Phage targeting protein molecules and their application in identification of Salmonella

Abstract

The present invention provides a bacteriophage targeting protein molecule and its application in identifying Salmonella, wherein the amino acid sequence of the bacteriophage targeting protein molecule is shown in SEQ ID NO: 1. The bacteriophage targeting protein molecule of the present invention can specifically identify Salmonella, can be used for Salmonella identification, has the advantages of being simple, fast and accurate, and has high application value.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to the application of bacteriophage targeting protein molecules in identifying Salmonella. Background Art

[0002] Salmonella is a foodborne pathogen that poses a major challenge to poultry farming, human health and food safety. According to statistics, there are about 93.8 million cases of gastroenteritis caused by Salmonella infection each year worldwide, and about 155,000 deaths. Among the many serotypes, Salmonella Enteritidis and Salmonella Typhimurium are recognized as the two serotypes most closely related to human diseases worldwide due to their strong host adaptability.

[0003] Currently, the detection of Salmonella mainly adopts methods such as PCR and whole genome sequencing, but these methods have disadvantages such as long detection cycle and high cost. Therefore, it is urgent to find a method for rapid identification of Salmonella with short detection cycle and low cost. Summary of the invention

[0004] The present invention aims to solve the technical problems existing in the prior art at least to a certain extent. To this end, the present invention proposes a phage targeting protein molecule, which can specifically identify Salmonella, can be used for Salmonella identification and enrichment, has the advantages of being simple, fast and accurate, and has high application value.

[0005] In one aspect of the present invention, the present invention provides a phage targeting protein molecule. According to an embodiment of the present invention, the amino acid sequence of the phage targeting protein molecule is shown in SEQ ID NO:1.

[0006] In another aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises: the aforementioned bacteriophage targeting protein molecule; and a marker, wherein the marker is connected to the bacteriophage targeting protein molecule and is used to label the bacteriophage targeting protein molecule.

[0007] In another aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the aforementioned bacteriophage targeting protein molecule.

[0008] In another aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the aforementioned nucleic acid molecule.

[0009] In another aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises at least one of the following: the aforementioned bacteriophage targeting protein molecule, the conjugate, the nucleic acid molecule and the expression vector.

[0010] In another aspect of the present invention, the present invention proposes the use of at least one of the aforementioned phage targeting protein molecules, the conjugates, the nucleic acid molecules, the expression vectors and the kits in identifying and / or enriching Salmonella for non-diagnostic purposes.

[0011] In another aspect of the present invention, a method for identifying Salmonella for non-diagnostic purposes is provided. According to an embodiment of the present invention, the method comprises: co-culturing the aforementioned bacteriophage targeting protein molecule with a microorganism to be detected; and determining whether the microorganism to be detected is Salmonella based on whether the bacteriophage targeting protein molecule binds to the microorganism to be detected.

[0012] In another aspect of the present invention, the present invention provides the use of a reagent in preparing a kit, wherein the reagent is selected from at least one of the aforementioned phage targeting protein molecule, the conjugate, the nucleic acid molecule and the expression vector. According to an embodiment of the present invention, the kit is used to diagnose diseases and / or symptoms caused by infection with Salmonella.

[0013] In another aspect of the present invention, a method for enriching Salmonella is provided. According to an embodiment of the present invention, the method comprises: co-culturing a sample to be processed containing Salmonella with the above-mentioned conjugate, wherein the marker in the conjugate comprises a magnetic bead probe; and adsorbing the magnetic bead probe by magnetic force, so as to enrich Salmonella from the sample to be processed.

[0014] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0016] Figure 1 Electropherograms are shown;

[0017] Figure 2 Different bacterial concentration-fluorescence intensity curves are shown;

[0018] Figure 3 The relative fluorescence intensity values ​​under different pH conditions are shown;

[0019] Figure 4 The relative fluorescence intensity values ​​under different temperature conditions are shown. DETAILED DESCRIPTION

[0020] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0021] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0022] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0023] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0024] Phage targeting protein molecules

[0025] In one aspect of the present invention, the present invention proposes a phage targeting protein molecule. According to an embodiment of the present invention, the phage targeting protein molecule has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% homology thereto. Thus, the phage targeting protein molecule of the present invention can quickly identify 24 strains of Enteritidis Salmonella and 22 strains of Salmonella typhimurium, but has no effect on Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecium, Clostridium perfringens, Staphylococcus aureus and Enterococcus faecalis. Thus, the use of the phage targeting protein molecule to specifically identify Salmonella is helpful for the study of pathogenic Salmonella, disease diagnosis and treatment, and is of great significance for the prevention and control of the spread of pathogenic Salmonella. In this article, the phage targeting protein molecule is named "phage targeting protein molecule proM".

[0026] (SEQ ID NO: 1)

[0027] Conjugates, nucleic acid molecules, expression vectors and kits

[0028] In another aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises: the aforementioned phage targeting protein molecule; a marker, the marker is connected to the phage targeting protein molecule and is used to mark the phage targeting protein molecule. Thus, the conjugate of the present invention can achieve the purpose of rapid and accurate identification and enrichment of Salmonella.

[0029] In some embodiments, the marker comprises at least one of eGFP protein, mCherry protein, FITC, TRITC, NHS-fluorescein, NHS-rhodamine, and a magnetic bead probe.

[0030] In another aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the aforementioned bacteriophage targeting protein molecule. Thus, the bacteriophage targeting protein molecule encoded by the nucleic acid molecule of the present invention can specifically identify Salmonella, thereby achieving rapid and accurate identification and enrichment of Salmonella.

[0031] According to an embodiment of the present invention, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 2 or a nucleotide sequence having at least 80%, 85%, 90%, 95% or 99% homology thereto.

[0032]

[0033] In another aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the aforementioned nucleic acid molecule. Thus, the expression vector of the present invention can express the nucleic acid molecule as a bacteriophage targeting protein molecule, thereby specifically binding to Salmonella, thereby achieving rapid and accurate identification and enrichment of Salmonella.

[0034] In another aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises at least one of the following: the aforementioned phage targeting protein molecule, the conjugate, the nucleic acid molecule and the expression vector. The kit of the present invention can specifically bind to Salmonella, thereby achieving rapid and accurate identification and enrichment of Salmonella.

[0035] It should be noted that the features and advantages described above for the phage targeting protein molecule are also applicable to the conjugate, nucleic acid molecule, expression vector and kit, and will not be repeated here.

[0036] application

[0037] In another aspect of the present invention, the present invention proposes the use of at least one of the aforementioned phage targeting protein molecules, the conjugates, the nucleic acid molecules, the expression vectors and the kits in identifying and / or enriching Salmonella for non-diagnostic purposes.

[0038] In another aspect of the present invention, the present invention provides the use of a reagent in preparing a kit, wherein the reagent is selected from at least one of the aforementioned phage targeting protein molecule, the conjugate, the nucleic acid molecule and the expression vector. According to an embodiment of the present invention, the kit is used to diagnose diseases and / or symptoms caused by infection with Salmonella.

[0039] As mentioned above, phage-targeted protein molecules can specifically bind to Salmonella, quickly and accurately identify and enrich Salmonella, which is helpful for pathogenic Salmonella research, disease diagnosis and treatment, and is of great significance for preventing and controlling the spread of pathogenic Salmonella.

[0040] According to an embodiment of the present invention, the disease and / or symptom is selected from at least one of the following: fever, nausea, vomiting, bacteremia, meningitis or osteomyelitis.

[0041] According to an embodiment of the present application, the Salmonella includes Salmonella Enteritidis or Salmonella Typhimurium.

[0042] It should be noted that the features and advantages described above for the phage targeting protein molecules, conjugates, nucleic acid molecules, expression vectors and kits are also applicable to this application and will not be repeated here.

[0043] method

[0044] In another aspect of the present invention, a method for identifying Salmonella is proposed. According to an embodiment of the present invention, the method comprises: co-culturing the aforementioned bacteriophage targeting protein molecule with the microorganism to be detected; determining whether the microorganism to be detected is Salmonella based on whether the bacteriophage targeting protein molecule binds to the microorganism to be detected. As mentioned above, the bacteriophage targeting protein molecule can specifically bind to Salmonella, thereby realizing rapid and accurate identification of Salmonella, which can be used for diagnosing diseases related to Salmonella, and can also be used for non-diagnostic purposes of Salmonella biology research.

[0045] According to an embodiment of the present invention, the bacteriophage targeting protein molecule binds to the microorganism to be detected, and the microorganism to be detected is determined to be Salmonella;

[0046] If the bacteriophage targeting protein molecule does not bind to the microorganism to be detected, it is determined that the microorganism to be detected is not Salmonella;

[0047] The bacteriophage targeting protein molecule is co-cultured with the microorganism to be detected in the form of the aforementioned conjugate, and the marker is detected to determine whether the bacteriophage targeting protein molecule is bound to the microorganism to be detected;

[0048] The Salmonella includes Salmonella Enteritidis or Salmonella Typhimurium;

[0049] The co-culture reaction has a pH value of 5-10 and a temperature of 4-42°C.

[0050] In another aspect of the present invention, a method for enriching Salmonella is provided. According to an embodiment of the present invention, the method comprises: co-culturing a sample to be processed containing Salmonella with the above-mentioned conjugate, wherein the marker in the conjugate comprises a magnetic bead probe; and adsorbing the magnetic bead probe by magnetic force so as to enrich Salmonella from the sample to be processed. As mentioned above, the phage targeting protein molecule can specifically bind to Salmonella, and quickly and accurately enrich Salmonella.

[0051] According to an embodiment of the present invention, the pH value of the co-culture reaction is 4-10, and the temperature is 4-42° C. Thus, the phage targeting protein molecule has higher biological activity and can bind to Salmonella more quickly and specifically.

[0052] It should be noted that the features and advantages described above for phage targeting protein molecules and conjugates are also applicable to this method and will not be repeated here.

[0053] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0054] In the following examples, the main test materials are as follows:

[0055] pET-28a-SUMO vector: purchased from Invitrogen, containing his tag sequence.

[0056] pET-28a-sumo-eGFP vector: This vector was saved in this laboratory and is a modified pET-28a-sumo vector carrying the eGFP gene.

[0057] BL21(DE3) competent cells: purchased from Invitrogen.

[0058] The formula of washing solution and eluent is: Tris 20mmol / L, NaCl 500mmol / L, imidazole 50-500mmol, glycerol 5%, Tween 0.05%.

[0059] Example 1 Construction and expression of recombinant protein

[0060] 1. Gene Amplification

[0061] The gene proM (shown as SEQ ID NO: 2) was synthesized and extended using the following primers.

[0062] Forward primer: 5'-C GAGCTC attgtgaacgtggcggatgctaag-3' (SEQ ID NO: 3) Sac I

[0063] Reverse primer: 5'-ccg CTCGAG ttatttggtgttctgccaaaattg-3' (SEQ ID NO: 4) Xho I

[0064] The above primer amplification system and amplification conditions are as follows:

[0065] Table 1 Amplification system

[0066]

[0067] PCR reaction program: 98°C pre-denaturation for 2 min; 98°C pre-denaturation for 20 s, 57°C annealing for 30 s, 72°C extension for 3 min, 32 cycles; finally 72°C extension for 10 min, and storage at 4°C.

[0068] 2. Construction of recombinant plasmid pET-28a-sumo-eGFP-proSM

[0069] (1) The target fragment recovered from gel and the pET-28a-sumo-eGFP vector after gel recovery were digested with restriction endonucleases Sac I and Xho I at 37°C for 1.5 h respectively;

[0070] (2) The digested products were ligated with T4 ligase overnight at 4°C. The ligation conditions were as follows:

[0071] Table 2 Connection system

[0072]

[0073] (3) Thaw the competent cells frozen at -80℃ on ice, take 100µL into a sterile 1.5mL EP tube, add 10µL of the ligation product, mix well, and place on ice for 30min.

[0074] (4) Heat shock in a 42°C water bath for 90 seconds, then return to ice for 2 minutes;

[0075] (5) Add 400 µL LB liquid medium and incubate at 37°C at 200 rpm for 35 min.

[0076] (6) Take 100 µL of bacterial solution and spread it on a Kanner (50 mg / mL) resistance plate and culture it at 37°C until a single colony appears.

[0077] The recombinant plasmid pET-28a-sumo-eGFP-proSM with correct base sequence was introduced into BL21 (DE3) competent cells according to conventional methods.

[0078] 3. Construction of recombinant plasmid pET-28a-sumo-proM

[0079] The construction method is the same as step 2, except that the constructed recombinant plasmid does not contain the eGFP gene. The other steps are the same.

[0080] 4. Expression of proSM and proM proteins

[0081] Recombinant BL21 (DE3) pET-28a-sumo-eGFP-proSM or pET-28a-sumo-proM was added to 1000 mL LB broth at a ratio of 1:100, and kanamycin was added at a final concentration of 50 μg / mL. The culture was shaken at 37°C (180 rpm) until OD600 = 0.6, and IPTG was added at a final concentration of 0.6 mM. The culture was shaken at 16°C (140 rpm) for 16 h.

[0082] The cells were collected and broken with an ultrasonic disruptor, with the ultrasonic setting of 3 seconds on and 4 seconds off, for a total of 20 minutes. Centrifuge at 12000 rpm (4°C) for 3 minutes, collect the supernatant, and store in a 4°C refrigerator.

[0083] 5. Purification and identification of proSM and proM proteins

[0084] (1) The supernatant was fully combined with nickel chromatography gel on a shaker at 4°C for two hours, passed through the column, washed three times with washing solution (imidazole concentration 20 mmol) (1 mL each time), and then eluted with eluents of different imidazole concentrations (50 mmol, 100 mmol, 150 mmol, 200 mmol, 250 mmol, 300 mmol, 350 mmol, 400 mmol, 450 mmol, 500 mmol). The supernatant, flow-through, and eluent were taken and run on SDS-PAGE respectively.

[0085] (2) Determine the expression level and size of the protein by configuring the gel.

[0086] (3) Purify the protein using a His-tag nickel column.

[0087] (4) Use PBS (pH = 7.2) to dialyze imidazole and concentrate the target protein using a 10 kDa protein concentration column.

[0088] 6. Western-Blot detection of recombinant protein

[0089] (1) Prepare SDS-PAGE gel according to the above steps.

[0090] (2) Use a transfer apparatus to transfer the membrane, setting the parameters to 400 mA and 20 min.

[0091] (3) Wash with PBST 3-5 times, 5 min each time, and add 5% skim milk to block for 2 h. After blocking, wash with PBST 3-5 times, 5 min each time, then add antibody and incubate at 4°C overnight.

[0092] (4) The next day, wash with PBST 3-5 times, 5 min each time, then add goat anti-rabbit secondary antibody and incubate at room temperature for 2 h. Wash with PBST 3-5 times, 5 min each time, and immediately add ECL chemiluminescent solution for observation.

[0093] The results are as follows Figure 1 As shown, the sizes of proSM (recombinant protein containing proM protein and eGFP) and proM protein after purification were 103.3 kDa and 76.4 kDa respectively. The results of Western-Blot detection were consistent with the expected results.

[0094] Example 2 Drawing of standard curve

[0095] Place 20 μL of magnetic beads (which can bind to his tags) in a 1.5 mL centrifuge tube and wash three times with 1 mL of sterile PBS (pH 7.2), then discard the supernatant. Add 100 μL (60 μg) of recombinant protein proM that is not labeled with a fluorescent tag to each tube. Then, add 1 mL of 2% bovine serum albumin (BSA) solution to each tube to block any remaining binding sites. After incubation for 30 minutes, wash the magnetic beads three times again with sterile PBS (pH 7.2). Subsequently, add 900 μL of different concentrations of bacteria (1×10 2 CFU to 1×10 8 CFU) solution and incubate for 15 min. For the control group, an equal volume of PBS (pH 7.2) was added. Afterwards, the centrifuge tube was placed on a magnetic separation rack, the supernatant was discarded, and the magnetic beads were washed once with sterile PBS (pH 7.2). In this way, the bacteria were enriched.

[0096] Next, add 100 μL (60 μg) of recombinant protein proSM to each tube and incubate for 15 min. Place the centrifuge tube on the magnetic separation stand again, discard the supernatant, and wash the magnetic beads three times with sterile PBS (pH 7.2). Finally, adjust the volume to 1 mL of sterile PBS (pH 7.2) buffer (at this point, the bacterial concentration range is 1 × 10 2 CFU / mL to 1 × 10 8 CFU / mL). The fluorescence intensity was measured, and the experiment was repeated three times to ensure the accuracy of the results.

[0097] The results are as follows Figure 2 As shown, the regression equation is y = a + bx + cx² (where y represents the fluorescence intensity value, x represents LogCFU / mL; a, b, c are fixed constants; R² = 0.997). The detection threshold was determined to be 67 ± 2.65, and the minimum detection limit was 10CFU / mL.

[0098] Example 3 pH stability of proSM protein

[0099] 1. Place 20 μL of magnetic beads in a 1.5 mL centrifuge tube and wash three times with 1 mL of sterile PBS (pH 7.2), then discard the supernatant. Add 100 μL (60 μg) of unlabeled fluorescent recombinant protein proM to each tube. Then, add 1 mL of 2% bovine serum albumin (BSA) solution to each tube to block any remaining binding sites. After incubation for 30 minutes, wash the magnetic beads three times again with sterile PBS (pH 7.2). Subsequently, add 900 μL of bacteria (1×10 5 CFU) and incubate for 15 min. An equal volume of PBS (pH 7.2) was added to the control group. Afterwards, the centrifuge tube was placed on a magnetic separation rack, the supernatant was discarded, and the magnetic beads were washed once with sterile PBS (pH 7.2).

[0100] 2. 100 μL of proSM protein was mixed with 900 μL of three different buffers, namely 0.2 M sodium acetate buffer (pH 2.0 to 6.0), 0.2 M sodium phosphate buffer (pH 7.0 to 8.0), and 0.2 M Tris-HCl buffer (pH 9.0 to 11.0), and incubated at 37 °C for 1 h.

[0101] 3. Add the magnetic beads obtained in step 1 to the recombinant protein proSM in step 2 and incubate for 15 minutes. Place the centrifuge tube on the magnetic separation rack again, discard the supernatant, and wash the magnetic beads three times with sterile PBS (pH 7.2). Then measure the fluorescence intensity and repeat the experiment three times. Calculate the relative fluorescence intensity (RFI) using the following formula: RFI = F X / F pH7.2 × 100%, where F X represents the fluorescence intensity of the different pH treatment groups, and F pH7.2 Represents the fluorescence intensity of the control group.

[0102] The results are as follows Figure 3 As shown, the activity of proSM protein is relatively stable between pH = 4-10, and at pH = 2 or 11, the protein has almost no activity.

[0103] Example 4 Temperature stability of proSM protein

[0104] 1. Place 20 μL of magnetic beads in a 1.5 mL centrifuge tube and wash three times with 1 mL of sterile PBS (pH 7.2), then discard the supernatant. Add 100 μL (60 μg) of unlabeled fluorescent recombinant protein proM to each tube. Then, add 1 mL of 2% bovine serum albumin (BSA) solution to each tube to block any remaining binding sites. After incubation for 30 minutes, wash the magnetic beads three times again with sterile PBS (pH 7.2). Subsequently, add 900 μL of bacteria (1×10 5 CFU) and incubate for 15 min. An equal volume of PBS (pH 7.2) was added to the control group. Afterwards, the centrifuge tube was placed on a magnetic separation rack, the supernatant was discarded, and the magnetic beads were washed once with sterile PBS (pH 7.2).

[0105] 2. Pretreat 100 μL of proSM protein at different temperatures (4°C, 25°C, 37°C, 42°C, 55°C, 65°C, 75°C and 80°C) for 1 h.

[0106] 3. Add the magnetic beads obtained in step 1 to the recombinant protein proSM in step 2 and incubate for 15 minutes. Place the centrifuge tube on the magnetic separation rack again, discard the supernatant, and wash the magnetic beads three times with sterile PBS (pH 7.2). Then measure the fluorescence intensity and repeat the experiment three times. Calculate the relative fluorescence intensity (RFI) according to the formula: RFI = F X / F C × 100%, where F X Represents the fluorescence intensity of the groups treated with different temperatures, F C Represents the fluorescence intensity of the control group.

[0107] The results are as follows Figure 4 As shown, the activity of protein proSM was relatively stable between 4-42°C, at 55-65°C, the activity of the protein decreased by more than 50%, and at 80°C, the protein had almost no activity.

[0108] Example 5

[0109] 1. Experimental strains

[0110] 24 strains of Salmonella Enteritidis, 22 strains of Salmonella Typhimurium and 9 strains of other bacteria, including Escherichia coli ATCC25922, Shigella flexneri ATCC 12022, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603, Acinetobacter baumannii ATCC BAA-1605, Enterococcus faecium ATCC 19434, Clostridium perfringens ATCC 13124, Staphylococcus aureus ATCC 27217 and Streptococcus faecalis ATCC 29212.

[0111] 2. Place 20 μL of magnetic beads in a 1.5 mL centrifuge tube and wash three times with 1 mL of sterile PBS (pH 7.2), then discard the supernatant. Add 100 μL (60 μg) of unlabeled fluorescent recombinant protein proM to each tube. Then, add 1 mL of 2% bovine serum albumin (BSA) solution to each tube to block any remaining binding sites. After incubation for 30 minutes, wash the magnetic beads three times again with sterile PBS (pH 7.2). Subsequently, add 900 μL of each bacteria (1×10 5 CFU) and incubated for 15 minutes. An equal volume of PBS (pH 7.2) was added to the control group. Afterwards, the centrifuge tube was placed on a magnetic separation stand, the supernatant was discarded, and the magnetic beads were washed once with sterile PBS (pH 7.2). The recombinant protein proSM was then added and incubated at 25°C in 0.2M sodium phosphate buffer with a pH of 7.2 for 15 minutes. The centrifuge tube was placed on a magnetic separation stand again, the supernatant was discarded, and the magnetic beads were washed three times with sterile PBS (pH 7.2). The fluorescence intensity was then measured and the experiment was repeated three times. The relative fluorescence intensity (RFI) was calculated using the following formula: RFI = ((F X -F PBS ) / (F SM07 -F PBS )) × 100%, where F X Represents the fluorescence intensity of the test sample, F PBS represents the fluorescence intensity of the PBS control group, F SM07 Represents the fluorescence intensity of Salmonella Enteritidis SM07.

[0112] The results are shown in Table 3. The recombinant protein proSM was effective against 24 strains of Enteritidis Salmonella and 22 strains of Salmonella Typhimurium, but had no effect on 9 other bacteria, including Escherichia coli ATCC 25922, Shigella flexneri ATCC 12022, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603, Acinetobacter baumannii ATCC BAA-1605, Enterococcus faecalis ATCC19434, Clostridium perfringens ATCC 13124, Staphylococcus aureus ATCC 27217 and Enterococcus faecalis ATCC 29212.

[0113] Table 3 Fluorescence detection results of different strains

[0114]

[0115] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A bacteriophage targeting protein molecule, characterized in that: The amino acid sequence of the phage targeting protein molecule is shown in SEQ ID NO:

1.

2. A conjugate, characterized in that The conjugate comprises: The phage targeting protein molecule according to claim 1; A marker is connected to the bacteriophage targeting protein molecule and is used to mark the bacteriophage targeting protein molecule.

3. The conjugate according to claim 2, characterized in that The marker is at least one of eGFP protein, mCherry protein, FITC, TRITC, NHS-fluorescein, NHS-rhodamine and magnetic bead probe.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the phage targeting protein molecule according to claim 1.

5. An expression vector, characterized in that: Comprising the nucleic acid molecule according to claim 4.

6. A kit, characterized in that: It comprises at least one of the following: the phage targeting protein molecule according to claim 1, the conjugate according to claim 2 or 3, the nucleic acid molecule according to claim 4 and the expression vector according to claim 5.

7. Use of at least one of the phage targeting protein molecule of claim 1, the conjugate of claim 2 or 3, the nucleic acid molecule of claim 4, the expression vector of claim 5 and the kit of claim 6 in identifying and / or enriching Salmonella for non-diagnostic purposes.

8. The use according to claim 7, characterized in that: The Salmonella includes Salmonella Enteritidis or Salmonella Typhimurium.

9. A method for identifying Salmonella for non-diagnostic purposes, characterized in that: include: Co-culturing the bacteriophage targeting protein molecule of claim 1 with the microorganism to be detected; Based on whether the bacteriophage targeting protein molecule binds to the microorganism to be detected, it is determined whether the microorganism to be detected is Salmonella.

10. The method for identifying Salmonella for non-diagnostic purposes according to claim 9, characterized in that: The bacteriophage targeting protein molecule binds to the microorganism to be detected, and the microorganism to be detected is determined to be Salmonella; If the bacteriophage targeting protein molecule does not bind to the microorganism to be detected, it is determined that the microorganism to be detected is not Salmonella; The bacteriophage targeting protein molecule is co-cultured with the microorganism to be detected in the form of the conjugate according to claim 2 or 3, and the marker is detected to determine whether the bacteriophage targeting protein molecule is bound to the microorganism to be detected; The Salmonella includes Salmonella Enteritidis or Salmonella Typhimurium; The co-culture reaction has a pH value of 5-10 and a temperature of 4-42°C.

11. Use of a reagent in preparing a kit, wherein the reagent is selected from at least one of the phage targeting protein molecule of claim 1, the conjugate of claim 2 or 3, the nucleic acid molecule of claim 4, and the expression vector of claim 5, characterized in that: The kit is used for diagnosing diseases and / or symptoms caused by infection with Salmonella.

12. The use according to claim 11, characterized in that Such illness and / or symptoms include fever, nausea, vomiting, bacteremia, meningitis, or osteomyelitis; The Salmonella includes Salmonella Enteritidis or Salmonella Typhimurium.

13. A method for enriching Salmonella, characterized in that: include: Co-culturing a sample to be treated containing Salmonella with the conjugate according to claim 2 or 3, wherein the marker in the conjugate is a magnetic bead probe; The magnetic bead probe is adsorbed by magnetic force so as to enrich the Salmonella from the sample to be processed.

14. The method for enriching Salmonella according to claim 13, characterized in that: The Salmonella includes Salmonella Enteritidis or Salmonella Typhimurium; The co-culture reaction has a pH value of 4-10 and a temperature of 4-42°C.

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