A nanobody against Listeria monocytogenes MurA protein and its application

Abstract

The present invention discloses a nanobody against Listeria monocytogenes MurA protein and its application, which relates to the field of biotechnology. The amino acid sequence of the nanobody is shown as SEQ ID NO.3. In the present invention, the MurA protein of Listeria monocytogenes expressed prokaryotically is used as an immunogen to immunize alpacas. Total RNA of peripheral blood lymphocytes is extracted, and the VHH fragment is amplified by reverse transcription and two rounds of nested PCR. The VHH fragment and the pComb3Xss vector are digested by enzymes, and after ligation, they are transformed into TG1 competent cells. Through three rounds of panning using phage display technology, a nanobody against Listeria monocytogenes MurA protein is successfully screened out. This nanobody can specifically recognize the MurA protein and whole Listeria monocytogenes. The immunochromatographic sensor based on the aggregation of thiolated phage-induced colloidal gold established by using it has strong specificity, high sensitivity, low detection limit, and can greatly shorten the detection time.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a nanobody against Listeria monocytogenes MurA protein and its applications. Background Art

[0002] Listeria monocytogenes (LM), as the pathogenic pathogen of listeriosis, is a Gram-positive facultative anaerobic bacillus. This bacterium has the characteristic of salt tolerance and can proliferate in low-temperature environments. It mainly causes diseases through the intestinal infection route. As one of the most lethal foodborne pathogens, LM is widely distributed in nature in soil, decaying plant bodies and sewage systems, and is transmitted through contaminated food media. Clinical studies have shown that pregnant women, newborns, the elderly and immunosuppressed patients are susceptible populations. Typical clinical manifestations include spontaneous abortion, sepsis, central nervous system infections such as meningitis, and febrile gastroenteritis syndrome.

[0003] Listeria monocytogenes can colonize in spoiled vegetables, agricultural products and food processing environments, especially ready-to-eat foods (RTE) stored and transported under refrigeration. High-risk food categories include fresh cheese, processed meat products (including cooked and frozen poultry, pork, etc.), hot dogs, cold-smoked aquatic products, pre-made salads and seafood products. Therefore, it is of great public health significance to establish a stable, sensitive and reproducible LM detection system.

[0004] MurA protein has been proven to be an extracellular membrane protein of Listeria monocytogenes cells. Encoded by the MurA gene, it is an autolysin produced by LM and has the function of hydrolyzing murein. Its N-terminus has a muramidase homology structure, and its C-terminus contains 4 repeated LysM motifs. Some studies have shown that MurA protein plays a crucial role in the proliferation and division of Listeria monocytogenes.

[0005] The current LM detection technology system covers traditional culture methods, immunological detection, molecular biology techniques, electrochemical sensing and microfluidic platforms, etc. Each method shows significant differences in terms of sensitivity and specificity. The current detection specification in China still takes the plate counting method as the gold standard, but this method has a long detection period, usually 5 - 7 days, and the operation steps are cumbersome, requiring multiple selective enrichments. These limitations easily lead to the mismatch between the detection timeliness and the requirements of food safety production. Immunological detection technology realizes rapid detection through the specific recognition of antigen-antibody. Compared with molecular biology techniques such as traditional PCR, it not only has higher specificity, can effectively reduce the false positive rate, but also has advantages such as good repeatability and easy standardization. Based on this, the newly developed immune detection platforms have shown good application potential.

[0006] In camelids (alpacas, dromedaries) and cartilaginous fish (sharks, rays), there exists a special type of heavy-chain antibodies (HCAbs), whose antigen-binding domain consists only of the variable region of the heavy chain. This structure is defined as nanobody (Nb) or variable domain of heavy chain of heavy-chain antibody (VHH). Nanobodies have significant advantages: with a molecular weight of only 15 kDa, they are more easily genetically engineered than traditional IgG antibodies (~150 kDa); they have outstanding thermal stability and chemical tolerance, and can withstand extreme pH and high temperatures (remaining active after 1 h at 80 °C); their unique CDR3 structure (about 30% longer than that of IgG) enables them to recognize cryptic antigenic epitopes and achieve high-affinity binding by penetrating into the hydrophobic clefts of folded proteins. These characteristics make nanobodies a key tool for breaking through the bottlenecks of existing immunoassay technologies.

[0007] In the field of detection of foodborne pathogens, immunosensor technology based on nanobodies has achieved high-sensitivity recognition of microbial targets, with a detection limit reaching the pg / mL level. Compared with the traditional ELISA method, the detection time can be shortened by 50% - 70%, and no complex instruments are required. Studies have confirmed that for nanobody sensors constructed against pathogens such as Salmonella and Escherichia coli O157:H7, the coefficient of variation (CV) between batches is less than 5%, significantly better than the conventional polyclonal antibody system (CV > 15%). This indicates that nanobody technology is expected to revolutionize the existing foodborne pathogen detection system, especially in the rapid screening of Listeria monocytogenes in cold-chain foods, where it has important application value.

[0008] Current immunoassays for Listeria monocytogenes still rely on traditional antibodies (monoclonal / polyclonal antibodies), which have obvious limitations: ① The production cycle is long and needs to be prepared through mammalian cell expression systems; ② Poor stability, with the titer decreasing by about 40% after half a year of storage at 4 °C; ③ Animal immunization involves ethical controversies, and 50 - 100 mice need to be immunized for each batch. Therefore, developing a new detection technology based on nanobodies can not only improve the detection sensitivity and shorten the detection time, but also achieve large-scale production through expression methods such as prokaryotic expression and phage display, effectively reducing costs. This has an urgent practical significance for improving the food safety guarantee system. Summary of the Invention

[0009] The object of the present invention is to provide a nanobody against Listeria monocytogenes MurA protein and its application to solve the problems existing in the above-mentioned prior art. This nanobody can specifically recognize MurA protein and whole Listeria monocytogenes bacteria. The immunochromatographic sensor based on the aggregation of thiolated phage-induced colloidal gold established by using it has strong specificity, high sensitivity, low detection limit, and can greatly shorten the detection time.

[0010] To achieve the above object, the present invention provides the following solutions:

[0011] The present invention provides a nanobody against Listeria monocytogenes MurA protein, and its amino acid sequence is shown as SEQ ID NO.3.

[0012] The present invention also provides the coding gene of the above nanobody.

[0013] Furthermore, the nucleotide sequence of the coding gene is shown as SEQ ID NO.2.

[0014] The present invention also provides a recombinant expression vector, including the above coding gene.

[0015] The present invention also provides a recombinant host cell, including the above recombinant expression vector.

[0016] The present invention also provides the application of the above coding gene, recombinant expression vector or recombinant host cell in the preparation of the above nanobody.

[0017] The present invention also provides the application of a thiolated recombinant phage in the preparation of a kit for detecting Listeria monocytogenes based on an immunochromatographic sensor, and the thiolated recombinant phage is obtained by expressing the above nanobody on the surface of M13K07 phage.

[0018] The present invention also provides a kit for detecting Listeria monocytogenes based on an immunochromatographic sensor, including a thiolated recombinant phage;

[0019] The thiolated recombinant phage is obtained by expressing the above nanobody on the surface of M13K07 phage.

[0020] The present invention discloses the following technical effects:

[0021] In the present invention, the MurA protein of Listeria monocytogenes expressed in prokaryotes was used as an immunogen to immunize alpacas. Total RNA of peripheral blood lymphocytes was extracted, and the VHH fragment was amplified by reverse transcription and two rounds of nested PCR. The VHH fragment and the pComb3Xss vector were digested by enzymes, and after ligation, they were transformed into TG1 competent cells. Through three rounds of panning using phage display technology, a nanobody MurANb4H8 against the MurA protein of Listeria monocytogenes was successfully screened out. Based on this, through phage display, this nanobody was successfully expressed on the surface of M13K07 phage, and the obtained recombinant phage was named Phage-MurANb4H8. The recombinant phage was purified, concentrated, and the pVIII protein on its surface was mercaptoylated. Listeria monocytogenes and mercaptoylated Phage-MurANb4H8 were added to the reaction system simultaneously for incubation, and then colloidal gold solution was added for reaction. Mercaptoylated phage will induce the aggregation of colloidal gold, while phage bound to Listeria monocytogenes will not be able to induce the aggregation of colloidal gold due to the change in steric hindrance. Thus, the established quantitative detection method can determine the concentration of Listeria monocytogenes bound to Phage-MurANb4H8 in the system by the degree of aggregation of colloidal gold. The immunocolorimetric sensor established based on the nanobody MurANb4H8 has high specificity, strong stability, good sensitivity, and a detection limit of 1.0×10 4 CFU / mL, with the advantage of high sensitivity.

[0022] In the present invention, an immunocolorimetric sensor was prepared using the nanobody MurANb4H8, and this sensor was used for the detection of Listeria monocytogenes, with a detection limit up to 1.0×10 4 CFU / mL, no cross-reaction with other foodborne pathogenic bacteria, strong specificity, and greatly shortened detection time. Brief Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 It is the SDS-PAGE protein electrophoresis identification diagram of MurA protein before and after purification; wherein, M: Marker; 1: MurA protein before purification; 2: MurA protein after purification;

[0025] Figure 2 It is the electrophoresis identification diagram of the first round of nested PCR amplification; wherein, M: Marker; 1: VH and VL regions of the heavy chains of IgG1, IgG2, and IgG3 antibodies;

[0026] Figure 3 It is the electrophoresis identification diagram for the second-round amplification of nested PCR; among them, M: Marker; 1: VHH sequence 1; 2: VHH sequence 2;

[0027] Figure 4 It is the electrophoresis identification diagram for the positive insertion rate of the nanobody library; among them, M: Marker; 1-24: PCR products of 24 randomly selected monoclonal colonies;

[0028] Figure 5 It is the specific detection result of the nanobody MurANb4H8 against Listeria monocytogenes;

[0029] Figure 6 It is the absorbance curve diagram obtained by reacting different concentrations of Listeria monocytogenes with thiolated Phage-MurANb4H8 recombinant phage;

[0030] Figure 7 It is the standard curve diagram for the method of detecting Listeria monocytogenes by an immunocolorimetric sensor. Detailed implementation manners

[0031] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of the present invention, various modifications and variations to the specific embodiments of the present invention specification will be apparent to those skilled in the art. Other embodiments obtained from the present invention specification will be apparent to those skilled in the art. The present invention specification and examples are merely exemplary.

[0035] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0036] Example 1

[0037] 1. Preparation of antigen

[0038] Select the MurA protein unique to the genus Listeria monocytogenes as the immunogen. The strain expressing the MurA protein is the BL21-pGEX4T-1-MurA prokaryotic expression strain pre-constructed in the present invention. By adding ampicillin with a final concentration of 100 μg / mL, culturing in a shaker at 37 °C with 220 r / min until the OD value reaches 0.5, adding isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 1 mM, inducing at 30 °C with 180 r / min for 6 h, collecting the bacterial liquid precipitate, resuspending it in PBS buffer, after ultrasonic disruption, taking the disrupted supernatant, and purifying the supernatant using the GSTPur Glutathione kit to obtain the purified MurA protein. The SDS-PAGE identification diagram of the purified MurA protein is shown in Figure 1 .

[0039] The construction method of the BL21-pGEX4T-1-MurA prokaryotic expression strain is as follows: Perform PCR amplification on the gene MurA encoding the MurA protein (the nucleotide sequence is shown in SEQ ID NO.1), introduce two restriction enzyme sites, BamH I and Xho I, and directionally clone it into the pGEX4T-1 vector (purchased from Yeasen Biotechnology (Shanghai) Co., Ltd.), and transform it into the Escherichia coli BL21 expression strain (purchased from Shanghai Weidi Biotechnology Co., Ltd.) to successfully construct the BL21-pGEX4T-1-MurA prokaryotic expression strain.

[0040] SEQ ID NO.1:

[0041]

[0042] 2. Alpaca immunization

[0043] Take 1 mL of purified MurA protein (concentration 1 mg / mL), mix it with an equal volume of Freund's complete adjuvant, and after thorough emulsification, inject it around the cervical lymph nodes of healthy adult male alpacas (purchased from the Experimental Animal Center of Dafang Nanobody Industry Research Institute, Jiangsu Academy of Agricultural Sciences). After the first immunization, boost the immunization every 14 days for a total of five immunizations. For the last four immunizations, replace Freund's complete adjuvant with Freund's incomplete adjuvant. Every 7 days after immunization, collect alpaca venous blood serum and monitor the antibody titer by ELISA. When the antibody titer reaches 1:51200, construct the nanobody library.

[0044] 3. Construction of the nanobody library

[0045] When the alpaca serum antibody titer reaches 1:51200, collect 50 mL of alpaca peripheral blood, use a kit to extract the total RNA of peripheral blood lymphocytes, reverse transcribe it into cDNA, design specific primers, and amplify it by two rounds of nested PCR. The target bands obtained in the first round of PCR are around 1000 bp and 700 bp (as Figure 2 shown). In the second round of PCR, two sticky ends, Sac I and Spe I, are introduced. Finally, a pure VHH sequence band (as Figure 3 shown) is obtained, and the band size is around 470 bp. Cut the target band from the gel and recover it. Use Fast Digest Sac I enzyme and Fast Digest Spe I enzyme to double-digest the VHH target fragment and the pComb3Xss vector (purchased from Beijing BioVector NTCC Inc.), and use T4 DNA ligase to ligate the VHH fragment to the pComb3Xss vector. Then, transform it into Escherichia coli TG-1 competent cells (purchased from Shanghai Vidy Biotechnology Co., Ltd.) by heat shock through the chemical transformation method. Collect the obtained single colonies to obtain the nanobody library, with a library capacity of 3×10 8 individuals, and the positive insertion rate is 100% (as Figure 4 shown).

[0046] 4. Rescue of the Listeria monocytogenes nanobody library

[0047] 1 mL of the nanobody library was added to 200 mL of 2×YT medium containing ampicillin at a final concentration of 100 μg / mL, and cultured with shaking at 37 °C and 220 rpm until the OD value reached 0.6. Helper phage M13K07 (purchased from Thermo Fisher Scientific, USA, catalog number: 18311019) was added at an MOI of 50:1, and the mixture was allowed to stand at room temperature for 30 min, then cultured at 37 °C and 180 rpm for 30 min. After centrifugation at 8000 rpm for 10 min, the supernatant was discarded. The bacterial pellet was resuspended in 200 mL of 2×YT medium containing 100 μg / mL of ampicillin and 75 μg / mL of kanamycin, and IPTG at a final concentration of 0.5 mM was added. Induction was carried out at 28 °C for 16 h. The induced bacterial solution was centrifuged at 8000 rpm for 30 min, and the supernatant was taken and 50 mL of autoclaved polyethylene glycol / sodium chloride (PEG / NaCl) solution was added. After incubation overnight on ice, centrifugation was carried out at 8000 rpm for 1 h after the ice bath ended. The precipitate was the recombinant phage containing the VHH sequence. The phage pellet was resuspended in 1 mL of sterile PBS, and centrifuged at 12000 rpm for 10 min to discard the residual bacterial pellet at the bottom of the centrifuge tube. The supernatant was the rescued phage.

[0048] 5. Screening of Listeria monocytogenes - specific recombinant phages

[0049] After verification by the checkerboard method, the optimal coating concentration of MurA protein was determined to be 10 μg / mL. The MurA protein was coated on the enzyme - linked immunosorbent assay (ELISA) plate, 100 μL per well, and incubated at 37 °C for 1 h. The plate was washed five times with 0.01% phosphate - buffered saline with Tween - 20 (PBST) at 5 - minute intervals, and then blocked with 5% non - fat milk powder for 1 h. The plate was washed five times with 0.01% PBST at 5 - minute intervals. The rescued phage was diluted to 1×10 11200 μL per well at a concentration of pfu / mL, incubated at 37 °C for 2 h, then shaken at a low speed of 200 rpm for 20 min at room temperature. Pour out the phages in the wells, wash the plates three times with 0.01% PBST, then wash the plates three times with 0.02% PBST, and then wash the plates four times with 0.03% PBST, with a 5-min interval each time. Finally, wash the plates 10 times with PBS, with a 3-min interval each time. Pat the washed ELISA plates dry. Add 150 μL of glycine-hydrochloric acid buffer (Glycine-HCl) with a pH of 2.2 filtered through a 0.22-μm filter to each well, shake at a high speed of 400 rpm at room temperature for 20 min to elute the phages adsorbed on the ELISA plates, collect the eluate, and quickly add a Tris-HCl neutralizing solution with a pH of 8.0 for neutralization. Infect 3.5 mL of Escherichia coli TG-1 competent cells shaken to an OD value of 0.6 with the neutralized recombinant phages, let stand at room temperature for 30 min, shake at 28 °C and 180 rpm for 30 min. Spread all the obtained bacterial liquid on a 2×YT solid medium with ampicillin resistance and incubate it upside down in an incubator at 37 °C overnight. Scrape the colonies to make a bacterial cell suspension, and re-add helper phage M13K07 at a ratio of MOI = 50:1. Repeat the processes of phage rescue and screening of specific recombinant phages, and perform three rounds of panning in total to obtain the P3-round specific recombinant phages against Listeria monocytogenes. After infecting all Escherichia coli TG-1 competent cells, spread them on a 2×YT solid medium with ampicillin resistance. Detect the obtained single colonies by Phage-ELISA. If the OD value obtained by ELISA is above the critical value (Cut-off), it is a positive result. Preserve the positive TG-1 strain and name it TG1-pComb3Xss-MurANb4H8. The recombinant plasmid inside the TG1-pComb3Xss-MurANb4H8 strain contains the specific VHH sequence against the MurA protein of Listeria monocytogenes. After sequencing, align and organize the sequences using the MAGA7 software. The sequenced sequence is the VHH sequence against the MurA protein of Listeria monocytogenes. The nanobody MurANb4H8 against Listeria monocytogenes was obtained by sequencing. The amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.

[0050] SEQ ID NO.2:

[0051] GATGTGCAGCTGGTGGAGTCTGGGGGAGGGTTGGTTCAGAATGGGGGGTCTCTGAGACTCTCCTGTGTAGCCTCCGGAATCACCGGAAGCACATTCAGTACCCACGCGTTGGCCTGGTTCCGCCAGGCTCCAGGAAAGCAGCGTGAATGGGTCGCAGACATTAATACTGATGGTAGTATAAGTTATGCAGAATCCGTGAGGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGTGTATTTGCAGATGAACAGCCTGAGAGATGAGGACACGGCCGTCTATTACTGTAATGCAAGGCGCTACGGTGCGAGTTATTGGGGTCAGGGGACCCAGGTCACCGTCTCCTCA.

[0052] SEQ ID NO.3:

[0053] DVQLVESGGGLVQNGGSLRLSCVASGITGSTFSTHALAWFRQAPGKQREWVADINTDGSISYAESVRGRFTISRDDAKNTVYLQMNSLRDEDTAVYYCNARRYGASYWGQGTQVTVSS.

[0054] 6. Preparation and mercapto modification of phage-MurANb4H8 phage against Listeria monocytogenes MurA protein

[0055] Construction of GCJ-MurANb4H8 strain:

[0056] The MurANb4H8 gene sequence (SEQ ID NO.2) was amplified by PCR, introducing two sticky ends of SpeI and SacI, and it was directionally cloned into the pComb3Xss vector and heat-shock transformed into the TG-1 strain to obtain an engineered strain GCJ-MurANb4H8 strain with the same function as the TG1-pComb3Xss-MurANb4H8 strain.

[0057] The GCJ-MurANb4H8 strain was inoculated into 200 mL of 2×YT liquid medium with ampicillin resistance, and the final concentration of ampicillin was 100 μg / mL. It was cultured at 37 °C and 220 rpm until the OD value reached 0.6. The helper phage M13K07 was added at an MOI of 50:1, and it was left standing for 30 min, then cultured at 37 °C and 180 rpm for 30 min. After centrifugation at 8000 rpm for 10 min, the supernatant was discarded. The bacterial pellet was resuspended in 200 mL of 2×YT medium, and 100 μg / mL of ampicillin and 75 μg / mL of kanamycin were pre-added to the medium. Then, IPTG with a final concentration of 1 mM was added, and induction was carried out at 28 °C for 16 h. The induced bacterial solution was centrifuged at 8000 rpm for 30 min, and the supernatant was taken. 200 mL of sterile polyethylene glycol / sodium chloride (PEG / NaCl) solution was added, and it was ice-bathed for more than 4 h. After the ice-bath, it was centrifuged at 12000 rpm for 1 h, and the precipitate was the recombinant phage Phage-MurANb4H8 containing the VHH sequence. The phage pellet was resuspended in 1 mL of sterile PBS, and centrifuged at 12000 rpm for 10 min to discard the residual bacterial pellet at the bottom of the centrifuge tube. The supernatant was the concentrated and purified recombinant phage Phage-MurANb4H8.

[0058] Preparation method of thiolated Phage-MurANb4H8 recombinant phage:

[0059] The obtained recombinant phage Phage-MurANb4H8 was chemically modified by coupling with cysteamine to add a thiol group. 10 12 PFU of phage was reacted with 1 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) for 30 minutes. 1 mM N-hydroxysuccinimide (NHS) and 1 mM cysteamine were mixed in a total volume of 2 mL and stirred at room temperature (RT). An equal amount of EDC was added every 30 minutes, and a total of two additions were made. After overnight reaction, the thiolated phage was dialyzed successively in PBS and ddH 2 O water, and finally precipitated with PEG / NaCl and resuspended in 1 mL of ddH 2 O.

[0060] Example 2 Specific verification of nanobody MurANb4H8

[0061] Listeria monocytogenes, Salmonella enteritidis, Escherichia coli O157:H7, Salmonella typhimurium, Vibrio parahaemolyticus, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterococcus faecium used in this example were purchased from the China General Microbiological Culture Collection Center.

[0062] The pVIII protein on the surface of the Phage-MurANb4H8 recombinant phage was thiolated, co-incubated with *Listeria monocytogenes*, and finally incubated with gold nanoparticles (AuNPs). The color change was observed to establish an immunocolorimetric sensor based on the aggregation of colloidal gold induced by thiolated nanobody phage.

[0063] Using *Listeria monocytogenes* and 8 other foodborne pathogenic bacteria (Salmonella enteritidis, Escherichia coli O157:H7, Salmonella typhimurium, Vibrio parahaemolyticus, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterococcus faecium) as test strains, the binding ability of the recombinant phage Phage-MurANb4H8 expressing nanobody to these 9 foodborne pathogenic bacteria was determined.

[0064] 50 μL of *Listeria monocytogenes*, Salmonella enteritidis, Escherichia coli O157:H7, Salmonella typhimurium, Vibrio parahaemolyticus, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterococcus faecium at a concentration of 10 9 CFU / mL were co-incubated with 100 μL of thiolated Phage-MurANb4H8 recombinant phage in an incubator at 37 °C for 120 min. Then 100 μL of AuNPs solution at a concentration of 2 nM was added to each system, and they were co-incubated at 37 °C for 100 min. 200 μL of the mixed solution was taken and added to an enzyme-linked immunosorbent assay (ELISA) plate. The ELISA plate was placed in an ELISA reader, and the OD values of each well were read at wavelengths of 524 / 682 nm to judge the specificity of the nanobody. The measurement results are as Figure 5 shown. It can be seen from Figure 5 that the nanobody Phage-MurANb4H8 has no cross-reaction with other foodborne pathogenic bacteria and has a strong binding ability to *Listeria monocytogenes*.

[0065] Example 3 Establishment of a method for detecting *Listeria monocytogenes* using an immunocolorimetric sensor based on the aggregation of colloidal gold induced by thiolated phage

[0066] Detection of *Listeria monocytogenes* by immunocolorimetric sensor:

[0067] Take 1.5 mL centrifuge tubes, and add 50 μL of *Listeria monocytogenes* diluted in concentration gradients of 10 3 ~10 8 CFU / mL to each tube. Add 100 μL of thiolated Phage-MurANb4H8 recombinant phage to the system, and incubate in an incubator at 37 °C for 120 min. Then continue to add 100 μL of AuNPs solution to each system, and incubate at 37 °C for 100 min. 200 μL of the reaction mixture was taken and added to an ELISA plate. The ELISA plate was placed in an ELISA reader and detected at wavelengths of 524 nm and 682 nm to obtain A 524 / A 682Value, and the measurement results are as Figure 6 shown. According to the obtained data, a standard curve is plotted, and the standard curve is as Figure 7 shown. The detection limit of this detection method is 1.0×10 4 CFU / mL.

[0068] Example 4

[0069] A Listeria monocytogenes detection kit based on an immunocolorimetric sensor, comprising the following components:

[0070] Mercapto-Phage-MurANb4H8 recombinant phage and AuNPs.

[0071] The above embodiments are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A nanobody against Listeria monocytogenes MurA protein, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

2. A gene encoding a Nanobody as claimed in claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that: Comprising the coding gene described in claim 2 or 3.

5. A recombinant host cell, characterized in that Comprising the recombinant expression vector according to claim 4.

6. Use of the encoding gene according to claim 2 or 3, the recombinant expression vector according to claim 4 or the recombinant host cell according to claim 5 in the preparation of the nanobody according to claim 1.

7. Use of a thiol-modified recombinant bacteriophage in the preparation of a kit for detecting Listeria monocytogenes based on an immunocolorimetric sensor, characterized in that: The thiol-modified recombinant phage is obtained by expressing the nanobody according to claim 1 on the surface of M13K07 phage.

8. A kit for detecting Listeria monocytogenes based on an immunocolorimetric sensor, characterized in that: including thiolated recombinant phage; The thiol-modified recombinant phage is obtained by expressing the nanobody according to claim 1 on the surface of M13K07 phage.

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