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

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, in particular, the present application relates to a protein molecule and its application in identifying various serotypes of avian pathogenic Escherichia coli. BACKGROUND

[0002] Avian pathogenic Escherichia coli (APEC) is the main pathogen causing avian colibacillosis, and the clinical manifestations include acute septicemia, pericarditis, perihepatitis and peritonitis, etc., which causes significant economic losses to the poultry industry. Among the many APEC serotypes, O2, O18 and O78 are widely reported as dominant epidemic serotypes, with high detection frequency in different regions and different poultry species, and wide spread, which are the key objects for prevention and control in poultry health breeding.

[0003] In recent years, many studies have shown that APEC serotypes O2, O18 and O78 not only have strong pathogenicity to poultry, but also may have potential risk of cross-species transmission. Genomic analysis shows that the strains of these serotypes are highly similar to human extraintestinal pathogenic Escherichia coli (such as UPEC and NMEC) in virulence gene spectrum, suggesting that there is a molecular basis for zoonotic transmission. Therefore, establishing a rapid and accurate detection method for O2, O18 and O78 serotypes not only helps to prevent and control poultry diseases, but also has positive significance for public health safety.

[0004] Currently, the identification of APEC still relies on traditional bacterial culture, serotyping and molecular biology methods such as PCR and sequencing technology. Although these methods have high accuracy, they are tedious, time-consuming and require high experimental conditions, which are not suitable for rapid screening in farms or primary sites.

[0005] Therefore, it is an urgent need in the industry to develop a high-specificity, simple-to-operate detection method based on protein-bacterium interaction to realize rapid identification of O2, O18 and O78 serotypes. SUMMARY

[0006] The present application aims to at least solve one of the technical problems in the prior art. To this end, the present application provides a protein molecule which can specifically recognize avian pathogenic Escherichia coli of O2, O18 and O78 antigen serotypes, can be used for identification and enrichment of avian pathogenic Escherichia coli of O2, O18 and O78 antigen serotypes, has the advantages of simplicity, speed and accuracy, and has high application value.

[0007] Therefore, in a first aspect, the present application provides a protein molecule. According to an embodiment of the present application, the amino acid sequence of the protein molecule is shown in SEQ ID No: 1. Thus, the protein molecule of the present application can specifically bind to avian pathogenic Escherichia coli of O2, O18 and O78 antigen serotypes, thereby realizing the detection and identification of such strains.

[0008] In a second aspect, the present application provides a conjugate. According to an embodiment of the present application, the conjugate comprises: the protein molecule of the first aspect; and a linking molecule connected to the protein molecule for labeling the protein molecule. Thus, the conjugate of the present application can realize the rapid and accurate identification and enrichment of avian pathogenic Escherichia coli of O2, O18 and O78 antigen serotypes.

[0009] In a third aspect, the present application provides a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the protein molecule of the first aspect. Thus, based on the above-mentioned nucleic acid molecule, the above-mentioned protein molecule can be efficiently expressed in vitro, simplifying the production process; in addition, the nucleic acid molecule is convenient for genetic engineering modification to obtain a protein molecule more suitable for practical application.

[0010] In a fourth aspect, the present application provides an expression vector. According to an embodiment of the present application, the expression vector carries the nucleic acid molecule of the third aspect, or expresses the protein molecule of the first aspect. Thus, after introducing the expression vector into a suitable recipient cell, the expression of the aforementioned protein molecule can be effectively realized under the mediation of a regulation system, thereby realizing the in vitro mass production of the protein molecule.

[0011] In a fifth aspect, the present application provides the use of at least one of the protein molecule of the first aspect, the conjugate of the second aspect, the nucleic acid molecule of the third aspect, and the expression vector of the fourth aspect in 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 described above, the protein molecule of the present application can specifically bind to avian pathogenic Escherichia coli of O2, O18 and O78 antigen serotypes, and thus can be used for the identification and enrichment of such strains, having the advantages of simple operation, accurate identification, etc.

[0012] In a sixth aspect, the present application provides a method for identifying avian pathogenic E. coli for non-diagnostic purposes. According to an embodiment of the present application, the method comprises: co-culturing the protein molecule of the first aspect with a microorganism to be tested; determining whether the microorganism to be tested is an avian pathogenic E. coli based on whether the protein molecule binds to the microorganism to be tested; wherein the avian pathogenic E. coli is of at least one of serotypes O2, O18, and O78. As described above, the protein molecule of the present application can specifically bind to avian pathogenic E. coli of serotypes O2, O18, and O78, and thus can be used for identification and enrichment of such strains, with the advantages of simple operation and accurate identification.

[0013] In a seventh aspect, the present application provides use of the protein molecule of the first aspect, the conjugate of the second aspect, the nucleic acid molecule of the third aspect, or the expression vector of the fourth aspect in the preparation of a kit for identifying avian pathogenic E. coli or diagnosing diseases and / or symptoms caused by infection with avian pathogenic E. coli, wherein the avian pathogenic E. coli is of at least one of serotypes O2, O18, and O78. As described above, the protein molecule of the present application can specifically bind to avian pathogenic E. coli of serotypes O2, O18, and O78, and thus can be used for diagnosis of diseases and / or symptoms caused by such strains, with the advantages of simple operation and accurate identification.

[0014] In an eighth aspect, the present application provides a method for enriching avian pathogenic E. coli. According to an embodiment of the present application, the method comprises: co-culturing a sample to be treated with the protein molecule of the first aspect, wherein the protein molecule is linked to a magnetic bead probe; and enriching avian pathogenic E. coli from the sample to be treated by magnetic adsorption of the magnetic bead probe; wherein the avian pathogenic E. coli is of at least one of serotypes O2, O18, and O78. As described above, the protein molecule of the present application can specifically bind to avian pathogenic E. coli of serotypes O2, O18, and O78, and thus can be used for identification and enrichment of such strains, with the advantages of simple operation and accurate identification.

[0015] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0016] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, taken in conjunction with the following drawings in which:

[0017] Figure 1 An electropherogram according to an embodiment of the present application is shown;

[0018] Figure 2 FIG. 4 shows a fluorescence image of recombinant protein PJNE213-2 adsorbed on the surface of host bacteria according to an embodiment of the present application;

[0019] Figure 3 FIG. 5 shows a scanning electron microscope image of recombinant protein PJNE213-2 bound on the surface of host bacteria according to an embodiment of the present application;

[0020] Figure 4 FIG. 6 shows a graph of the results of a competitive adsorption experiment according to an embodiment of the present application;

[0021] Figure 5 FIG. 7 shows a schematic diagram of a specificity analysis of recombinant protein PJNE213-2 according to an embodiment of the present application. DETAILED DESCRIPTION

[0022] Embodiments of the present application are described in detail herein. The embodiments described herein are merely exemplary and are not to be construed as limiting the scope of the present application.

[0023] It is to be understood that the terms "first", "second", etc. are used herein only to describe various conditions, and cannot be construed as indicating or implying relative importance or a specific number of the technical features indicated. Thus, the features defined with "first", "second", etc. can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise specified, "a plurality of" means two or more.

[0024] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges or values should be construed to be roughly about the ranges or values. For ranges, the endpoints are included as well as intervening points which are not specifically recited. For values, the value includes the recited value as well as approximately the recited value.

[0025] In this document, the terms "comprises" or "comprising" are open-ended, that is, they mean including, but not limited to, the recited elements.

[0026] Protein molecule

[0027] Therefore, in a first aspect, the present application provides a protein molecule. According to an embodiment of the present application, the protein molecule has an amino acid 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. Thus, the protein molecule of the present application can specifically recognize avian pathogenic E. coli of O2, O18 and O78 antigen serotypes, rapidly and accurately identify and enrich avian pathogenic E. coli of O2, O18 and O78 antigen serotypes, and is helpful for the research, disease diagnosis and treatment of avian pathogenic E. coli, and is of great significance for the prevention and control of the spread of avian pathogenic E. coli. In this paper, the protein molecule is named "protein molecule PJNE213-2".

[0028] SSVQAQALRYHKHAGGFGEHDNAGAFGNTANSGYVGTRKGLDWDNRSYYTNDGFETTDPWNRDGHTTLNSEGLIANETRPWTMSVLFIIKV (SEQ ID No: 1).

[0029] Conjugate, nucleic acid molecule and expression vector

[0030] In a second aspect, the present application provides a conjugate. According to an embodiment of the present application, the conjugate comprises: the protein molecule of the first aspect; and a linking molecule connected to the protein molecule, for labeling the protein molecule. Thus, the conjugate of the present application can be used to rapidly and accurately identify and enrich avian pathogenic E. coli of O2, O18 and O78 antigen serotypes.

[0031] 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.

[0032] In a third aspect, the present application provides a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the protein molecule of the first aspect. Thus, based on the above-mentioned nucleic acid molecule, the above-mentioned protein molecule can be efficiently expressed in vitro, simplifying the production process; in addition, the nucleic acid molecule is convenient for genetic engineering modification to obtain a protein molecule more suitable for practical application.

[0033] In some embodiments, the nucleic acid molecule has a nucleotide sequence as set forth in SEQ ID No: 2 or a nucleotide sequence having at least 80%, 85%, 90%, 95% or 99% homology thereto.

[0034] tcttccgttcaagcccaagcattacgttatcataaacatgctggtggttttggtgaacatgataatgcgggtgccttcggtaacaccgctaactccggatatgttggtactcgtaaaggccttgactgggataaccgtagttattataccaacgacggttttgagaccaccgatccttggaaccgtgatggacataccacattgaatagtgaaggtcttattgccaacgaaacacgtccatggacaatgtccgttctttttattattaaagta (SEQ ID No: 2).

[0035] In some embodiments, the nucleic acid molecule is DNA.

[0036] It should be noted that, for the nucleic acid molecules mentioned in the present specification and claims, a person skilled in the art should understand that, either one of the complementary double strands, or both, are actually included. For the convenience, in the present specification and claims, although only one strand is given in most cases, the other complementary strand is actually disclosed. In addition, the nucleic acid sequences in the present application include DNA form or RNA form, and the disclosure of one means the disclosure of the other.

[0037] In the fourth aspect of the present application, an expression vector is provided. According to embodiments of the present application, the expression vector carries the nucleic acid molecule of the third aspect, or expresses the protein molecule of the first aspect. Thus, after the expression vector is introduced into a suitable recipient cell, the expression of the aforementioned protein molecule can be effectively realized under the mediation of the regulatory system, and the in vitro mass production of the protein molecule can be realized.

[0038] It should be noted that, when the aforementioned nucleic acid molecule is connected to a vector, the nucleic acid molecule 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 molecule, etc. Of course, these control elements can be directly from the vector itself, or can be exogenous, i.e., not from the vector itself. Of course, the nucleic acid molecule can be operably connected to the control elements.

[0039] In some embodiments, the vector can refer to a cloning vector, or can refer to an expression vector, which can be obtained by operably connecting the nucleic acid to a commercially available vector (such as a plasmid or a viral vector).

[0040] In the present disclosure, the term "operably linked" refers to the linkage of an exogenous gene to a vector, so that the control elements within the vector, such as the amino acid sequences for transcription control and the amino acid sequences for translation control, and the like, can exert their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, viral vectors, plasmids, bacteriophages, and the like. After the expression vector according to some embodiments of the present disclosure is introduced into a suitable recipient cell, the expression of the nucleic acid molecule as described above can be effectively realized under the mediation of the regulation system, and thus the in vitro mass acquisition of the protein encoded by the nucleic acid molecule can be realized.

[0041] Applications and methods

[0042] In a fifth aspect of the present disclosure, there is provided a use of at least one of the protein molecule of the first aspect, the conjugate of the second aspect, the nucleic acid molecule of the third aspect, and the expression vector of the fourth aspect for identifying and / or enriching avian pathogenic E. coli for non-diagnostic purposes; wherein the avian pathogenic E. coli has at least one of serotypes O2, O18, and O78. As described above, the protein molecule of the present disclosure can specifically bind to avian pathogenic E. coli of serotypes O2, O18, and O78, and thus can be used for identifying and enriching the strains, with the advantages of simple operation and accurate identification.

[0043] In a sixth aspect of the present disclosure, there is provided a method for identifying avian pathogenic E. coli for non-diagnostic purposes. According to an embodiment of the present disclosure, the method comprises: co-culturing the protein molecule of the first aspect with a microorganism to be tested; and determining whether the microorganism to be tested is avian pathogenic E. coli based on whether the protein molecule binds to the microorganism to be tested; wherein the avian pathogenic E. coli has at least one of serotypes O2, O18, and O78. As described above, the protein molecule of the present disclosure can specifically bind to avian pathogenic E. coli of serotypes O2, O18, and O78, and thus can be used for rapidly and accurately identifying avian pathogenic E. coli of serotypes O2, O18, and O78, and can be used for diagnosing diseases related to avian pathogenic E. coli of serotypes O2, O18, and O78, and can also be used for biological research on avian pathogenic E. coli of serotypes O2, O18, and O78 for non-diagnostic purposes.

[0044] In some embodiments, the protein molecule binds to the microorganism to be tested, and it is determined that the microorganism to be tested is avian pathogenic E. coli.

[0045] In some embodiments, the protein molecule does not bind to the microorganism to be tested, and it is determined that the microorganism to be tested is not avian pathogenic E. coli.

[0046] In some embodiments, a label molecule is attached to the protein molecule, and the protein molecule is determined to bind to the microorganism to be detected by detecting the label molecule.

[0047] In a seventh aspect of the present application, the protein molecule of the first aspect, the conjugate of the second aspect, the nucleic acid molecule of the third aspect, or the expression vector of the fourth aspect is used for preparing a kit for identifying avian pathogenic E. coli or diagnosing diseases and / or symptoms caused by infection of avian pathogenic E. coli, wherein the avian pathogenic E. coli is at least one of serotypes O2, O18, and O78. As described above, the protein molecule of the present application can specifically bind to avian pathogenic E. coli of serotypes O2, O18, and O78, and thus can be used for identifying avian pathogenic E. coli or diagnosing diseases and / or symptoms caused by the strain, with the advantages of simple operation and accurate identification.

[0048] In some embodiments, the diseases and / or symptoms are at least one of acute septicemia, pericarditis, perhepatitis, and peritonitis.

[0049] In an eighth aspect of the present application, a method for enriching avian pathogenic E. coli is provided. According to an embodiment of the present application, the method comprises: co-culturing a sample to be treated with the protein molecule of the first aspect, wherein the protein molecule is attached with a magnetic bead probe; and enriching avian pathogenic E. coli from the sample to be treated by magnetic adsorption of the magnetic bead probe, wherein the avian pathogenic E. coli is at least one of serotypes O2, O18, and O78. As described above, the protein molecule of the present application can specifically bind to avian pathogenic E. coli of serotypes O2, O18, and O78, and thus can be used for identifying and enriching the strain, with the advantages of simple operation and accurate identification.

[0050] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only, and are not to be construed as limiting the present application. Unless otherwise defined, technical or scientific terms used in the embodiments have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated, the products used in the embodiments are commercially available or are prepared according to the procedures described in the literature. Unless otherwise indicated, the reagents or instruments used in the embodiments are commercially available or are conventional products.

[0051] In the following examples, the main test materials are as follows:

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

[0053] pET-28a-sumo-eGFP vector: stored in the laboratory.

[0054] BL21(DE3) competent cells: purchased from Invitrogen Corporation.

[0055] Washing liquid eluent formula: Tris 20 mmol / L, NaCl 500 mmol / L, imidazole 50-500 mmol, glycerol 5%, Tween 0.05%.

[0056] Example 1: Construction and expression of recombinant protein

[0057] 1. Synthesize the target gene PJNE213-2 (nucleotide sequence as shown in SEQ ID No: 2) and extend according to the following primers:

[0058] Forward primer: 5'-CAAGCTTAAGCTTTCTTCCGTTCAAGCCCAA-3' (SEQ ID No: 3).

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

[0060] The above primer amplification system is shown in Table 1:

[0061] Table 1

[0062]

[0063] The PCR reaction program is shown in Table 2:

[0064] Table 2

[0065]

[0066] 2. Construction of recombinant plasmid pET-28a-sumo-eGFP-PJNE213-2

[0067] (1) The recovered target fragment and the recovered pET-28a-sumo-eGFP vector were respectively subjected to restriction enzyme Sac I and Hind III incubation in a 37°C water bath for 3 h, and the enzyme digestion system is shown in Table 3:

[0068] Table 3

[0069]

[0070] (2) The enzyme-digested vector pET-28a-sumo-eGFP and the target fragment PJNE213-2 were connected under the action of T4 ligase at 4°C overnight to obtain the recombinant product. The reaction system is shown in Table 4:

[0071] Table 4

[0072]

[0073] (3) Take the competent DH5a from the ultra-low temperature freezer storage box and place it on ice for thawing. Mix 10 μL of the recombination product with 100 μL of the competent DH5a gently, and then place it on ice for reaction for 30 min, and then place it in a water bath at 42°C for heat shock for 90 s, and then place it on ice for 5 min, add 900 μL of LB liquid medium, place it on a constant temperature shaker for incubation for 40 min, centrifuge at 5000 rpm at room temperature for 5 min, discard 900 μL of the supernatant, resuspend the remaining 100 μL and spread it on an LB solid culture dish (containing 50 mg / mL kanamycin), and place it in a constant temperature incubator at 37°C for overnight culture.

[0074] (4) The recombination plasmid pET-28a-sumo-eGFP-PJNE213-2 with correct base sequence is introduced into BL21 (DE3) competent cells according to the conventional method.

[0075] 3. Expression of the PJNE213-1 protein

[0076] (1) Take a single colony of the plasmid heat-shocked into BL21 (DE3) to 5 mL of LB liquid medium (containing 50 mg / mL kanamycin) for culture, and then transfer the bacterial liquid to 600 mL of LB liquid medium (containing 50 mg / mL kanamycin) at a ratio of 1:100, and culture in a constant temperature shaker at 37°C at 160 rpm until the OD600=0.4-0.6. At this time, add 600 μL of 0.6 M IPTG solution (isopropyl-β-D-thiogalactoside, final concentration 0.6 mM) to the culture system, and immediately transfer the culture bottle to a low-temperature constant temperature shaker at 16°C, and continue to shake at 130 rpm for 12-16 h to complete the protein induction expression.

[0077] (2) Transfer the bacterial liquid to a 50 mL sharp-bottom centrifuge tube precooled in advance, centrifuge at 10,000 rpm at 4°C for 3 min, discard the supernatant and collect the bacterial pellet. Resuspend the bacterial pellet with 40 mL of pre-cooled sterile PBS buffer (pH 7.4) gently, and repeat the washing three times under the same centrifugation conditions to remove the residual culture medium. Finally, resuspend the bacterial pellet with 15 mL of PBS buffer, and fix it on ice for ultrasonic crushing. Use a 6 mm titanium alloy probe, set the power to 150 W, the pulse cycle to 3 s working / 4 s intermittent, and continue crushing for 30 min. Immediately after the end, stand on ice for standby use. After ultrasonic crushing is completed, use a 4°C centrifuge to centrifuge at 10,000 rpm for 10 min, and reserve the supernatant.

[0078] 4. Purification and identification of PJNE213-2 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 PJNE213-2 protein has a size of 46.97 kDa.

[0081] Example 2: Fluorescence Microscopy Observation

[0082] Take 200 μL of recombinant protein PJNE213-2 (0.4 μg / μL) and 200 μL of freshly cultured host bacteria (wild-type Escherichia coli O2 serotype) (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, this indicates that recombinant PJNE213-2 can adsorb onto the surface of the host bacteria.

[0084] Example 3: Scanning Electron Microscopy

[0085] The recombinant protein PJNE213-2 (0.4 μg / μL) was mixed with host bacteria (wild-type Escherichia coli O78) culture (1×10⁻⁶). 8 The protein was mixed in equal volumes (CFU / mL) and the adsorption of the protein to the host bacteria was observed using scanning electron microscopy.

[0086] The results are as follows Figure 3 As shown, this indicates that PJNE213-2 can specifically bind to the surface of the host bacteria.

[0087] Example 4: Competitive Adsorption Experiment

[0088] Freshly cultured wild-type Escherichia coli O78 bacterial suspension (1×10⁻⁶) 7 CFU / mL), bacteriophages (1×10⁻⁶) 6 The phage titer was determined by mixing PFU / mL and recombinant protein PJNE213-2 (0.4 μg / μL) in equal proportions, centrifuging at 4000 rpm for 10 min at 4℃, collecting the supernatant, and measuring the phage titer using the double-layer agar method.

[0089] In the test group, there are equal proportions of bacteria solution, bacteriophage and recombinant protein PJNE213-2; in the control group, there are equal proportions of bacteriophage and bacteria solution.

[0090] The results, as shown in Table 1, show that the residual rate of bacteriophage in the supernatant of recombinant protein PJNE213-2 has a significant difference (P < 0.01) from that of the control group, indicating that recombinant protein PJNE213-2 can hinder the adsorption of bacteriophage. Figure 4 Example 5: ELISA detection

[0091] First, centrifuge commercially purchased E. coli solutions with different serotypes (O2, O18, O78, O82, O83, O86, O88, O91) (12,882 x g, 5 min), resuspend the bacterial bodies with PBS buffer after discarding the supernatant, and adjust the concentration of the bacteria solution to not less than 1 x 10 8 CFU / mL. Add 2% paraformaldehyde, fix at room temperature for 30 min, and wash the bacterial bodies with PBS buffer after fixation.

[0092] Subsequently, dilute the bacteria by 10 times, and dilute the recombinant protein PJNE213-2 (30 μg / mL) by 2 times. Take 200 μL of the bacterial suspension to coat the ELISA plate wells, and incubate at 37°C for 2 h. After incubation, add 200 μL of PBST (phosphate buffer containing Tween 20) to each well, stand for 1 min, and then discard it. Repeat the washing for 5 times. Then add 200 μL of 5% BSA (bovine serum albumin) solution, block at 37°C for 1 h to block non-specific binding. After washing with PBST again, add recombinant protein PJNE213-2 (final concentration 3 μg / mL) to each well, and incubate at 37°C for 1-2 h. After the reaction, wash with PBST for three times, immediately read the fluorescence signal using a fluorescence enzyme labeling detection system, with an excitation wavelength of 488 nm and an emission wavelength window of 520-530 nm.

[0093] The negative control (PBS) is set in the experiment, and at least 3 duplicate wells are set in each group to ensure the repeatability of the results. P > 0.05 is considered to have no statistical significance, and P < 0.01 is considered to have a significant difference.

[0094] The results, as shown in Table 1, show that the residual rate of bacteriophage in the supernatant of recombinant protein PJNE213-2 has a significant difference (P < 0.01) from that of the control group, indicating that recombinant protein PJNE213-2 can hinder the adsorption of bacteriophage.

[0095] Figure 5 ​As shown, it is indicated that the recombinant protein PJNE213-2 has specificity for E. coli strains of serotypes O2, O18 and O78, and the fluorescence signal is significantly higher than that of the control group (P<0.01). It has no specificity for other 5 different serotypes of E. coli, including O82, O83, O86, O88 and O91, and the fluorescence signal has no difference with the control group (P>0.05).

[0096] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0097] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. The use of at least one of a protein molecule, or a nucleic acid molecule encoding said protein molecule, or an expression vector expressing said protein molecule 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.

2. A method for identifying pathogenic avian Escherichia coli for non-diagnostic purposes, characterized in that, include: Co-culture protein molecules 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 a protein molecule, or a nucleic acid molecule encoding said protein molecule, or an expression vector expressing said protein molecule in the preparation of a kit for identifying avian pathogenic *Escherichia coli*, wherein, The serotype of the avian pathogenic Escherichia coli is at least one of O2, O18, and O78, and the amino acid sequence of the protein molecule is shown in SEQ ID No:

1.

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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