A nanobody against Escherichia coli, its preparation method and application

Abstract

This invention provides an anti-Escherichia coli nanobody, its preparation method, and its applications, belonging to the fields of molecular biology, phage display technology, and proteomics. The anti-Escherichia coli nanobody has the function of specifically binding to Escherichia coli. This invention discloses the nanobody, the gene sequence encoding the nanobody, the expression vector and host cell for the nanobody, and the method for producing the nanobody. The anti-Escherichia coli nanobody provided by this invention has advantages such as small size, high expression efficiency, good solubility, and strong stability, and has good application prospects in the detection of Escherichia coli in food.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology, phage display technology, and proteomics, specifically to an anti-Escherichia coli nanobody, its preparation method, and its application. Background Technology

[0002] Escherichia coli ( Escherichia coli Escherichia coli (E. coli), a Gram-negative bacillus belonging to the Enterobacteriaceae family, is an important foodborne pathogen. This bacterium has a strong invasive ability and is commonly found in various foods such as meat, eggs, dairy products, and vegetables. Infection with E. coli can cause serious diseases such as hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura, posing a threat to human health. In low- and middle-income countries and regions with poor sanitation, unsafe drinking water, and weak medical infrastructure, the incidence of diarrheal diseases caused by E. coli is particularly high, significantly increasing the burden on local public health.

[0003] Currently, the microbiological detection of Escherichia coli mainly employs the multiple-tube fermentation method. This method relies on culture characteristics and phenotypic identification, has a cumbersome procedure, and typically takes 4 to 7 days to obtain definitive results, making it difficult to meet the needs of rapid diagnosis of modern pathogens. Therefore, establishing a rapid, accurate, highly sensitive, and highly specific bacterial pathogen identification technology is of significant practical importance for the effective prevention and treatment of bacterial infections.

[0004] Immunoassays, based on the specific reaction between antigens and antibodies, offer advantages such as high sensitivity, ease of operation, and relatively low cost, making them a crucial technical approach for pathogen detection. The core element of this type of method is the antibody. While monoclonal and polyclonal antibodies against Escherichia coli are available on the market, polyclonal antibodies suffer from poor batch-to-batch uniformity, while monoclonal antibodies have complex, time-consuming, and costly preparation processes, limiting their widespread application. Therefore, developing novel antibodies with high specificity, good stability, and ease of large-scale preparation is of paramount importance.

[0005] Notably, a special type of antibody, heavy-chain antibodies, naturally exists in camels. These antibodies consist solely of heavy chains and contain no light chains. Through genetic engineering, nanobodies (NBs) containing only the variable region (VHH) of the heavy chain can be obtained. These nanobodies have a molecular weight of approximately 15 kDa, about one-tenth that of traditional IgG antibodies, and are characterized by their small size and structural stability. Despite the size reduction, their complementarity-determining regions (especially CDR3) can still form a complete antigen-binding interface, achieving high affinity and specificity for target antigens and possessing the ability to neutralize pathogens. Furthermore, nanobodies have shorter gene sequences, making them easy to clone, modify, and fuse for expression. They also exhibit good solubility, tissue penetration, and high expression yield, demonstrating significant advantages in engineering modification.

[0006] However, to date, no nanobodies specifically targeting Escherichia coli have been reported. Therefore, developing nanobodies capable of efficiently recognizing and neutralizing Escherichia coli is not only of significant scientific value but also provides a new technological approach for the rapid detection and control of this bacterium. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention provides an anti-Escherichia coli nanobody, its preparation method, and its application. The nanobody can efficiently recognize and neutralize Escherichia coli, providing a new technical approach for the rapid detection and prevention of Escherichia coli.

[0008] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0009] In a first aspect, the present invention provides an anti-Escherichia coli (Escherichia coli) Escherichia coli The nanobody comprises a complementary variable region CDR1, CDR2 and CDR3.

[0010] Wherein, CDR1 is selected from the following sequences: the sequence shown in SEQ ID NO:7; a sequence having more than 90% homology with SEQ ID NO:7; CDR2 is selected from the following sequences: the sequence shown in SEQ ID NO:8; a sequence having more than 90% homology with SEQ ID NO:8; CDR3 is selected from the following sequences: the sequence shown in SEQ ID NO:9; a sequence having more than 90% homology with SEQ ID NO:9.

[0011] Furthermore, the amino acid sequence of the nanobody further includes framework regions FR1, FR2, FR3, and FR4; wherein, FR1 is selected from one of the following sequences: the sequence shown in SEQ ID NO:3; or a sequence having more than 90% homology with SEQ ID NO:3; FR2 is selected from one of the following sequences: the sequence shown in SEQ ID NO:4; or a sequence having more than 90% homology with SEQ ID NO:4; FR3 is selected from one of the following sequences: the sequence shown in SEQ ID NO:5; or a sequence having more than 90% homology with SEQ ID NO:5; and FR4 is selected from one of the following sequences: the sequence shown in SEQ ID NO:6; or a sequence having more than 90% homology with SEQ ID NO:6.

[0012] Furthermore, the amino acid sequence of the nanobody is selected from one of the following sequences: the sequence shown in SEQ ID NO:1; or a sequence having more than 90% homology with SEQ ID NO:1.

[0013] Secondly, the present invention provides a polynucleotide encoding the amino acid sequence of the aforementioned anti-Escherichia coli nanobody.

[0014] Furthermore, the polynucleotide is selected from the following sequences: the sequence shown in SEQ ID NO:2; or a sequence having more than 90% homology with SEQ ID NO:2.

[0015] Thirdly, the present invention provides a recombinant vector carrying polynucleotides containing the aforementioned anti-Escherichia coli nanoantibody.

[0016] Fourthly, the present invention provides a host cell containing the polynucleotide of the anti-Escherichia coli nanobody or the recombinant vector.

[0017] Fifthly, the present invention provides a method for producing the aforementioned anti-Escherichia coli nanobody, comprising the following steps:

[0018] The host cells are cultured to obtain a culture medium containing the anti-Escherichia coli nanobody, and the anti-inflammatory Escherichia coli nanobody is isolated and purified from the culture medium.

[0019] Sixthly, the present invention provides the application of the nanobody described herein for the detection of Escherichia coli in food.

[0020] Furthermore, the food products mentioned include, but are not limited to, drinking water, milk, beverages, and fruit juice.

[0021] In a seventh aspect, the present invention provides a kit for detecting Escherichia coli, the kit containing the aforementioned anti-Escherichia coli nanobody.

[0022] Compared with the prior art, the advantages of the present invention are:

[0023] (1) The anti-Escherichia coli nanobody provided by the present invention has a unique variable region sequence, which enables the antibody to have specific recognition and binding ability against Escherichia coli.

[0024] (2) The anti-Escherichia coli nanobody provided by the present invention has the advantages of easy expression and high expression efficiency.

[0025] (3) The anti-Escherichia coli nanobody provided by the present invention has the advantages of high affinity and strong specificity.

[0026] (4) The anti-Escherichia coli nanobody provided by the present invention has the advantage of strong stability.

[0027] (5) Based on the anti-Escherichia coli nanobody provided by the present invention, a rapid detection method for Escherichia coli in food can be established. Attached Figure Description

[0028] Figure 1 This is an electrophoresis image of the VHH gene amplified in the first round of PCR in Example 1.

[0029] Figure 2 This is an electrophoresis image of the VHH gene amplified in the second round of PCR in Example 1.

[0030] Figure 3 The identification results of the anti-Escherichia coli bacteriophage display nanobody in Example 2;

[0031] Figure 4 This is an SDS-PAGE electrophoresis image of the anti-Escherichia coli nanobody NB5-20 in Example 3;

[0032] Figure 5 The results show the specificity analysis of the anti-Escherichia coli nanobody NB5-20 in Example 4;

[0033] Figure 6 The results of the thermal stability analysis of the anti-Escherichia coli nanobody NB5-20 in Example 5 are shown.

[0034] Figure 7 The standard binding curves of Escherichia coli in food based on nanobody are shown. Detailed Implementation

[0035] To enable those skilled in the art to clearly and completely understand the technical solution of the present invention, the present invention will be further described in detail below with reference to embodiments. Obviously, the embodiments described herein are only for explaining the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0036] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in the art.

[0037] Definitions:

[0038] Nanobody: The variable region of camel heavy chain antibody;

[0039] The amino acid sequence framework region FR1 is the first constant region sequence of the nanobody.

[0040] The amino acid sequence framework region FR2 is the second constant region sequence of the nanobody.

[0041] The amino acid sequence framework region FR3 is the third constant region sequence of the nanobody;

[0042] The amino acid sequence framework region FR4 is the fourth constant region sequence of the nanobody;

[0043] The amino acid sequence complementarity-determining region CDR1 is the first variable region sequence of the nanobody.

[0044] The amino acid sequence complementarity-determining region CDR2 is the second variable region sequence of the nanobody.

[0045] The amino acid sequence complementarity-determining region CDR3 is the third variable region sequence of the nanobody.

[0046] The anti-Escherichia coli nanobody of the present invention can be used in food, such as the detection of Escherichia coli in liquid foods such as drinking water, milk, beverages, and fruit juice.

[0047] Example 1: Construction of an anti-Escherichia coli phage-displaying nanobody library

[0048] 1. Camel Immunization: Select a camel that has not been immunized against any antigens. Emulsify inactivated Escherichia coli and Freund's complete adjuvant at a 1:1 ratio, and then... 6Camels were immunized with CFU / mL via subcutaneous multiple-point injection, with booster immunizations every 2 weeks. Subsequent immunizations were performed using Freund's incomplete adjuvant and emulsified with inactivated Escherichia coli, for a total of 5 immunizations. One week after each immunization, blood samples were collected from the camels to test serum titers.

[0049] 2. Extraction of total RNA from blood: After the fifth immunization, peripheral blood was collected from camels, and lymphocytes were separated from the blood and total RNA was extracted according to the standard procedures in this technical field.

[0050] 3. Obtain cDNA through reverse transcription:

[0051] Using the obtained total RNA as a template and oligo(dT)15 as a primer, reverse transcription was performed to synthesize the first strand of cDNA and obtain a cDNA library.

[0052] 4. Amplification of the nanobody (VHH) gene fragment:

[0053] Using the synthesized cDNA as a template, the first round of PCR amplification was performed using the forward primer CALL001 and the reverse primer CALL002.

[0054] The reaction system for the first round of PCR amplification is as follows:

[0055]

[0056] The reaction mixture was vortexed and briefly centrifuged before PCR amplification was performed under the following conditions:

[0057] (1) 94 ℃ for 2 min;

[0058] (2) 94 ℃ for 30 s;

[0059] (3) 55 ℃ for 30 s;

[0060] (4) 68 ℃ for 1 min;

[0061] (2)~(4) Amplify for 30 cycles;

[0062] (5) 68℃ for 5 min.

[0063] The sequences of the forward primer CALL001 and the reverse primer CALL002 used in the first round of PCR amplification are as follows:

[0064]

[0065] The PCR products obtained from the first round of PCR amplification were separated by 1% agarose gel electrophoresis, and the 700 bp DNA fragment was purified and recovered using a kit. This fragment was used for the first round of PCR amplification of the VHH gene. See the electrophoresis identification image below. Figure 1 In the figure, M represents the DL 2000 marker, and 1 represents the VHH gene product amplified by the first round of PCR.

[0066] Using the VHH gene product from the first round of PCR amplification as a template, a second round of PCR amplification was performed using the forward primer CAM-FOR and the reverse primer CAM-BACK.

[0067] The reaction system for the second round of PCR amplification is as follows:

[0068]

[0069] Vortex the reaction mixture, briefly centrifuge, and then perform PCR amplification under the following conditions:

[0070] (1) 94 ℃ for 2 min;

[0071] (2) 94 ℃ for 30 s;

[0072] (3) 55 ℃ for 30 s;

[0073] (4) 68 ℃ for 1 min;

[0074] (2)~(4) Amplify for 20 cycles;

[0075] (5) 68 ℃ for 5 min.

[0076] The sequences of the forward primer CAM-FOR and the reverse primer CAM-BACK used in the second round of PCR amplification are as follows:

[0077]

[0078] The PCR products obtained from the second round of PCR amplification were separated by 1% agarose gel electrophoresis, and the 400 bp DNA fragment, namely the VHH fragment, was purified and recovered using a kit. See the electrophoresis diagram below. Figure 2 , Figure 2 In the diagram, M represents the DL 2000 marker, and 1 represents the product of the VHH gene amplified by the second round of PCR.

[0079] 4. Construction of the carrier

[0080] pComb3xss was digested using the following reaction system:

[0081]

[0082] After the enzyme digestion products were separated by 1% agarose gel electrophoresis, the 3400 bp vector fragment was purified and recovered using a kit.

[0083] The VHH gene product amplified by the second round of PCR was ligated into the double-digested pComb3xss vector, and in-fusion ligation was performed according to the following system:

[0084]

[0085] The reaction system was incubated overnight at 16 °C for 16 h, and the DNA was recovered using an agarose gel DNA purification kit and stored at -20 °C for later use.

[0086] 5. Electroconversion of the bonding products

[0087] Add 3 μL of the ligation product to 50 μL of E. coli ER2738 electroporation competent cells, mix well, and then transfer to a pre-chilled 0.1 cm electroporation cuvette (Bio-RAD). Perform electroporation on a Bio-RAD electroporator under the following conditions: 1.8 kV, 200 Ω, 25 μF. Immediately after electroporation, add 1 mL of preheated SOC liquid medium to the cuvette, pipette, and transfer to a clean, sterile 15 mL shake tube. Perform ten electroporations using the same method. Combine the results of the ten electroporations and incubate at 37 °C with gentle shaking for 1 h to allow the cells to recover.

[0088] 6. Construction of an anti-Escherichia coli phage-display nanobody library

[0089] The revived bacterial culture was transferred to 200 mL of SB medium and shaken at 37 ℃ and 250 rpm until OD reached. 600 When the value is 0.5, add 1 mL of 1×10 12 PFU helper phage M13KO7 was incubated at 37 °C for 1 h, followed by shaking for 2 h. Kanamycin was added to a final concentration of 70 μg / mL, and the mixture was shaken overnight. The next day, the overnight bacteria were centrifuged at 10,000 rpm for 15 min at 4 °C. The supernatant was transferred to a sterile centrifuge bottle, and 1 / 4 volume of 5× PEG / NaCl was added. After incubating on ice for 2 h, the mixture was centrifuged at 12,000 rpm for 20 min at 4 °C. The precipitate was dissolved in 10 mL of sterile resuspension solution (PBS buffer containing 1× protease inhibitor, 0.02% NaN3, and 0.5% BSA) to obtain the amplified anti-Escherichia coli phage-displaying nanobody library.

[0090] Example 2: Panning and Identification of Anti-Escherichia coli Nanobodies

[0091] 1. Selection of nanobodies against Escherichia coli

[0092] Positive Escherichia coli phage clones were obtained using a biopanning method of "adsorption-elution-amplification". The specific steps are as follows: On the first night, Escherichia coli were fixed into 96-well microplates; the phages were diluted to 10 μL with PBS. 8 100 μL / well of CFU / mL inactivated Escherichia coli culture was added to each well of an ELISA plate, coating four wells. The plate was incubated overnight at 4 °C. The next day, excess wells were blocked: unimmobilized coating solution was removed, the plate was washed three times with 0.05% PBST, and then 300 μL / well of blocking agent was added. After discarding excess blocking solution, the plate was washed again, and 150 μL of phage library was added. The plate was incubated at 37 °C for 2 h to allow the phages to fully bind to the immobilized antigen. The plate was then tapped to remove unbound phage solution, and 300 μL / well of 0.05% PBST was added. After washing the plate thoroughly to remove free phages, the phages bound to the immobilized antigen were eluted with 100 μL / well of Gly-HCl elution buffer (pH 2.2). The elution buffer was collected in a clean centrifuge tube, and Tris-HCl neutralization buffer was added to bring the pH of the solution to 7. Finally, the neutralized eluent was amplified, completing the first round of selection.

[0093] The second to fourth rounds of screening are basically the same as the first round, except that: (1) the blocking solution used in each round is different. The blocking solutions used in the first to fourth rounds are 3% skim milk powder, 3% BSA, 2% OVA and 3% skim milk powder respectively. (2) In the second to fourth rounds of screening, in order to remove the non-specific phages that were bound to the blocking solution in the previous round, the phage library is first added to the wells coated with the blocking solution in the previous round before adding it to the wells containing the blocking solution, so as to remove the non-specifically adsorbed phages.

[0094] 2. Identification of anti-Escherichia coli nanobodies

[0095] The selected phage-displaying nanobodies were identified using ELISA. The specific procedure was as follows: The ELISA plate was coated with a specific concentration of inactivated *Escherichia coli*, blocked with 3% skim milk powder, and then 100 μL of phage supernatant was added. The plate was incubated at 37 °C for 1 h, the supernatant was discarded, and the plate was washed 6 times with 0.05% PBST solution. Then, 100 μL of enzyme-labeled anti-M13 secondary antibody was added, and the plate was incubated at 37 °C for 1 h. After washing, TMB substrate was added for color development, and the plate was incubated for 15 min. Finally, stop solution was added, and the OD values ​​of each well were read using a microplate reader.

[0096] Using a direct ELISA method, phages capable of binding to Escherichia coli were selected to display nanobodies, such as... Figure 3As shown, the phage-displaying nanobody NB5-20, which exhibits strong binding to *Escherichia coli*, is a positive phage and was sent to a sequencing company for gene sequencing. The sequencing results were analyzed using Bioedit software. The antibody gene sequence was analyzed on the IMGT website (www.imgt.org / ) to determine the frame region and complementarity-determining region of the antibody sequence. The analysis results are as follows:

[0097] The amino acid sequence of the framework region FR1 of the anti-Escherichia coli nanobody NB5-20 is shown in SEQ ID NO:3;

[0098] The amino acid sequence of the framework region FR2 of the anti-Escherichia coli nanobody NB5-20 is shown in SEQ ID NO:4;

[0099] The amino acid sequence of the framework region FR3 of the anti-Escherichia coli nanobody NB5-20 is shown in SEQ ID NO:5;

[0100] The amino acid sequence of the framework region FR4 of the anti-Escherichia coli nanobody NB5-20 is shown in SEQ ID NO:6;

[0101] The amino acid sequence of the complementarity-determining region CDR1 of the anti-Escherichia coli nanobody NB5-20 is shown in SEQ ID NO:7.

[0102] The amino acid sequence of the complementarity-determining region CDR2 of the anti-Escherichia coli nanobody NB5-20 is shown in SEQ ID NO:8.

[0103] The amino acid sequence of the complementarity-determining region CDR3 of the anti-Escherichia coli nanobody NB5-20 is shown in SEQ ID NO:9.

[0104] The amino acid sequence of the anti-Escherichia coli nanobody NB5-20 is shown in SEQ ID NO:1;

[0105] The DNA sequence of the anti-Escherichia coli nanobody NB5-20 is shown in SEQ ID NO:2.

[0106] Example 3: Expression of anti-Escherichia coli nanobodies

[0107] The plasmid of the phage-displaying nanobody NB5-20 was extracted and transformed into host cells TOP10F' by heat shock. TOP10F' cells containing the nucleotide sequence of the nanobody NB5-20 were cultured overnight. The next day, the bacterial cells were precipitated, the cell walls were disrupted by sonication, and the nanobody was purified by nickel column chromatography. The purification effect was evaluated using SDS-PAGE, and the results are shown below. Figure 4 , Figure 4 In the middle, M represents the takara premix protein marker; Figure 4 The number 1 indicates the anti-Escherichia coli nanobody NB5-20. The concentration of the nanobody was determined using the Bradford method, and the expression efficiency of the nanobody was calculated to be 5 mg / L of culture medium.

[0108] Example 4: Specificity analysis of anti-Escherichia coli nanobodies

[0109] The interaction between the anti-Escherichia coli nanobody NB5-20 and five different foodborne pathogens was determined by ELISA. Escherichia coli nanobodies (NB5-20) were coated onto ELISA plates. Escherichia coli ), Bacillus cereus ( Bacillus cereus ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Shigella flexneri ( Shigella flexneri Salmonella enteritidis ( ) Salmonella Enteritidis ), Enterobacter sakazakii ( Enterobacter sakazakii ), Streptococcus pyogenes ( Streptococcus pyogenes Listeria monocytogenes ( ) Listeria monocytogenes Salmonella typhimurium ( Salmonella typhimurium Nine pathogenic bacteria were blocked, and 100 μL of NB5-20 nanobody solution was added. The mixture was incubated at 37 ℃ for 1 h, washed three times with PBST, and then anti-HA-HRP secondary antibody was added. The mixture was incubated at 37 ℃ for 1 h, washed six times with PBST, and then TMB solution was added for color development for 15 min. The reaction was terminated by adding sulfuric acid solution. The specificity of the nanobody was determined by measuring the absorbance of each well at 450 nm. Results are as follows: Figure 5 As shown, only the OD value of the wells coated with Escherichia coli was high, while the OD values ​​of the other wells were comparable to those of the blank, indicating that the nanobody NB5-20 of the present invention has strong specificity against Escherichia coli.

[0110] Example 5: Thermal stability analysis of anti-Escherichia coli nanobodies

[0111] The anti-Escherichia coli nanobody NB5-20 was incubated at 70 °C for 0.5 h, 1 h, 1.5 h, and 2 h, respectively. The activity changes of the nanobody were detected by ELISA and compared with the activity of the untreated nanobody. The results are as follows: Figure 6 As shown, the nanobody retains 60% of its activity after being treated at 70 °C for 1 h, indicating that the nanobody has high thermal stability.

[0112] Example 6: Detection method for Escherichia coli in food

[0113] Based on the screening and pairing, anti-Escherichia coli monoclonal antibody 2B4 (disclosed in CN107688094A) was selected as the capture antibody, and NB5-20-HRP was used as the detection antibody for double-antibody sandwich immunoassay to detect Escherichia coli. The capture antibody 2B4 was coated onto a 96-well ELISA plate at a concentration of 10 μg / mL per well and incubated overnight at 4 °C. The next day, the supernatant was discarded, the plate was washed three times with 0.05% PBST, and each well was blocked with 3% skim milk powder. Ten saturated pans were then prepared. 2 ~10 8 CFU / mL Escherichia coli solution was added to each well along with 50 μL of standard bacterial solution and 50 μL of detection antibody (NB5-20 nanobody), and incubated at 37 °C for 1 h. The plate was washed six times with 0.05% PBST, and TMB substrate chromogenic solution was added. The plate was incubated at room temperature for 15 min, and the reaction was terminated by adding 2M sulfuric acid solution. The OD value of each well was measured at 450 nm, and a standard curve was plotted. The standard curve is shown below. Figure 7 As shown. The detection limit of this method is 10. 4 CFU / mL.

[0114] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. An antibacterial agent against Escherichia coli (E. coli) Escherichia coli The nanobody is characterized by, The amino acid sequence of the nanobody includes complementary variable regions CDR1, CDR2, and CDR3; wherein, CDR1 is selected from the following sequences: the sequence shown in SEQ ID NO:7; or a sequence having more than 90% homology with SEQ ID NO:7; CDR2 is selected from the following sequences: the sequence shown in SEQ ID NO:8; or a sequence having more than 90% homology with SEQ ID NO:8; and CDR3 is selected from the following sequences: the sequence shown in SEQ ID NO:9; or a sequence having more than 90% homology with SEQ ID NO:

9.

2. The nanobody against Escherichia coli according to claim 1, characterized in that, The amino acid sequence of the nanobody further includes framework regions FR1, FR2, FR3, and FR4; wherein, FR1 is selected from the following sequences: the sequence shown in SEQ ID NO:3; or a sequence having more than 90% homology with SEQ ID NO:3; FR2 is selected from the following sequences: the sequence shown in SEQ ID NO:4; or a sequence having more than 90% homology with SEQ ID NO:4; FR3 is selected from the following sequences: the sequence shown in SEQ ID NO:5; or a sequence having more than 90% homology with SEQ ID NO:5; and FR4 is selected from the following sequences: the sequence shown in SEQ ID NO:6; or a sequence having more than 90% homology with SEQ ID NO:

6.

3. The nanobody against Escherichia coli according to claim 2, characterized in that, The amino acid sequence of the nanobody is selected from one of the following sequences: the sequence shown in SEQ ID NO:1; or a sequence having more than 90% homology with SEQ ID NO:

1.

4. A polynucleotide, characterized in that, The amino acid sequence of the anti-Escherichia coli nanobody according to any one of claims 1-3 is encoded.

5. A polynucleotide according to claim 4, characterized in that, The polynucleotide is selected from the following sequences: the sequence shown in SEQ ID NO:2; or a sequence having more than 90% homology with SEQ ID NO:

2.

6. A recombinant vector, characterized in that, The recombinant vector carries polynucleotides containing the anti-Escherichia coli nanobody as described in claim 4 or 5.

7. A host cell, characterized in that, The host cell contains a polynucleotide carrying the anti-Escherichia coli nanobody as described in claim 4 or 5, or a recombinant vector as described in claim 6.

8. The application of the nanobody according to any one of claims 1-3 for the detection of Escherichia coli in food.

9. The application according to claim 8, characterized in that, The food items mentioned include, but are not limited to, drinking water, milk, beverages, and fruit juice.

10. A kit for detecting Escherichia coli, characterized in that, The kit contains the anti-Escherichia coli nanobody as described in any one of claims 1-3.

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