecule. According to an embodiment of the invention, the amino acid sequence of the phage targeting protein molecule is as shown in SEQ ID NO: 1.
In another aspect of the invention, the invention provides a conjugate. According to an embodiment of the invention, the conjugate comprises: a phage targeting protein molecule as described previously; and a linker molecule linked to the phage targeting protein molecule for labeling the phage targeting protein molecule.
In yet another aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes a phage targeting protein molecule as described previously.
In yet another aspect of the invention, the invention provides an expression vector. According to an embodiment of the invention, the expression vector comprises the nucleic acid molecule as described above.
In yet another aspect of the invention, the invention provides a kit. According to an embodiment of the invention, the kit comprises at least one of the following: the phage targeting protein molecule, the conjugate, the nucleic acid molecule, and the expression vector described previously.
In a further aspect of the invention, the invention proposes the use of at least one of the phage targeting protein molecules, the conjugates, the nucleic acid molecules, the expression vectors and the kit as described previously in shiga toxin-producing escherichia coli for the identification and/or enrichment of O103 antigen serotypes for non-diagnostic purposes.
In yet another aspect of the invention, the invention provides a method of identifying shiga toxin-producing escherichia coli of the O103 antigen serotype for non-diagnostic purposes. According to an embodiment of the invention, the method comprises: co-culturing the phage targeting protein molecule; determining whether the microorganism to be tested is shiga toxin-producing escherichia coli of an O103 antigen serotype based on whether the phage targeting protein molecule binds to the microorganism to be tested.
In a further aspect of the invention, the invention proposes the use of a reagent selected from at least one of the phage targeting protein molecules, the conjugates, the nucleic acid molecules and the expression vectors described previously in the preparation of a kit. According to an embodiment of the invention, the kit is used for diagnosing diseases and/or symptoms caused by shiga toxin-producing escherichia coli infected with the O103 antigen serotype.
In yet another aspect of the invention, the invention provides a method of enriching a shiga toxin-producing escherichia coli of an O103 antigen serotype. According to an embodiment of the invention, the method comprises: co-incubating a shiga toxin-producing escherichia coli sample containing an O103 antigen serotype with a conjugate as described previously, wherein the linking molecule is selected from the group consisting of magnetic bead probes; the magnetic bead probe is adsorbed by magnetic force so as to enrich the shiga toxin-producing escherichia coli of the O103 antigen serotype from the sample to be treated.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows an electrophoretogram;
FIG. 2 shows the OD of ppO protein 103 at different pH values;
FIG. 3 shows the OD of ppO protein 103 under different temperature conditions;
FIG. 4 shows STEC O103 bacterial OD values at different bacterial concentrations;
FIG. 5 shows STEC O103 bacterial OD values at various concentrations of different ppO103 proteins;
FIG. 6 shows a schematic diagram of ppO protein-103 specific assay.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
Phage targeting protein molecules
In one aspect of the invention, the invention provides a phage targeting protein molecule. According to an embodiment of the invention, the phage targeting protein molecule has the sequence as set forth in SEQ ID NO:1 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% homology thereto. Therefore, the phage targeting protein molecule can specifically identify the shiga toxin-producing escherichia coli of the O103 antigen serotype, rapidly and accurately identify and enrich the shiga toxin-producing escherichia coli of the O103 antigen serotype, is beneficial to research on pathogenic escherichia coli, disease diagnosis and treatment, and has important significance in defending and controlling pathogenic escherichia coli transmission. This phage targeting protein molecule is herein designated as "phage targeting protein molecule ppO103".
IVNLANAVDDGDALSFGQVKTMNQNSWQARNESLQFRNEAETFRNQAEGFKNESGTNATNTYKWRNEAEGFRDEAEQFKNTATQQATDAGNSAAAAHQSEVNAENSATASANSATLAEQQADRAEREADKLGNWNALAGTIQDVSGEDVTWRGIQTAKGFQLIEGANKPSYIVGRADNSNSWYVGKGTAGSPEVALHSYDLDTSILLKSDHIVLNKPLNVNGEVRTLNSALQNDGDIRGGRWGGTLGDTIQRNRVNYQRVFSGTWLQGQTVTFTDAPIGRQFIMIGVNTGAAIEYVSACTPVAGETFWVNIGNWSYKIGTSQDGWGAVIHASQDIHGGVPGAIAEVYVIRTDLDK（SEQ ID NO：1）
Conjugates, nucleic acid molecules, expression vectors and kits
In another aspect of the invention, the invention provides a conjugate. According to an embodiment of the invention, the conjugate comprises: a phage targeting protein molecule as described previously; and a linker molecule linked to the phage targeting protein molecule for labeling the phage targeting protein molecule. Therefore, the conjugate can be used for rapidly and accurately identifying and enriching the shiga toxin-producing escherichia coli of the O103 antigen serotype.
In some embodiments, the linking molecule is selected from at least one of the following: GFP protein, mCherry protein, FITC, TRITC, NHS-fluorescein and NHS-rhodamine.
In yet another aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes a phage targeting protein molecule as described previously. Therefore, the phage targeting protein molecule coded by the nucleic acid molecule can specifically identify the shiga toxin-producing escherichia coli of the O103 antigen serotype, and the purposes of rapid and accurate identification and enrichment are achieved.
According to an embodiment of the invention, the nucleic acid molecule has the sequence as set forth in SEQ ID NO:2 or a nucleotide sequence having at least 80%, 85%, 90%, 95% or 99% homology thereto.
atcgtgaacctagcgaacgctgtggatgatggtgatgctctttcatttggtcaagttaagaccatgaaccagaactcatggcaagcacgcaatgaatccttacagttccgcaatgaggctgagaccttccgtaaccaagcggagggctttaagaatgagtccggaactaacgctacgaatacctataagtggcgtaatgaggctgaggggttccgtgatgaggccgaacagttcaagaacactgcgacacaacaggctaccgatgctggtaactctgcggctgctgctcaccaatccgaggtgaacgctgagaactcagctaccgcttccgctaactctgctaccttggcagaacagcaggcagaccgtgcggaacgtgaagcagacaagttgggtaactggaatgctcttgcagggacgatacaagatgtttctggagaggatgttacttggagaggaatacagacagcaaaggggttccaattgattgagggtgctaataaaccatcgtacatagttggacgggcggataacagtaactcttggtacgtcggtaagggcactgcaggttctccagaggtggccttacacagttatgacctcgacacctcaattctcttaaaaagtgaccacattgtattaaacaaaccgctaaatgtgaatggggaggttaggaccctgaactccgcattacaaaacgacggtgatatacgaggcggcagatgggggggtacactaggtgataccattcaaagaaatcgtgtgaattatcaaagggtcttttcgggtacgtggttgcaaggtcaaacagttacattcacagatgcgccaataggacgacaattcattatgataggagtcaatacaggagcagccatagagtatgtatcagcatgtacgccagtcgctggggaaacattctgggtaaatatcgggaactggagctacaaaataggcacttcacaggatggttggggggccgttatacacgcatcacaggatatccatggcggagtccctggcgcaattgcggaggtctacgttatcaggactgatttagataaatag（SEQ ID NO：2）
In yet another aspect of the invention, the invention provides an expression vector. According to an embodiment of the invention, the expression vector comprises the nucleic acid molecule as described above. Therefore, the expression vector can express the nucleic acid molecule as a phage targeting protein molecule, so that the shiga toxin-producing escherichia coli of the O103 antigen serotype can be specifically combined, and the shiga toxin-producing escherichia coli of the O103 antigen serotype can be rapidly and accurately identified and enriched.
In yet another aspect of the invention, the invention provides a kit. According to an embodiment of the invention, the kit comprises at least one of the following: the phage targeting protein molecule, the conjugate, the nucleic acid molecule, and the expression vector described previously. The kit can specifically combine with the shiga toxin-producing escherichia coli of the O103 antigen serotype, and can rapidly and accurately identify and enrich the shiga toxin-producing escherichia coli of the O103 antigen serotype.
It should be noted that the features and advantages described above for phage targeting protein molecules are equally applicable to the conjugates, nucleic acid molecules, expression vectors and kits, and are not described in detail herein.
Application of
In a further aspect of the invention, the invention proposes the use of at least one of the phage targeting protein molecules, the conjugates, the nucleic acid molecules, the expression vectors and the kit as described previously in shiga toxin-producing escherichia coli for the identification and/or enrichment of O103 antigen serotypes for non-diagnostic purposes.
In a further aspect of the invention, the invention proposes the use of a reagent selected from at least one of the phage targeting protein molecules, the conjugates, the nucleic acid molecules and the expression vectors described previously in the preparation of a kit. According to an embodiment of the invention, the kit is used for diagnosing diseases and/or symptoms caused by shiga toxin-producing escherichia coli infected with the O103 antigen serotype.
As described above, the phage targeting protein molecule can specifically bind to the shiga toxin-producing escherichia coli of the O103 antigen serotype, rapidly and accurately identify and enrich the shiga toxin-producing escherichia coli of the O103 antigen serotype, is beneficial to research, disease diagnosis and treatment of pathogenic escherichia coli, and has important significance in defending and controlling pathogenic escherichia coli transmission.
According to an embodiment of the invention, the disease and/or condition is selected from at least one of the following: diarrhea, colitis and uremia.
It should be noted that the features and advantages described above for phage targeting protein molecules, conjugates, nucleic acid molecules, expression vectors and kits are equally applicable to this application and are not described here in detail.
Method
In yet another aspect of the invention, the invention provides a method of identifying shiga toxin-producing escherichia coli of the O103 antigen serotype. According to an embodiment of the invention, the method comprises: co-culturing the phage targeting protein molecule; determining whether the microorganism to be tested is shiga toxin-producing escherichia coli of an O103 antigen serotype based on whether the phage targeting protein molecule binds to the microorganism to be tested. As described above, the phage targeting protein molecule can specifically bind to the shiga toxin-producing escherichia coli of the O103 antigen serotype, thereby realizing rapid and accurate identification of the shiga toxin-producing escherichia coli of the O103 antigen serotype, being applicable to diagnosis of related diseases of the shiga toxin-producing escherichia coli of the O103 antigen serotype and also being applicable to biological research of the shiga toxin-producing escherichia coli of the O103 antigen serotype for non-diagnostic purposes.
In yet another aspect of the invention, the invention provides a method of enriching a shiga toxin-producing escherichia coli of an O103 antigen serotype. According to an embodiment of the invention, the method comprises: co-incubating a shiga toxin-producing escherichia coli sample containing an O103 antigen serotype with a conjugate as described previously, wherein the linking molecule is selected from the group consisting of magnetic bead probes; the magnetic bead probe is adsorbed by magnetic force so as to enrich the shiga toxin-producing escherichia coli of the O103 antigen serotype from the sample to be treated. As described above, the phage targeting protein molecule can specifically bind to the shiga toxin-producing escherichia coli of the O103 antigen serotype, and rapidly and accurately enrich the shiga toxin-producing escherichia coli of the O103 antigen serotype.
According to the embodiment of the invention, the pH value of the co-culture reaction is 5-10, and the temperature is 4-37 ℃. Therefore, the phage targeting protein molecule has higher bioactivity, and can be more rapidly and specifically combined with the shiga toxin-producing escherichia coli of the O103 antigen serotype.
It should be noted that the features and advantages described above for phage-targeted protein molecules and conjugates are equally applicable to this method and are not described here in detail.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the following examples, the main test materials are as follows:
pET-28a-sumo vector: purchased from Invitrogen.
pET-28a-sumo-eGFP vector: the laboratory was kept.
BL21 (DE 3) competent cells: purchased from Invitrogen.
The formula of the eluent of the washing liquid comprises: tris 20mmol/L, naCl mmol/L, imidazole 50-500mmol, glycerol 5%, tween0.05%.
EXAMPLE 1 construction and expression of recombinant proteins
1. According to SEQ ID NO:2 to obtain the target fragment gene ppO103.
2. Construction of recombinant plasmid pET-28a-sumo-eGFP-pO103
(1) The target fragment and pET-28a-sumo-eGFP vector after gel recovery are respectively subjected to restriction enzymeSacI andXhoi, enzyme cutting at 37 ℃ for 1.5 hours;
(2) The products after enzyme digestion are connected at 4 ℃ overnight under the action of T4 ligase, and the connection system conditions are as follows:
(3) After thawing competent cells frozen at-80 ℃ on ice, taking 100 mu L of the cells in a sterile 1.5mL EP tube, adding 10 mu L of a connection product, uniformly mixing, and placing the cells on ice for 30min;
(4) Heat-beating in a water bath at 42 ℃ for 90s, and putting back on ice for 2min;
(5) Adding 400 mu L of LB liquid medium, and carrying out shake culture at 37 ℃ and 200rpm for 35min;
(6) 100 mu L of bacterial liquid is coated on a Kan (50 mg/mL) resistance plate, and the bacterial liquid is cultured at 37 ℃ until single colony appears.
The recombinant plasmid pET-28a-sumo-eGFP-pO103, which had no misidentification of the base sequence, was introduced into BL21 (DE 3) competent cells according to the conventional method.
3. Expression of ppO103 protein
pET-28a-sumo-eGFP-pO103 of recombinant BL21 (DE 3) was added in a 1:100 ratio to 1000mL LB broth, and kana antibiotic was added at a final concentration of 50. Mu.g/mL, and when cultured with shaking at 37℃at 180rpm to OD 600=0.6, IPTG was added at a final concentration of 0.6mM, and cultured with shaking at 16℃at 140rpm for 14h.
And collecting thalli, crushing the thalli by using an ultrasonic crusher, and setting ultrasonic waves for 3 seconds to stop for 4 seconds, wherein the total time is 20 minutes. 12000rpm (4 ℃) and centrifuging for 3min, collecting supernatant, and storing in a refrigerator at 4 ℃.
4. purification and identification of ppO103 protein
(1) The supernatant was combined with a nickel chromatography gel in a shaker at 4℃for two hours, the mixture was passed through the column, and after washing three times (1 mL each time) with a washing solution (20 mmol of imidazole), the eluates were eluted (50 mmol, 100mmol, 150mmol, 200mmol, 250mmol, 300mmol, 350mmol, 400mmol, 450mmol, 500 mmol) with different imidazole concentrations, and SDS-PAGE was performed separately from the supernatant, the flow through, the eluate.
(2) The expression amount and the size of the protein are determined by configuring the gel.
(3) Proteins were purified using His-tag nickel columns.
(4) Imidazole was dialyzed against PBS (ph=9.0) and the protein of interest was concentrated using a 50-kDa protein concentration column.
5. Western-Blot detection of recombinant proteins
(1) SDS-PAGE gels were prepared as described above.
(2) And (3) transferring the film by using a film transfer instrument, and setting parameter current to 400mA for 20min.
(3) Washing with PBST for 3-5 times each for 5min, adding 5% skimmed milk after washing for 2h, washing with PBST for 3-5 times each for 5min after blocking, adding anti-eGFP antibody, and incubating overnight at 4deg.C.
(4) The next day, PBST is washed 3-5 times for 5min each time, then goat anti-rabbit secondary antibody is added, and the mixture is incubated for 2h at room temperature. The ECL chemiluminescent solution was added immediately and observed after washing 3-5 times with PBST for 5min each.
The results showed that the ppO103 protein was 76.3kDa in size after purification (FIG. 1). The detection result of the ppO protein purity is 413 mug/mL respectively. Western-Blot detection ppO protein 103 size was consistent with expected results.
Example 2 pH stability of the ppo103 protein
（1）180μL 10 5 CFU/mL STEC O103, 20. Mu.L of the coating solution was added, and incubated overnight in a 4℃refrigerator. The control group was sterile PBS (ph=9.0) buffer.
(2) The next day, the wells were discarded and the water was drained, 200 μl of PBST buffer was added to each well, the liquid was decanted, and repeated 3-5 times. 200. Mu.L of 5% skim milk was added to each well and blocked at 37℃for 2h.
(3) The wells were discarded, 200. Mu.L of PBST buffer was added to each well, the liquid was decanted, and the procedure was repeated 3-5 times.
(4) mu.L of ppO103 protein (520. Mu.g) was taken, dissolved in 900. Mu.L of buffer (pH=2, 3,4,5,6,7,8,9,10, 11) and incubated for 1h at 37℃in an incubator. mu.L of ppO103 protein was added to each well and incubated for 2h at 37 ℃.
(5) The wells were discarded, and 200. Mu.L of PBST buffer was added to each well to pour out the liquid, and the procedure was repeated 3-5 times. mu.L of GFP antibody diluent (1:5000) was added to each well and incubated for 1h at 37 ℃.
(6) One anti-dilution was discarded, 200. Mu.L of PBST buffer was added to each well and the solution was poured off and repeated 3-5 times. mu.L of goat anti-rabbit secondary antibody diluent (1:7000) was added to each well and incubated for 1h at 37℃in an incubator.
(7) The secondary antibody dilutions were discarded, 200 μl of PBST buffer was added to each well and the solution was decanted and repeated 3-5 times. mu.L of a color development solution was added to each well, and incubated in a 37℃incubator for 30min in the absence of light.
(8) The reaction was stopped by adding 50. Mu.L of 2moL/L concentrated sulfuric acid to each well, and OD450 was measured.
The results showed that recombinant protein ppO103 remained highly active at pH5-10 (fig. 2).
Example 3 temperature stability of the ppo103 protein
The sensitivity test of recombinant protein ppO103 to different temperatures was consistent with the pH test method described above. Recombinant protein ppO103 was incubated at 4 ℃, 25 ℃,37 ℃, 55 ℃, 65 ℃, 75 ℃ and 85 ℃ respectively for 1h. The OD450 was finally determined.
The results showed that protein ppO103 was relatively stable in activity between 4-37 ℃, decreasing protein activity by more than 30% at 42 ℃ and little protein activity at 75-85 ℃ (figure 3).
Example 4 detection of the lowest concentration of bacteria by the ppo103 protein
（1）180μL（0.6×10 8 —0.6×10 1 CFU), i.e. 3.3x10 8 —3.3×10 0 CFU/mL was added with 20. Mu.L of coating solution, and incubated overnight in a 4℃refrigerator. The control group was sterile PBS (ph=9.0) buffer.
(2) The next day, the wells were discarded and the water was drained, 200 μl of PBST buffer was added to each well, the liquid was decanted, and repeated 3-5 times. 200. Mu.L of 5% skim milk was added to each well and blocked at 37℃for 2h.
(3) The wells were discarded, 200. Mu.L of PBST buffer was added to each well, the liquid was decanted, and the procedure was repeated 3-5 times.
(4) ppO103 protein 103 (52. Mu.g) was added to each well and incubated for 2h at 37 ℃.
(5) The wells were discarded, and 200. Mu.L of PBST buffer was added to each well to pour out the liquid, and the procedure was repeated 3-5 times. mu.L of GFP antibody diluent (1:5000) was added to each well and incubated for 1h at 37 ℃.
(6) One anti-dilution was discarded, 200. Mu.L of PBST buffer was added to each well and the solution was poured off and repeated 3-5 times. mu.L of goat anti-rabbit secondary antibody diluent (1:7000) was added to each well and incubated for 1h at 37℃in an incubator.
(7) The secondary antibody dilutions were discarded, 200 μl of PBST buffer was added to each well and the solution was decanted and repeated 3-5 times. mu.L of a color development solution was added to each well, and incubated in a 37℃incubator for 30min in the absence of light.
(8) The reaction was stopped by adding 50. Mu.L of 2moL/L concentrated sulfuric acid to each well, and OD450 was measured. prism 8.0 software compares FL differences between groupsP＜0.001）。
The results showed that recombinant protein ppO103 showed significant differences from the PBS control group when the detected bacterial concentration was 33 CFU/mL (fig. 4).
Example 5 lowest detection concentration of ppo103 protein
In accordance with the method of example 4 above (180. Mu.L of 10 was added to each well) 5 CFU/mL STEC O103), except that 100. Mu.L ppO protein 103 (413-0.4. Mu.g/mL) was added at various concentrations and incubated for 2h at 37℃in an incubator. The OD450 was finally determined. prism 8.0 software compares FL differences between groupsP＜0.001）。
The results showed that ppO protein at a concentration of 0.8 μg/mL still detected STEC O103 bacteria, with a significant difference from the PBS control group (fig. 5).
Example 6 specificity of the ppo103 protein
(1) 180. Mu.L of 10 was added to each well 5 CFU/mL of O1, O2, O3, O4, O5, O6, O15, O16, O17, O18, O26, O27, O29, O33, O36, O45, O58, O64, O83, O87, O91, O99, STEC O103, O105, O111, O116, O123, O128, O131, O145, O157, O173, O180, E.coli ATCC25922, salmonella ATCC14028, shigella flexneri ATCC12022, pseudomonas aeruginosa ATCC27853, klebsiella pneumoniae ATCC700603, acinetobacter baumannii ATCC BAA-1605, enterococcus faecium ATCC19434, clostridium perfringens ATCC13124, staphylococcus aureus ATTCC27217, enterococcus faecalis ATCC 29212 were added with 20. Mu.L of coating solution per well, and incubated overnight in a refrigerator at 4 ℃. The control group was sterile PBS (ph=9.0) buffer.
(2) The next day, the wells were discarded and the water was drained, 200 μl of PBST buffer was added to each well, the liquid was decanted, and repeated 3-5 times. 200. Mu.L of 5% skim milk was added to each well and blocked at 37℃for 2h.
(3) The wells were discarded, 200. Mu.L of PBST buffer was added to each well, the liquid was decanted, and the procedure was repeated 3-5 times.
(4) mu.L of ppO103 protein (52. Mu.g) was added to each well and incubated for 2h at 37 ℃.
(5) The wells were discarded, and 200. Mu.L of PBST buffer was added to each well to pour out the liquid, and the procedure was repeated 3-5 times. mu.L of GFP antibody diluent (1:5000) was added to each well and incubated for 1h at 37 ℃.
(6) One anti-dilution was discarded, 200. Mu.L of PBST buffer was added to each well and the solution was poured off and repeated 3-5 times. mu.L of goat anti-rabbit secondary antibody diluent (1:7000) was added to each well and incubated for 1h at 37℃in an incubator.
(7) The secondary antibody dilutions were discarded, 200 μl of PBST buffer was added to each well and the solution was decanted and repeated 3-5 times. mu.L of a color development solution was added to each well, and incubated in a 37℃incubator for 30min in the absence of light.
(8) The reaction was stopped by adding 50. Mu.L of 2moL/L concentrated sulfuric acid to each well, and OD450 was measured.
As a result, it was revealed that recombinant protein ppO acted only on STEC O103, and did not act on E.coli (O1, O2, O3, O4, O5, O6, O15, O16, O17, O18, O26, O27, O29, O33, O36, O45, O58, O64, O83, O87, O99, O105, O116, O123, O128, O131, O145, O157, O173, O180) and 10 other bacteria (E.coli ATCC25922, salmonella ATCC14028, shigella flexneri ATCC12022, pseudomonas aeruginosa ATCC27853, klebsiella pneumoniae ATCC700603, acinetobacter baumannii ATCC BAA-1605, enterococcus faecium ATCC19434, clostridium perfringens ATCC13124, staphylococcus aureus ATTCC27217, enterococcus faecalis ATCC 29212) of the other 30 strains of different O-antigens (FIG. 6).
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (12)
1. A phage targeting protein molecule, wherein the amino acid sequence of the phage targeting protein molecule is as set forth in SEQ ID NO: 1.
2. A conjugate, the conjugate comprising:
the phage targeting protein molecule of claim 1;
and a linker molecule linked to the phage targeting protein molecule for labeling the phage targeting protein molecule.
3. A nucleic acid molecule encoding the phage-targeted protein molecule of claim 1.
4. An expression vector comprising the nucleic acid molecule of claim 3.
5. A kit comprising at least one of the following: the phage targeting protein molecule of claim 1, the conjugate of claim 2, the nucleic acid molecule of claim 3, and the expression vector of claim 4.
6. Use of at least one of the phage targeting protein molecule of claim 1, the conjugate of claim 2, the nucleic acid molecule of claim 3, the expression vector of claim 4 and the kit of claim 5 for identifying and/or enriching for non-diagnostic purposes shiga toxin-producing escherichia coli of the O103 antigen serotype.
7. A method for non-diagnostic identification of shiga toxin-producing escherichia coli of the O103 antigen serotype, comprising:
co-culturing the phage targeting protein molecule of claim 1 with a microorganism to be tested;
determining whether the microorganism to be tested is shiga toxin-producing escherichia coli of an O103 antigen serotype based on whether the phage targeting protein molecule binds to the microorganism to be tested.
8. The method for non-diagnostic identification of shiga toxin-producing escherichia coli of O103 antigen serotype as defined in claim 7, wherein the phage targeting protein molecule binds to the microorganism to be tested and determines that the microorganism to be tested is shiga toxin-producing escherichia coli of O103 antigen serotype;
the phage targeting protein molecule is not combined with the microorganism to be detected, and the microorganism to be detected is determined not to be shiga toxin-producing escherichia coli of O103 antigen serotype;
the phage targeting protein molecule is co-cultured with a test microorganism in the form of the conjugate of claim 2 by detecting the linker molecule to determine whether the phage targeting protein molecule binds to the test microorganism.
9. Use of a reagent selected from at least one of the phage targeting protein molecule of claim 1, the conjugate of claim 2, the nucleic acid molecule of claim 3 and the expression vector of claim 4 for the preparation of a kit for the diagnosis of a disease and/or condition caused by shiga toxin-producing escherichia coli infected with the O103 antigen serotype.
10. The use according to claim 9, wherein the disease and/or condition is selected from at least one of the following: diarrhea, colitis and uremia.
11. A method of enriching a shiga toxin-producing escherichia coli for an O103 antigen serotype comprising:
co-incubating a shiga toxin-producing escherichia coli sample containing an O103 antigen serotype with the conjugate of claim 2, wherein the linking molecule is selected from the group consisting of magnetic bead probes;
the magnetic bead probe is adsorbed by magnetic force so as to enrich the shiga toxin-producing escherichia coli of the O103 antigen serotype from the sample to be treated.
12. The method for identifying shiga toxin-producing escherichia coli of O103 antigen serotype for non-diagnostic purposes according to claim 7 or 8 or the method for enriching shiga toxin-producing escherichia coli of O103 antigen serotype according to claim 11, wherein the co-culture has a reaction pH of 5-10 and a temperature of 4-37 ℃.
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