Protein molecules and their use in identifying multiple serotypes of avian pathogenic e. coli

Abstract

The application belongs to the field of biology and specifically provides a protein molecule and application thereof in identifying avian pathogenic E. coli of multiple serotypes, wherein the amino acid sequence of the protein molecule is shown as SEQ ID No: 1. The protein molecule of the application can specifically recognize avian pathogenic E. coli of O2, O18 and O78 antigen serotypes, and can be used for identification and enrichment of avian pathogenic E. coli of O2, O18 and O78 antigen serotypes, and has the advantages of simplicity, rapidness and accuracy, and has high application value.

Description

Technical Field

[0001] This application relates to the field of biology, and more specifically, to protein molecules and their application in identifying various serotypes of pathogenic avian Escherichia coli. Background Technology

[0002] Escherichia coli is a Gram-negative bacterium widely found in the intestines of animals. Most are symbiotic, but some pathogenic strains can cause serious diseases. Avian pathogenic Escherichia coli (APEC) is one of the important pathogens in poultry farming. It is mainly transmitted through the respiratory and digestive tracts, causing diseases such as acute septicemia and peritonitis in poultry, resulting in significant economic losses to the poultry industry.

[0003] APEC strains exhibit diverse serotypes, with O2, O18, and O78 being common prevalent serotypes, consistently showing high detection rates in poultry across different regions. Recent studies have revealed a high degree of similarity between APEC and human extraintestinal pathogenic Escherichia coli (ExPEC) in virulence and resistance genes. In particular, strains of serotypes O2, O18, and O78 may possess cross-species transmission potential, posing a potential threat to public health. Therefore, establishing rapid and specific detection methods for these key serotypes is of great significance for poultry disease control and food safety.

[0004] Currently, APEC serotyping mainly relies on techniques such as bacterial isolation and culture, serotyping, PCR, and whole-genome sequencing. These methods have limitations, including being cumbersome, time-consuming, requiring sophisticated equipment, or being costly, making them unsuitable for rapid on-site testing. Therefore, developing a highly specific, simple, and rapid APEC serotyping technology based on protein recognition is particularly urgent. Summary of the Invention

[0005] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application proposes a protein molecule that can specifically recognize avian pathogenic Escherichia coli serotypes O2, O18, and O78, and can be used for the identification and enrichment of avian pathogenic Escherichia coli serotypes O2, O18, and O78. It has the advantages of being simple, rapid, and accurate, and has high application value.

[0006] Therefore, in a first aspect of this application, a protein molecule is provided. According to embodiments of this application, the amino acid sequence of the protein molecule is shown in SEQ ID No: 1. Thus, the protein molecule of this application can specifically bind to avian pathogenic Escherichia coli serotypes O2, O18, and O78, and the protein can competitively inhibit the adsorption of bacteriophages to avian pathogenic Escherichia coli possessing the aforementioned antigenic serotypes. Therefore, it can be used for the specific detection and identification of characteristic serotypes of Escherichia coli, and has the advantages of simple operation and accurate identification.

[0007] In a second aspect of this application, a conjugate is proposed. According to an embodiment of this application, the conjugate comprises: the protein molecule described in the first aspect; and a linker molecule linked to the protein molecule for labeling the protein molecule. Thus, the conjugate of this application enables rapid and accurate identification and enrichment of avian pathogenic Escherichia coli serotypes O2, O18, and O78.

[0008] In a third aspect of this application, a nucleic acid molecule is proposed. According to an embodiment of this application, the nucleic acid molecule encodes the protein molecule described in the first aspect. Therefore, based on the aforementioned nucleic acid molecule, the aforementioned protein molecule can be efficiently expressed in vitro, simplifying the production process; furthermore, the nucleic acid molecule is easily genetically engineered to obtain protein molecules more suitable for practical applications.

[0009] In a fourth aspect, this application provides an expression vector. According to embodiments of this application, the expression vector carries the nucleic acid molecule described in the third aspect, or expresses the protein molecule described in the first aspect. Thus, after the expression vector is introduced into suitable recipient cells, the aforementioned protein molecule can be effectively expressed under the mediation of a regulatory system, thereby achieving the large-scale in vitro preparation of the protein molecule.

[0010] In a fifth aspect of this application, the application proposes the use of at least one of the protein molecule described in the first aspect, the conjugate described in the second aspect, the nucleic acid molecule described in the third aspect, and the expression vector described in the fourth aspect for the identification and / or enrichment of avian pathogenic Escherichia coli for non-diagnostic purposes; wherein the serotype of the avian pathogenic Escherichia coli is at least one of O2, O18, and O78. As mentioned above, the protein molecule of this application can specifically bind to avian pathogenic Escherichia coli of serotypes O2, O18, and O78, and therefore can be used for the identification and enrichment of this strain, with advantages such as simple operation and accurate identification.

[0011] In a sixth aspect of this application, a method for identifying avian pathogenic Escherichia coli for non-diagnostic purposes is proposed. According to an embodiment of this application, the method includes: co-culturing the protein molecule described in the first aspect with a test microorganism; determining whether the test microorganism is an avian pathogenic Escherichia coli based on whether the protein molecule binds to the test microorganism; wherein the serotype of the avian pathogenic Escherichia coli is at least one of O2, O18, and O78. As mentioned above, the protein molecule of this application can specifically bind to avian pathogenic Escherichia coli of serotypes O2, O18, and O78, and therefore can be used for the identification and enrichment of such strains, offering advantages such as ease of operation and accurate identification.

[0012] In a seventh aspect of this application, the use of the protein molecule described in the first aspect, the conjugate described in the second aspect, the nucleic acid molecule described in the third aspect, or the expression vector described in the fourth aspect in the preparation of a kit for identifying avian pathogenic Escherichia coli or diagnosing diseases and / or symptoms caused by infection with avian pathogenic Escherichia coli, wherein the serotype of the avian pathogenic Escherichia coli is at least one of O2, O18, and O78. As described above, the protein molecule of this application can specifically bind to avian pathogenic Escherichia coli antigen serotypes O2, O18, and O78, and therefore can be used to diagnose related diseases and / or symptoms caused by such strains, with advantages such as ease of operation and accurate identification.

[0013] In an eighth aspect of this application, a method for enriching avian pathogenic Escherichia coli is provided. According to an embodiment of this application, the method includes: co-culturing a sample to be treated with the protein molecule described in the first aspect, wherein the protein molecule is attached to a magnetic bead probe; and magnetically adsorbing the magnetic bead probe to enrich avian pathogenic Escherichia coli from the sample to be treated; wherein the serotype of the avian pathogenic Escherichia coli is at least one of O2, O18, and O78. As described above, the protein molecule of this application can specifically bind to avian pathogenic Escherichia coli of serotypes O2, O18, and O78, and therefore can be used for the identification and enrichment of such strains, offering advantages such as ease of operation and accurate identification.

[0014] Additional aspects and advantages of this application 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 this application. Attached Figure Description

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

[0016] Figure 1 An electrophoresis pattern according to an embodiment of this application is shown;

[0017] Figure 2 The image shows a fluorescence pattern of recombinant protein PJNE213-1 adsorbed on the surface of a host bacterium according to an embodiment of this application;

[0018] Figure 3 The image shows a scanning electron microscope image of recombinant protein PJNE213-1 bound to the surface of a host bacterium according to an embodiment of this application;

[0019] Figure 4 The diagram shows the results of a competitive adsorption experiment according to one embodiment of this application;

[0020] Figure 5 A schematic diagram of specificity analysis of recombinant protein PJNE213-1 according to an embodiment of this application is shown. Detailed Implementation

[0021] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0022] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0023] The endpoints and any values ​​of the ranges disclosed herein 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 the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0025] protein molecules

[0026] Therefore, in a first aspect of this application, a protein molecule is proposed. According to embodiments of this application, the amino acid sequence of the protein molecule is as shown in SEQ ID No: 1 or has at least 80%, 85%, 90%, 95%, or 99% homology with it. Thus, the protein molecule of this application can specifically recognize avian pathogenic Escherichia coli with serotypes O2, O18, and O78 antigens, and the protein can competitively inhibit the adsorption of bacteriophages to avian pathogenic Escherichia coli with the aforementioned antigen serotypes. This allows for rapid and accurate identification and enrichment of avian pathogenic Escherichia coli with serotypes O2, O18, and O78 antigens, which is helpful for the research, diagnosis, and treatment of avian pathogenic Escherichia coli and is of great significance for preventing and controlling the spread of avian pathogenic Escherichia coli. In this document, this protein molecule is named "protein molecule PJNE213-1" or "recombinant protein PJNE213-1".

[0027] (SEQ ID No: 1).

[0028] Conjugates, nucleic acid molecules and expression vectors

[0029] In a second aspect of this application, a conjugate is proposed. According to an embodiment of this application, the conjugate comprises: the protein molecule described in the first aspect; and a linker molecule linked to the protein molecule for labeling the protein molecule. Thus, the conjugate of this application enables rapid and accurate identification and enrichment of avian pathogenic Escherichia coli serotypes O2, O18, and O78.

[0030] In some embodiments, the linker molecule is selected from at least one of the following: GFP protein, mCherry protein, FITC, TRITC, NHS-luciferin, and NHS-rhodamine.

[0031] In a third aspect of this application, a nucleic acid molecule is proposed. According to an embodiment of this application, the nucleic acid molecule encodes the protein molecule described in the first aspect. Therefore, based on the aforementioned nucleic acid molecule, the aforementioned protein molecule can be efficiently expressed in vitro, simplifying the production process; furthermore, the nucleic acid molecule is easily genetically engineered to obtain protein molecules more suitable for practical applications.

[0032] In some embodiments, 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 with it.

[0033]

[0034] In some embodiments, the nucleic acid molecule is DNA.

[0035] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned in this specification and claims actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is actually disclosed as well. In addition, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0036] In a fourth aspect, this application provides an expression vector. According to embodiments of this application, the expression vector carries the nucleic acid molecule described in the third aspect, or expresses the protein molecule described in the first aspect. Thus, after the expression vector is introduced into suitable recipient cells, the aforementioned protein molecule can be effectively expressed under the mediation of a regulatory system, thereby achieving the large-scale in vitro preparation of the protein molecule.

[0037] It should be noted that when linking the aforementioned nucleic acid molecules to the vector, the nucleic acid molecules can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. These control elements can originate directly from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the connection between the nucleic acid molecules and the control elements must be operably established.

[0038] In some embodiments, the vector may refer to a cloning vector or an expression vector, which can be obtained by operatively linking the nucleic acid to a commercially available vector (such as a plasmid or viral vector).

[0039] In this document, the term "operably ligated" refers to ligating a foreign gene to a vector such that the control elements within the vector, such as amino acid sequences controlling transcription and amino acid sequences controlling translation, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include viral vectors, plasmids, bacteriophages, etc. After the expression vectors according to some specific embodiments of this application are introduced into suitable recipient cells, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of the proteins encoded by the nucleic acid molecules.

[0040] Applications and methods

[0041] In a fifth aspect of this application, the application proposes the use of at least one of the protein molecule described in the first aspect, the conjugate described in the second aspect, the nucleic acid molecule described in the third aspect, and the expression vector described in the fourth aspect for the identification and / or enrichment of avian pathogenic Escherichia coli for non-diagnostic purposes; wherein the serotype of the avian pathogenic Escherichia coli is at least one of O2, O18, and O78. As mentioned above, the protein molecule of this application can specifically bind to avian pathogenic Escherichia coli of serotypes O2, O18, and O78, and therefore can be used for the identification and enrichment of this strain, with advantages such as simple operation and accurate identification.

[0042] In a sixth aspect of this application, a method for identifying avian pathogenic Escherichia coli for non-diagnostic purposes is proposed. According to an embodiment of this application, the method includes: co-culturing the protein molecule described in the first aspect with a test microorganism; determining whether the test microorganism is avian pathogenic Escherichia coli based on whether the protein molecule binds to the test microorganism; wherein the serotype of the avian pathogenic Escherichia coli is at least one of O2, O18, and O78. As described above, the protein molecule of this application can specifically bind to avian pathogenic Escherichia coli of serotypes O2, O18, and O78, thereby achieving rapid and accurate identification of avian pathogenic Escherichia coli of serotypes O2, O18, and O78. This method can be used for diagnosing diseases related to avian pathogenic Escherichia coli of serotypes O2, O18, and O78, and can also be used for biological research on avian pathogenic Escherichia coli of serotypes O2, O18, and O78 for non-diagnostic purposes.

[0043] In some embodiments, when the protein molecule binds to the microorganism to be tested, the microorganism to be tested is identified as avian pathogenic Escherichia coli.

[0044] In some embodiments, if the protein molecule does not bind to the microorganism to be tested, then the microorganism to be tested is determined not to be avian pathogenic Escherichia coli.

[0045] In some embodiments, a marker molecule is attached to the protein molecule, and the marker molecule is detected to determine whether the protein molecule binds to the microorganism to be tested.

[0046] In a seventh aspect of this application, the use of the protein molecule described in the first aspect, the conjugate described in the second aspect, the nucleic acid molecule described in the third aspect, or the expression vector described in the fourth aspect in the preparation of a kit for identifying avian pathogenic Escherichia coli or diagnosing diseases and / or symptoms caused by infection with avian pathogenic Escherichia coli, wherein the serotype of the avian pathogenic Escherichia coli is at least one of O2, O18, and O78. As described above, the protein molecule of this application can specifically bind to avian pathogenic Escherichia coli antigen serotypes O2, O18, and O78, and therefore can be used to identify avian pathogenic Escherichia coli or diagnose related diseases and / or symptoms caused by such strains, with advantages such as simple operation and accurate identification.

[0047] In some embodiments, the disease and / or symptoms are selected from at least one of the following: acute sepsis, pericarditis, perihepatitis, and peritonitis.

[0048] In an eighth aspect of this application, a method for enriching avian pathogenic Escherichia coli is provided. According to an embodiment of this application, the method includes: co-culturing a sample to be treated with the protein molecule described in the first aspect, wherein the protein molecule is attached to a magnetic bead probe; and magnetically adsorbing the magnetic bead probe to enrich avian pathogenic Escherichia coli from the sample to be treated; wherein the serotype of the avian pathogenic Escherichia coli is at least one of O2, O18, and O78. As described above, the protein molecule of this application can specifically bind to avian pathogenic Escherichia coli of serotypes O2, O18, and O78, and therefore can be used for the identification and enrichment of such strains, offering advantages such as ease of operation and accurate identification.

[0049] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0050] Main test materials:

[0051] pET-28a-sumo-eGFP vector: Store in our laboratory.

[0052] pET-28a-sumo vector: purchased from Invitrogen.

[0053] BL21(DE3) competent cells: purchased from Invitrogen.

[0054] Elution buffer formulation: Tris 20 mmol / L, NaCl 500 mmol / L, imidazole 50-500 mmol, glycerol 5%, Tween 0.05%.

[0055] Example 1 Construction and expression of recombinant proteins

[0056] 1. Gene amplification

[0057] The gene PSE31 (as shown in SEQ ID No: 1) was synthesized and amplified using the following primers.

[0058] Forward primer: 5'-GACGAGCTGTACAAGGAGCTCATGAAACAAGACTTAAAA-3' (SEQ ID NO: 3).

[0059] Reverse primer: 5'-CTCGAGTGCGGCCGCAAGCTTTTATGCAATCCTCATCCA-3' (SEQ ID NO: 4).

[0060] The primer amplification system and amplification conditions are as follows:

[0061] Table 1 Primer amplification system

[0062]

[0063] Table 2 PCR reaction procedure

[0064]

[0065] 2. Construct the recombinant plasmid pET-28a-sumo-eGFP-PJNE213-1

[0066] (1) The target fragment PJNE213-1 and the pET-28a-sumo-eGFP vector recovered from the gel were incubated with restriction endonucleases Sac I and Hind III in a water bath at 37°C for 3 h. The enzyme digestion system is shown in Table 3.

[0067] Table 3 Enzyme digestion system

[0068]

[0069] (2) The digested vector pET-28a-sumo-eGFP and the target fragment PJNE213-1 were prepared according to the instructions of the ClonExpressUltraOneStepCloningKit V2. The reaction system is shown in Table 4.

[0070] Table 4 Reaction System

[0071]

[0072] (3) Remove the competent DH5α cells from the cryogenic storage box and thaw them on ice. Take 10 μL of the recombinant product and mix it gently with 100 μL of competent cells. React on ice for 30 min, then heat shock in a water bath at 42℃ for 90 s, and then place on ice for 5 min. Add 900 μL of LB liquid medium, and incubate on a constant temperature shaker for 40 min. Centrifuge at 5000 rpm at room temperature for 5 min, discard 900 μL of supernatant, resuspend the remaining 100 μL and spread it on an LB solid culture dish (containing kanamycin), and incubate overnight in a constant temperature incubator at 37℃.

[0073] (4) The recombinant plasmid pET-28a-sumo-eGFP-PJNE213-1 with the base sequence confirmed was introduced into BL21(DE3) competent cells using conventional methods.

[0074] 3. Expression of PJNE213-1 protein

[0075] (1) Select a single colony of the plasmid in BL21 that has been heat-shocked and culture it in 5 mL of LB liquid medium (containing 50 mg / mL kanamycin). Then, transfer the bacterial culture to 600 mL of LB liquid medium (containing 50 mg / mL kanamycin) at a ratio of 1:100 and culture it in a constant temperature shaker at 37°C and 160 rpm until OD600 = 0.4-0.6. At this time, add 600 μL of 0.6 M IPTG solution (final concentration 0.6 mM) to the culture system and immediately transfer the culture flask to a low temperature constant temperature shaker at 16°C and culture it continuously at 130 rpm for 12-16 h to complete the protein induction expression.

[0076] (2) Transfer the bacterial culture to a pre-cooled 50 mL centrifuge tube and centrifuge at 4°C and 10,000 rpm for 3 min. Discard the supernatant and collect the bacterial pellet. Gently resuspend the bacterial pellet in 40 mL of pre-cooled sterile PBS buffer (pH 7.4) and wash three times under the same centrifugation conditions to remove residual culture medium.

[0077] (3) The bacterial pellet was resuspended in 15 mL of PBS buffer and fixed on ice for sonication. A 6 mm titanium alloy probe was used, with a power of 150 W and a pulse period of 3 s working and 4 s intermittent, for 30 min of continuous sonication. After sonication, the pellet was immediately placed on ice for later use. After sonication, the pellet was centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was collected.

[0078] 4. Purification of PJNE213-1 protein

[0079] The supernatant was filtered through a 0.22 μm filter membrane and added to an equilibrated affinity chromatography column. The mixture was slowly stirred to ensure adequate contact between the sample and the packing material. The flow buffer was collected, and gradient elution was performed sequentially using buffers containing 50 mM and 100 mM imidazole to gradually remove non-specifically bound proteins. Finally, the target protein was collected using elution buffers containing 150-300 mM imidazole, and the elution effect of different imidazole concentrations was verified by SDS-PAGE electrophoresis.

[0080] The results are as follows Figure 1 As shown, the purified PJNE213-1 protein has a size of 103.4 kDa.

[0081] Example 2: Fluorescence Microscopy Observation

[0082] Take 200 μL of recombinant protein PJNE213-1 (0.4 μg / μL) and 200 μL of freshly cultured Escherichia coli (1×10⁻⁶). 8 Mix the samples in equal proportions (CFU / mL) and incubate at 37°C in a constant-temperature shaker for 30 min. Centrifuge at 5000 rpm for 5 min at room temperature, discard the supernatant, and observe under a fluorescence microscope.

[0083] The results are as follows Figure 2 As shown, recombinant PJNE213-1 can adsorb onto the surface of the host bacteria.

[0084] Example 3 Scanning Electron Microscopy

[0085] Recombinant protein PJNE213-1 (0.4 μg / μL) was mixed with host bacterial culture (1×10⁻⁶). 6 Equal volumes of PFU / mL were mixed, transported at low temperature to the testing institution, and the adsorption of the protein on the host bacteria was observed using scanning electron microscopy.

[0086] The results are as follows Figure 3 As shown, PJNE213-1 can specifically bind to the surface of the host bacteria.

[0087] Example 4 Competitive Adsorption Experiment

[0088] Experimental group: Freshly cultured E. coli solution (1×10⁻⁶) 7 CFU / mL), bacteriophages (1×10⁻⁶) 6 Equal volumes of PFU / mL and recombinant protein PJNE213-1 (0.4 μg / μL) were mixed and centrifuged at 4000 rpm for 10 min at 4℃. The supernatant was collected and the phage titer was determined using the double-layer agar method.

[0089] Control group: The difference from the experimental group is that the recombinant protein PJNE213-1 is not added, and the bacterial culture and bacteriophage are mixed in equal volumes.

[0090] The results are as follows Figure 4 As shown, the residual rate of phage in the supernatant of recombinant protein PJNE213-1 was significantly different from that in the control group (P<0.01), indicating that recombinant protein PJNE213-1 can inhibit phage adsorption.

[0091] Example 5: ELISA Detection

[0092] Commercially available E. coli bacterial suspensions with different serotypes were treated as follows:

[0093] Centrifuge (12,882 × g, 5 min), discard the supernatant, resuspend the bacterial cells in PBS buffer, and adjust the bacterial concentration to no less than 1 × 10⁻⁶. 8 CFU / mL. Add 2% paraformaldehyde and fix at room temperature for 30 min. After fixation, wash the bacterial cells with PBS buffer.

[0094] The bacterial cells were serially diluted 10-fold, and the recombinant protein PJNE213-1 was serially diluted 2-fold. 200 μL of the bacterial suspension was used to coat the wells of an ELISA plate and incubated at 37°C for 2 h. After incubation, 200 μL of PBST was added to each well, and the solution was discarded after standing for 1 min. This washing process was repeated 5 times. Then, 200 μL of 5% BSA solution was added, and the plate was blocked at 37°C for 1 h to prevent non-specific binding. After washing with PBST again, recombinant protein PJNE213-1 (final concentration 3 μg / mL) was added to each well, and the plate was incubated at 37°C for 1–2 h. After the reaction, the plate was washed three times with PBST, and the fluorescence signal was immediately read using a fluorescent enzyme-linked immunosorbent assay (ELISA) system. The excitation wavelength was 488 nm, and the emission wavelength window was 520–530 nm.

[0095] A negative control (PBS) was included in the experiment, and each group had three replicates to ensure the reproducibility of the results. P > 0.05 was considered statistically insignificant, and P < 0.01 was considered statistically significant.

[0096] The results are as follows Figure 5 As shown, the recombinant protein PJNE213-1 is specific for Escherichia coli serotypes O2, O18, and O78, and its fluorescence signal is significantly higher than that of the control group (P<0.01). It is not specific for the other five different serotypes of Escherichia coli, including O82, O83, O86, O88, and O91, and their fluorescence signals are not different from those of the control group (P>0.05).

[0097] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. The use of at least one of protein molecules, conjugates, nucleic acid molecules and expression vectors in the identification and / or enrichment of pathogenic avian Escherichia coli for non-diagnostic purposes; in, The serotype of the avian pathogenic Escherichia coli is at least one of O2, O18 and O78; The amino acid sequence of the protein molecule is shown in SEQ ID No: 1; The conjugate contains the protein molecule; The nucleic acid molecule encodes the protein molecule; The expression vector carries the nucleic acid molecule or protein molecule; The conjugate includes: The protein molecule; A linker molecule, which is attached to the protein molecule and used to label the protein molecule; The linker molecule is selected from at least one of the following: GFP protein, mCherry protein, FITC, TRITC, NHS-luciferin, and NHS-rhodamine.

2. A method for identifying pathogenic avian Escherichia coli for non-diagnostic purposes, characterized in that, include: The protein molecules are co-cultured with the microorganisms to be tested; Based on whether the protein molecule binds to the microorganism to be tested, it is determined whether the microorganism to be tested is avian pathogenic Escherichia coli; The serotype of the avian pathogenic Escherichia coli is at least one of O2, O18 and O78; The amino acid sequence of the protein molecule is shown in SEQ ID No:

1.

3. The method according to claim 2, characterized in that, If the protein molecule binds to the microorganism to be tested, then the microorganism to be tested is identified as avian pathogenic Escherichia coli. If the protein molecule does not bind to the microorganism to be tested, then the microorganism to be tested is determined not to be avian pathogenic Escherichia coli. The protein molecule is attached to a marker molecule, and the marker molecule is detected to determine whether the protein molecule binds to the microorganism to be tested.

4. The use of protein molecules, conjugates, nucleic acid molecules, or expression vectors in the preparation of a kit for identifying pathogenic avian Escherichia coli, wherein, The serotype of the avian pathogenic Escherichia coli is at least one of O2, O18 and O78; The amino acid sequence of the protein molecule is shown in SEQ ID No: 1; The conjugate contains the protein molecule; The nucleic acid molecule encodes the protein molecule; The expression vector carries the nucleic acid molecule or protein molecule; The conjugate includes: The protein molecule; A linker molecule, which is attached to the protein molecule and used to label the protein molecule; The linker molecule is selected from at least one of the following: GFP protein, mCherry protein, FITC, TRITC, NHS-luciferin, and NHS-rhodamine.

5. A method for enriching pathogenic avian Escherichia coli, characterized in that, include: The sample to be processed is co-cultured with protein molecules, wherein magnetic bead probes are attached to the protein molecules. The magnetic bead probe is magnetically adsorbed to enrich avian pathogenic Escherichia coli from the sample to be treated; The serotype of the avian pathogenic Escherichia coli is at least one of O2, O18 and O78; The amino acid sequence of the protein molecule is shown in SEQ ID No: 1.

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