EGFP-LysFEC14CBD fluorescent fusion protein as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and relates to an EGFP-LysFEC14CBD fluorescent fusion protein as well as a preparation method and application thereof. In order to solve the technical problem that CBD of target lyase LysFEC14 has potential insolubility, fusion expression is innovatively carried out on the target lyase LysFEC14 and high-solubility EGFP protein, and the EGFP-LysFEC14 CBD fluorescent fusion protein is successfully constructed. The fusion protein disclosed by the invention has remarkable targeting specificity, can recognize various escherichia coli strains from different sources, has extremely low cross reaction to other bacteria (such as vibrio and staphylococcus aureus), and effectively avoids the problem of false positive; the sensitivity is high, the detection limit can be as low as 100 CFU, and the requirements for early warning and detection of trace pollution in a sample can be met; the method can be used for preparing commercial kits, is simple and convenient to operate, is rapid and convenient, and has wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the EGFP-Lys_FEC14_CBD fluorescent fusion protein, its preparation method, and its applications. Background Technology

[0002] Escherichia coli ( Escherichia coli Escherichia coli (E. coli) is one of the most important pathogens in clinical medicine and food safety. It is not only a major cause of various infectious diseases such as diarrhea, urinary tract infections, and sepsis, but also a key indicator for evaluating food hygiene. With the widespread use of antibiotics, E. coli strains producing extended-spectrum β-lactamases (ESBLs) and carbapenem-resistant bacteria are constantly emerging, posing a serious challenge to global public health. Therefore, rapid and accurate detection of E. coli is crucial for early disease diagnosis, epidemic tracing, and food safety monitoring.

[0003] Currently, methods for detecting E. coli mainly include traditional microbial culture methods, molecular biology techniques, and immunological techniques. While traditional culture methods are considered the "gold standard," the process involves multiple steps such as enrichment, isolation, and biochemical identification, typically requiring 2-3 days to obtain results. This is time-consuming and labor-intensive, far from meeting the demands of modern public health for immediate response. Molecular detection technologies, such as polymerase chain reaction (PCR), significantly shorten detection time and improve sensitivity, but they rely on expensive, sophisticated instruments and specialized operators, and cannot distinguish between the nucleic acids of dead and live bacteria, potentially leading to false positives, thus limiting their application in rapid on-site screening. Immunological methods, such as enzyme-linked immunosorbent assay (ELISA), rely on antibodies as biorecognition elements. Although they offer high specificity, antibody preparation is time-consuming and costly, and their stability is easily affected by environmental factors, limiting their widespread and economical application in the detection field.

[0004] Endolysins are key proteins synthesized by bacteriophages during the lysis of host bacteria, efficiently hydrolyzing the cell wall peptidoglycan layer to release progeny phages. This unique bactericidal mechanism makes them ideal candidates as alternatives to antibiotics. Endolysins typically consist of a catalytic domain (EAD) responsible for lysis and a cell wall-binding domain (CBD) responsible for recognition. CBDs can recognize and bind to specific bacterial cell wall surface components with extremely high specificity and affinity. Their protein properties are stable and easily expressed through large-scale recombinant genetic engineering, making them ideal diagnostic probes to replace antibodies, thus expanding the function of lysins from "treatment" to "diagnosis." However, current CBD-based detection technologies are still in their early stages, facing technical bottlenecks such as limited identification of bacterial strains and the need to improve detection sensitivity and accuracy.

[0005] Therefore, developing a novel and efficient detection method for Escherichia coli using CBD is of great significance for overcoming the limitations of existing technologies. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing Escherichia coli detection technologies, such as long time consumption, high cost, and complex operation, and to provide a novel fluorescent fusion protein based on phage lyase CBD, and to establish a method and reagents for rapid, highly sensitive, and highly specific detection of Escherichia coli using this probe.

[0007] The fusion protein provided by the present invention includes an N-terminal enhanced green fluorescent protein (EGFP), a C-terminal cell wall binding domain (Lys_FEC14_CBD) of the phage lysin Lys_FEC14, and a flexible linker connecting the two. Its amino acid sequence is shown in SEQ ID NO:1, and the nucleic acid sequence encoding the amino acid is shown in SEQ ID NO:2.

[0008] SEQ ID NO:1: MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGGGSAILKLGNRGSEVKALQQSLNKIGFSLTADGIFGKATENAVKSVQAGAGLVIDGIAGPKTFYAIRNAGDAHQEHLTEADLVDAARELGV SEQ ID NO:2: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGTGGCGGTGGCTCCGCCATTCTAAAACTTGGCAACCGAGGTTCTGAAGTCAAAGCACTTCAACAAAGCCTCAACAAAATCGGTTTCTCTCTTACAGCCGATGGCATATTTGGTAAGGCAACAGAGAATGCCGTCAAATCCGTTCAGGCAGGTGCTGGATTGGTTATTGATGGTATTGCTGGGCCAAAGACCTTCTATGCTATCCGCAACGCTGGAGACGCTCACCAGGAACATCTGACCGAAGCGGACTTGGTTGACGCAGCACGTGAACTTGGTGTT The addition of a linker sequence ensures that the two proteins in the fusion protein form the correct spatial structure, thus enhancing their biological activity. Typically, the choice of linker is closely related to the adapter sequences of the two components in the fusion protein. This invention investigated the selection of the linker sequence in experiments. The results show that its core function is to provide sufficient spatial separation between the N-terminal EGFP protein and the C-terminal Lys_FEC14_CBD domain, ensuring that both can independently undergo correct spatial folding and avoiding steric interference between them.

[0009] In this invention, a flexible linker is preferably used. This is because: 1) A flexible linker (such as a sequence rich in glycine and serine) provides a high degree of freedom of movement for the two functional domains, allowing the Lys_FEC14_CBD terminus to flexibly adjust its spatial orientation without being limited by the large size of EGFP, thereby efficiently recognizing and binding to specific target sites on the surface of the E. coli cell wall in optimal conformation. This is key to achieving high specificity and high affinity binding. 2) Flexible linking can minimize non-specific interactions and structural stress between the two domains, which is beneficial to the stable expression and solubility of the entire fusion protein.

[0010] In contrast, using a rigid linker (such as an α-helical linker) fixes the two domains at a specific spatial distance and angle. Without precise structural information to guide the process, this rigid connection is highly likely to cause the CBD binding site to be incorrectly oriented or kept at an suboptimal distance from the target, thus severely weakening or even completely losing its binding ability, resulting in a significant decrease in detection sensitivity and specificity.

[0011] Therefore, by employing a flexible linker, such as the sequence GGGGS, this invention successfully solves the problem of the collaborative operation of two functional domains, which is one of the technical guarantees for the excellent detection performance of the fusion protein of this invention.

[0012] The nucleic acid encoding this linker sequence is GGTGGCGGTGGCTCC (SEQ ID NO:5). The present invention also provides a nucleic acid encoding the above-mentioned fusion protein.

[0013] The present invention also provides an expression vector containing the above-mentioned nucleic acid.

[0014] The expression vector described in this invention refers to a DNA molecule capable of expressing a target gene in a suitable host. This invention does not limit the backbone of the plasmid vector, as long as it can be expressed in host cells, it is within the scope of protection of this invention. In some embodiments, the backbone vector of the expression vector is a pET series vector. Specifically, pET28a is used as the backbone vector.

[0015] The present invention also provides a recombinant host that transforms or transfects the above expression vector.

[0016] Transfection is the process by which eukaryotic cells acquire a new phenotype by actively or passively introducing exogenous DNA fragments under certain conditions. Transformation is the phenomenon in which a cell of one genotype absorbs DNA from the surrounding medium of another genotype, resulting in a corresponding change in its genotype and phenotype. The recombinant host can be a microorganism, plant cell, or animal cell. Preferably, the recombinant host is a microorganism, and more preferably, a prokaryote. In some embodiments, the host of the recombinant host is *Escherichia coli*. In some specific embodiments, the *Escherichia coli* is BL21.

[0017] The method for preparing the fusion protein of the present invention includes culturing the above-mentioned recombinant host and inducing the expression of the fusion protein.

[0018] Specific culture and induction methods include: culturing recombinant Escherichia coli at 37°C in LB medium until OD (Organic Degree) is reached. 600 The concentration was 0.6, and IPTG was added as an inducer; expression was induced at 16℃ for 18-24 hours to obtain a culture containing the fusion protein.

[0019] The present invention also provides the application of the above-mentioned fusion protein in the preparation of an Escherichia coli detection kit.

[0020] The fusion protein described above exists in bacterial cells and can be used directly as a detection reagent.

[0021] The present invention also provides a detection reagent for detecting Escherichia coli, comprising any one of the following I) to II): I) The aforementioned fusion protein; II) The aforementioned recombinant host.

[0022] The present invention also provides a method for detecting Escherichia coli, wherein any one of the following I) to II) is used. Contact the analyte with the sample and determine whether a reaction occurs based on the fluorescence: I) The aforementioned fusion protein; II) The aforementioned recombinant host.

[0023] The fluorescence is the fluorescence at the 488nm excitation source.

[0024] In some embodiments, the detection method is as follows: the analyte is contacted with a probe solution containing the fusion protein of the present invention, centrifuged, the precipitate is washed, and the fluorescence of the precipitate is detected at 488 nm. If there are obvious green fluorescent bacteria, or if the ELISA reader reading is significantly higher than that of the negative control group, the analyte is determined to be positive for Escherichia coli.

[0025] Implementing this invention has the following beneficial effects: This invention pioneers a novel approach for the specific detection of *E. coli* using the phage lyase CBD as a fluorescent probe. Addressing the potential insolubility of CBD in the target lyase Lys_FEC14, this invention innovatively fuses it with the highly soluble EGFP protein. This not only successfully solves the solubility and stability issues of CBD but also cleverly utilizes EGFP as a reporter molecule to construct a structurally stable and functionally efficient single molecular probe, avoiding complex steps such as antibody labeling.

[0026] The fusion protein of this invention exhibits excellent detection performance: 1) High specificity: The fluorescent fusion protein of this invention exhibits excellent targeting specificity, capable of recognizing a variety of Escherichia coli strains from different sources, while showing extremely low cross-reactivity with other genera (such as Vibrio and Staphylococcus aureus), effectively avoiding the problem of false positives.

[0027] 2) High sensitivity: The fluorescent fusion protein of this invention has high sensitivity and the detection limit can be as low as 100 CFU, which can meet the needs of early warning and detection of trace contamination in samples.

[0028] 3) Fast and convenient: The fluorescent fusion protein detection process of this invention can be completed within 2 hours, which is dozens of times more efficient than the traditional culture method (2-3 days) and greatly shortens the detection cycle.

[0029] The fusion protein of this invention has enormous application potential and high economic value: This invention is a recombinant protein that can be produced on a large scale and at low cost using a mature *E. coli* expression system. The results can be directly converted into commercial reagent kits, which are easy to operate and require minimal equipment and personnel. They are particularly suitable for rapid screening in food production sites, primary healthcare institutions, and environmental monitoring scenarios, possessing broad market application prospects and significant socio-economic value. Attached Figure Description Figure 1 This is a schematic diagram of the design of the fusion protein EGFP-Lys_FEC14_CBD of the present invention and an analysis of the hydrophobicity of the original lysin Lys_FEC14.

[0030] Figure 2This is an agarose gel electrophoresis image of the linearized pET-28a plasmid amplified by reverse PCR. In the image, M represents DNAmark 10000, 1 represents the PCR product of the original plasmid concentration, and 2 represents the PCR product of a 10-fold dilution of the plasmid.

[0031] Figure 3 This is an agarose gel electrophoresis image of PCR amplification of the EGFP and Lys_FEC14_CBD genes. In the image, 1 represents the EGFP gene amplification product, 2 represents the Lys_FEC14_CBD gene amplification product, and M represents DNA maker 2000.

[0032] Figure 4 This is an agarose gel electrophoresis image of the EGFP-Lys_FEC14_CBD fusion gene. In the image, M represents DNA maker2000, and 1 represents the amplified product of the EGFP-Lys_FEC14_CBD fusion gene.

[0033] Figure 5 This is an SDS-PAGE electrophoresis image of the purified fusion protein EGFP-Lys_FEC14_CBD of this invention. In the image, M represents the protein marker, 1 represents the cell lysis supernatant, and 2 represents the purified EGFP-Lys_FEC14_CBD protein.

[0034] Figure 6 This is a fluorescence micrograph showing the binding specificity of the fusion protein of this invention to different bacterial species (Escherichia coli, Vibrio, Staphylococcus aureus).

[0035] Figure 7 The fluorescence intensity of the recognition spectrum of the fusion protein of this invention against 13 different strains (9 Escherichia coli, 3 Vibrio, and 1 Staphylococcus aureus) is shown.

[0036] Figure 8 The fluorescence intensity of the binding between the fusion protein of this invention and Escherichia coli DH5α at different incubation times is shown.

[0037] Figure 9 The fluorescence intensity of the fusion protein of the present invention at different concentrations bound to Escherichia coli DH5α is shown.

[0038] Figure 10 The fluorescence intensity represents the detection sensitivity (detection limit) of the fusion protein of this invention against different concentrations of Escherichia coli DH5α. Detailed Implementation

[0039] To better understand the present invention, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, those skilled in the art will understand that the following embodiments are not intended to limit the scope of protection of the present invention, and any changes and variations made on the basis of the present invention are within the scope of protection of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0042] In this invention, enhanced green fluorescent protein is abbreviated as EGFP; In this invention, the Escherichia coli phage lyase is abbreviated as Lys_FEC14; In this invention, the cell wall binding domain is abbreviated as CBD.

[0043] Example 1: Construction, expression, and purification of the fluorescent probe fusion protein EGFP-Lys_FEC14_CBD This invention aims to construct a fluorescent probe for the detection of *E. coli*. Hydrophobicity prediction of the *E. coli* phage lysin Lys_FEC14 (NCBI accession number: ATW66849.1) using ProtScale (https: / / web.expasy.org / protscale / ) revealed that its cell wall binding domain (CBD) sequence exhibits potential insolubility. To improve its stability and functionality as a probe, this invention selected to link this CBD with highly soluble enhanced green fluorescent protein (EGFP) via a flexible linker (GGGGS), designing an EGFP-Lys_FEC14_CBD fusion protein, the structural diagram of which is shown below. Figure 1 As shown, its amino acid sequence is shown in SEQ ID NO:1, and the nucleic acid sequence encoding the amino acid is shown in SEQ ID NO:2.

[0044] SEQ ID NO:1: MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGGGSAILKLGNRGSEVKALQQSLNKIGFSLTADGIFGKATENAVKSVQAGAGLVIDGIAGPKTFYAIRNAGDAHQEHLTEADLVDAAERELGV SEQ ID NO:2: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGTGGCGGTGGCTCCGCCATTCTAAAACTTGGCAACCGAGGTTCTGAAGTCAAAGCACTTCAACAAAGCCTCAACAAAATCGGTTTCTCTCTTACAGCCGATGGCATATTTGGTAAGGCAACAGAGAATGCCGTCAAATCCGTTCAGGCAGGTGCTGGATTGGTTATTGATGGTATTGCTGGGCCAAAGACCTTCTATGCTATCCGCAACGCTGGAGACGCTCACCAGGAACATCTGACCGAAGCGGACTTGGTTGACGCAGCACGTGAACTTGGTGTT The recombinant expression plasmid pET-28a-EGFP-Lys_FEC14_CBD was constructed using seamless cloning technology. First, using the pET-28a plasmid as a template, reverse PCR was performed using the pET-28a-F / R primers listed in Table 2 to obtain a linearized plasmid of 5310 bp. Figure 2 The PCR reaction system is shown in Table 1. The PCR amplification conditions were: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 61.8℃ annealing for 15 s, 72℃ extension for 5 min, for a total of 35 cycles; and 72℃ extension for another 5 min.

[0045] Subsequently, the pET-28a-Lys_FEC14 and pET-28a-EGFP glycerol bacteria preserved in the laboratory were streaked onto LB solid medium containing Kan and cultured overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing Kan and cultured at 37°C with a shaker at 200 rpm for 6-8 h. Plasmids were extracted according to the instructions of the plasmid miniprep kit. Using pET-28a-EGFP and pET-28a-Lys_FEC14 plasmids as templates, amplification was performed using the primers listed in Table 2. EGFP Gene (whose nucleotide sequence is shown in SEQ ID NO:3) and Lys_FEC14_CBD The gene (its nucleotide sequence is shown in SEQ ID NO:4). EGFP-F carries the homologous arm of the linearized plasmid, and EGFP-R carries... Lys_FEC14_CBD Homologous arms and flexible linkers (GGGGS). Lys_FEC14_CBD-F features EGFP The homologous arms of the plasmid and the flexible linker (GGGGS) were included. Lys_FEC14_CBD-R contained the homologous arms of the linearized plasmid. The PCR reaction system is shown in Table 3. PCR amplification conditions: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 61.8℃ annealing for 15 s, 72℃ extension for 5 min, for a total of 35 cycles; followed by a final extension at 72℃ for 5 min. Figure 3 As shown, target bands of 772 bp and 289 bp in size were successfully amplified. The target bands were purified and recovered using a DNA gel extraction kit.

[0046] Table 1. PCR reaction system for linearized pET-28a plasmid

[0047] Table 2 Primer Information

[0048] Table 3 Lys_FEC14_CBD and EGFP PCR reaction system for gene amplification

[0049] The EGFP and Lys_FEC14_CBD fragments were ligated by overlap extension PCR to obtain a fusion gene fragment of 1026 bp. Figure 4 The PCR reaction system is shown in Table 4, and was performed in two steps: Step 1 reaction conditions: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 58.7℃ annealing for 15 s, 72℃ extension for 40 s, for a total of 10 cycles; followed by a final extension at 72℃ for 5 min. Step 2 reaction conditions were the same as above, except the annealing temperature was changed to 64.75℃, for a total of 35 cycles. According to the seamless cloning reaction system in Table 5, the seamless cloning kit was used to thoroughly mix all components, react at a constant temperature of 37℃ for 30 min, and the recombinant product was transformed into E. coli DH5α. Positive clones were screened and verified by sequencing, and the recombinant expression plasmid pET-28a-EGFP-Lys_FEC14_CBD was successfully obtained.

[0050] Table 4 EGFP-Lys_FEC14_CBD PCR reaction system for fusion genes

[0051] The verified recombinant plasmid was transformed into the *E. coli* BL21 expression strain, and single colonies were picked and cultured to the logarithmic growth phase (OD2). 600 =0.6). The culture was expanded at a ratio of 1:100, with a total volume of 1000 mL. When OD... 600 When the concentration reached 0.6, 0.1 mmol / L IPTG was added, and expression was induced at 16℃ for 18-24 hours. The bacterial cells were collected by centrifugation at 10000 r / min for 5 min at 4℃, washed three times with PBS, and resuspended. The cells were sonicated for 15 min (40% power, 3 s on, 4 s off) until the liquid was clear. The bacterial culture was centrifuged at 10000 r for 30 min at 4℃, the bacterial cells were collected, sonicated, and the supernatant was collected and purified. The purified protein was detected by SDS-PAGE electrophoresis, and the results are shown below. Figure 5 As shown, a single protein band with high purity was observed at approximately 33.46 kDa, which is consistent with the theoretical molecular weight of EGFP-Lys_FEC14_CBD, indicating that the fusion protein was successfully expressed in a highly soluble manner.

[0052] SEQ ID NO:3: EGFP nucleotide sequence ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG SEQ ID NO:4: Nucleotide sequence of Lys_FEC14_CBD GCCATTCTAAAACTTGGCAACCGAGGTTCTGAAGTCAAAGCACTTCAACAAAGCCTCAACAAAATCGGTTTCTCTCTTACAGCCGATGGCATATTTGGTAAGGCAACAGAGAATGCCGTCAAATCCGTTCAG GCAGGTGCTGGATTGGTTATTGATGGTATTGCTGGGCCAAAGACCTTCTATGCTATCCGCAACGCTGGAGACGCTCACCAGGAACATCTGACCGAAGCGGACTTGGTTGACGCAGCACGTGAACTTGGTGTT Example 2: Application and performance evaluation of EGFP-Lys_FEC14_CBD in bacterial detection To verify the binding specificity of the fusion protein probe, it was used to treat *Escherichia coli* (Gram-negative bacteria), *Vibrio* (Gram-negative bacteria), and *Staphylococcus aureus* (Gram-positive bacteria), respectively. 25 μL of EGFP-Lys_FEC14_CBD, 25 μL of 5 mM EDTA, and 200 μL of bacterial suspension (OD200) were added. 600 (≈0.8) Place in the dark at room temperature for 3 h, centrifuge at 5000 r for 10 min at 4℃, remove the supernatant, resuspend in PBS, repeat washing twice, and observe the smear under a fluorescence microscope. Figure 6 Fluorescence microscopy results showed that EGFP-Lys_FEC14_CBD emitted bright green fluorescence after binding to *E. coli* cells, indicating strong binding ability; the fluorescence was weaker after binding to *Vibrio*; and there was no binding at all to the Gram-positive bacterium *Staphylococcus aureus*, with no fluorescence signal in the field of view. Quantitative detection using an ELISA reader further evaluated the probe's recognition spectrum for 13 different bacterial strains. Figure 7 As shown, the probe produced strong fluorescent signals for all nine Escherichia coli strains from different sources tested, weaker signals for three Vibrio strains, and no signal for Staphylococcus aureus. This result is consistent with microscopic observation and directly demonstrates that the fusion protein probe has high specificity for Escherichia coli.

[0053] To determine the optimal detection time, fluorescence intensity (488 nm excitation / 520 nm emission) was measured at different time points (0.5 h, 1 h, 2 h, and 3 h) after treating *E. coli* DH5α with the probe. Figure 8 As shown, the fluorescence signal reached its peak and stabilized after 1 hour of incubation, thus determining the optimal incubation time to be 1 hour. To evaluate the sensitivity of the detection method, a fixed concentration of *E. coli* was treated with different concentrations of probe (1000, 500, 100, 50, 10, 5 μg / mL). Figure 9As shown, even a low concentration of the probe, 10 μg / mL, can produce an effective fluorescence signal. Subsequently, different concentrations of *E. coli* (10 to 10 μg / mL) were detected using the 10 μg / mL probe. 8 (CFU / mL), results are as follows Figure 10 As shown, when the bacterial concentration is as low as 10 2 Even at 100 CFU, a fluorescence signal significantly higher than the background can still be detected, indicating that the detection limit (LOD) of this method is 100 CFU. In summary, the EGFP-Lys_FEC14_CBD fusion protein constructed in this invention is a highly efficient and specific fluorescent probe for *E. coli*. The detection method based on this probe can be completed within 2 hours, with a detection limit as low as 100 CFU, exhibiting excellent performance in terms of speed, sensitivity, and specificity.

[0054] Example 3: Components and Usage of the EGFP-Lys_FEC14_CBD-Based Rapid Detection Kit for Escherichia coli This embodiment provides a commercially available testing kit based on the research results of Embodiments 1 and 2.

[0055] This test kit contains the following components: 1) Core recognition probe: EGFP-Lys_FEC14_CBD fusion protein in lyophilized powder form (prepared as described in Example 1), 100 µg per tube.

[0056] 2) Probe dissolution solution: 1 mL / tube of PBS buffer (pH 7.4).

[0057] 3) Reaction buffer: 50 mL / bottle of PBS buffer (pH 7.4) for sample dilution and washing (optional).

[0058] 4) Auxiliary reagent: 1 mL / tube of 50 mM EDTA solution.

[0059] 5) Negative control: Buffer solution without the target bacteria.

[0060] 6) Positive control: Inactivated Escherichia coli standard strain (optional).

[0061] 7) Instruction manual: It describes in detail the steps for using the kit, the criteria for interpreting results, and precautions.

[0062] Instructions for using the kit (taking food sample testing as an example) 1) Probe preparation: Before use, add 1 mL of probe dissolving solution to a tube of lyophilized EGFP-Lys_FEC14_CBD fusion protein, dissolve it completely, and prepare a probe storage solution of 100 µg / mL.

[0063] 2) Sample preparation: Take 1 gram of food sample, add it to 10 mL of reaction buffer, homogenize and let stand, and take the supernatant as the original solution of the sample to be tested.

[0064] 3) Incubation reaction: Add 200 µL of sample stock solution, 25 µL of EDTA solution (final concentration 5 mM), and 25 µL of probe stock solution (final concentration 10 µg / mL) to a centrifuge tube, and incubate at room temperature in the dark for 1 hour. A negative control tube should also be prepared.

[0065] 4) Washing procedure: After incubation, centrifuge at 4℃ and 5000 r / min for 10 minutes and discard the supernatant. Add 1 mL of reaction buffer to resuspend the precipitate and repeat the washing twice to completely remove unbound fluorescent probes.

[0066] 5) Result Detection and Interpretation: Resuspend the final precipitate in 200 µL of reaction buffer, drop an appropriate amount onto a glass slide, and observe using a fluorescence microscope; or transfer it to a black 96-well plate and detect the fluorescence intensity using a fluorescence microplate reader (excitation wavelength 488 nm, emission wavelength 520 nm). If obvious green fluorescent bacteria are observed under the microscope, or the microplate reader reading is significantly higher than that of the negative control group, the sample is considered positive for E. coli.

[0067] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A fusion protein, characterized in that: The amino acid sequence of the fusion protein is shown as SEQ ID NO:

1.

2. A nucleic acid encoding the fusion protein of claim 1.

3. The nucleic acid of claim 2, wherein, The sequence of the nucleic acid is shown as SEQ ID NO:

2.

4. An expression vector containing the nucleic acid of claim 2 or 3.

5. A recombinant host transformed or transfected with the expression vector of claim 4.

6. A method of producing the fusion protein of claim 1, characterized by, culturing the recombinant host of claim 5, inducing expression of the fusion protein.

7. Use of the fusion protein of claim 1 in the preparation of an E. coli detection kit.

8. A detection reagent for detecting Escherichia coli, characterized by comprising the polypeptide according to claim 1 or 2. comprising any one of the following I)~II): I) the fusion protein of claim 1; II) the recombinant host of claim 5.

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