Anti-Listeria monocytogenes MurA protein nano antibody and application thereof

Abstract

The invention discloses a nano antibody for resisting a listeria monocytogenes MurA protein and application of the nano antibody, and relates to the technical field of biology. The amino acid sequence of the nano antibody is as shown in SEQ ID NO.3. MurA protein of prokaryotically expressed listeria monocytogenes is used as an immunogen to immunize alpaca, total RNA of peripheral blood lymphocytes is extracted, VHH fragments are amplified through reverse transcription and two rounds of nested PCR, the VHH fragments and a pComb3Xss vector are converted into TG1 competent cells after enzyme digestion and connection, and a phage display technology is used for three rounds of elutriation, so that the total RNA of the listeria monocytogenes is obtained. The nanometer antibody for resisting the listeria monocytogenes MurA protein is successfully screened out. The nano antibody can specifically recognize MurA protein and whole listeria monocytogenes, and the immunocolorimetric sensor based on thiolated bacteriophage induced colloidal gold aggregation, which is established by utilizing the nano antibody, is strong in specificity, high in sensitivity and low in detection limit, and can greatly shorten the detection time.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a nano antibody against Listeria monocytogenes MurA protein and application thereof. Background Art

[0002] Listeria monocytogenes (LM), the causative pathogen of listeriosis, is a Gram-positive facultative anaerobic rod. The bacterium is salt-tolerant and can proliferate in low-temperature environments, mainly causing disease through intestinal infection. As one of the most deadly foodborne pathogens, LM is widely distributed in soil, decaying plants and sewage systems in nature, and is transmitted through contaminated food media. Clinical studies have shown that pregnant women, newborns, the elderly and immunosuppressed patients are susceptible populations, and typical clinical manifestations include spontaneous abortion, sepsis, central nervous system infections such as meningoencephalitis, and febrile gastroenteritis syndrome.

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

[0004] MurA protein has been proven to be an extracellular membrane protein of Listeria monocytogenes, encoded by the MurA gene. It is an autolysin produced by LM and has the function of hydrolyzing cell wall substances. Its N-terminus has a homologous structure of cell wall acid enzymes, and its C-terminus contains 4 repeated LysM motifs. Studies have shown that MurA protein plays a vital role in the proliferation and division of Listeria monocytogenes.

[0005] The current LM detection technology system covers traditional culture methods, immunological detection, molecular biology technology, electrochemical sensing and microfluidic platforms, etc., and each method shows significant differences in sensitivity and specificity. my country's current detection specifications still use the plate count method as the gold standard, but this method has a long detection cycle, usually 5-7 days, cumbersome operation steps, and requires multiple selective bacterial enrichment. These limitations can easily lead to a mismatch between the timeliness of detection and the needs of food production safety. Immunological detection technology achieves rapid detection through antigen-antibody specific recognition. Compared with traditional molecular biology technologies such as PCR, it not only has higher specificity and can effectively reduce the false positive rate, but also has the advantages of good repeatability and easy standardization. The new immunoassay platform developed based on this has shown good application potential.

[0006] There is a special type of heavy-chain antibodies (HCAbs) in camelids (alpacas, dromedaries) and cartilaginous fish (sharks, rays), whose antigen-binding domain is composed only of the variable region of the heavy chain. This structure is defined as nanobody (Nbs) or single-domain antibody (VHH). Nanobody has significant advantages: with a molecular weight of only 15 kDa, it is easier to genetically engineer than traditional IgG antibodies (~150 kDa); outstanding thermal stability and chemical tolerance, can withstand extreme pH and high temperature (80℃ for 1 h and still remain active); unique CDR3 structure (about 30% longer than IgG) enables it to recognize hidden antigen epitopes and achieve high affinity binding by penetrating into the hydrophobic cleft of folded proteins. These characteristics make nanobody a key tool to break through the bottleneck of existing immunoassay technology.

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

[0008] At present, immune detection for Listeria monocytogenes still relies on traditional antibodies (monoclonal / polyclonal antibodies), which have obvious limitations: ① Long production cycle, need to be prepared through mammalian cell expression system; ② Poor stability, the potency drops by about 40% in half a year when stored at 4℃; ③ Animal immunization involves ethical disputes, and 50-100 mice need to be immunized for each batch. Therefore, the development of new detection technology based on nano-antibodies can not only improve detection sensitivity and shorten detection time, but also achieve large-scale production through prokaryotic expression, phage display and other expression methods, effectively reducing costs, which is of urgent practical significance for improving the food safety guarantee system. Summary of the invention

[0009] The purpose 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. The nanobody can specifically recognize MurA protein and whole Listeria monocytogenes bacteria, and the immunocolorimetric sensor based on thiol-modified bacteriophage-induced colloidal gold aggregation established by using the nanobody 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 in SEQ ID NO.3.

[0012] The present invention also provides a gene encoding the above-mentioned nanobody.

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

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

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

[0016] The present invention also provides the use of the above-mentioned encoding gene, recombinant expression vector or recombinant host cell in the preparation of the above-mentioned nanobody.

[0017] The present invention also provides an application of a thiolated recombinant bacteriophage in preparing a kit for detecting Listeria monocytogenes based on an immunocolorimetric sensor. The thiolated recombinant bacteriophage is obtained by expressing the above-mentioned nano antibody on the surface of M13K07 phage.

[0018] The present invention also provides a kit for detecting Listeria monocytogenes based on an immunocolorimetric sensor, comprising a thiol-modified recombinant bacteriophage;

[0019] The thiol-modified recombinant phage is obtained by expressing the above-mentioned nanobody on the surface of M13K07 phage.

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

[0021] The present invention uses the MurA protein of Listeria monocytogenes expressed in prokaryotes as an immunogen to immunize alpacas, extracts total RNA from peripheral blood lymphocytes, amplifies VHH fragments through reverse transcription and two rounds of nested PCR, digests the VHH fragments with the pComb3Xss vector through enzyme digestion, connects and transforms into TG1 competent cells, and successfully screens out a nano antibody MurANb4H8 against the MurA protein of Listeria monocytogenes through three rounds of panning using phage display technology. Based on this, this nano antibody is successfully expressed on the surface of M13K07 phage through phage display, and the resulting recombinant phage is named Phage-MurANb4H8, which is purified and concentrated, and the pVIII protein on its surface is thiolated. Listeria monocytogenes and thiolated Phage-MurANb4H8 are added to the reaction system at the same time for co-incubation, and then a colloidal gold solution is added for reaction. The thiolated phage will induce the aggregation of colloidal gold, while the phage bound to Listeria monocytogenes will not induce the aggregation of colloidal gold due to the change of steric hindrance. Therefore, the quantitative detection method established can judge the concentration of Listeria monocytogenes bound to Phage-MurANb4H8 in the system by the aggregation degree of colloidal gold. The immunocolorimetric sensor established based on the nanoantibody MurANb4H8 has high specificity, strong stability, good sensitivity, and a detection limit of 1.0×10 4 CFU / mL, which has the advantage of high sensitivity.

[0022] The present invention uses the nanoantibody MurANb4H8 to prepare an immunocolorimetric sensor, which is used to detect Listeria monocytogenes with a detection limit of 1.0×10 4 CFU / mL, no cross-reaction with other foodborne pathogens, strong specificity, and greatly shortened the detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 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 This is the electrophoresis identification diagram of the first round of nested PCR amplification; where M: Marker; 1: VH and VL regions of the heavy chain of IgG1, IgG2, and IgG3 antibodies;

[0026] Figure 3 This is the electrophoresis identification diagram of the second round of nested PCR amplification; where M: Marker; 1: VHH sequence 1; 2: VHH sequence 2;

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

[0028] Figure 5 The specific detection results of the nanoantibody MurANb4H8 against Listeria monocytogenes;

[0029] Figure 6 The absorbance curves obtained after different concentrations of Listeria monocytogenes reacted with the thiol-modified Phage-MurANb4H8 recombinant phage;

[0030] Figure 7 Standard curve of the immunocolorimetric sensor method for detecting Listeria monocytogenes. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1

[0037] 1. Preparation of Antigen

[0038] The MurA protein unique to Listeria monocytogenes was selected as the immunogen, and the strain expressing the MurA protein was the BL21-pGEX4T-1-MurA prokaryotic expression strain pre-constructed in the present invention. Ampicillin with a final concentration of 100 μg / mL was added, and the culture was cultured in a 37°C shaker at 220 r / min until the OD value was 0.5. Isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 1 mM was added, and the culture was induced at 30°C 180 r / min for 6 hours. The bacterial precipitate was collected and resuspended in PBS buffer. After ultrasonic disruption, the supernatant was taken and purified using the GSTPur Glutathione kit to obtain the purified MurA protein. The electrophoresis 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: PCR amplification of the gene MurA encoding the MurA protein (nucleotide sequence as shown in SEQ ID NO.1), introduction of two restriction sites, BamH I and Xho I, directionally cloning it into the pGEX4T-1 vector (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.), and transformation into the Escherichia coli BL21 expression strain (purchased from Shanghai Weidi Biotechnology Co., Ltd.), and successfully constructed the BL21-pGEX4T-1-MurA prokaryotic expression strain.

[0040] SEQ ID NO.1:

[0041]

[0042] 2. Alpaca Immunity

[0043] 1 mL of purified MurA protein (concentration 1 mg / mL) was mixed with an equal volume of Freund's complete adjuvant. After sufficient emulsification, it was injected around the cervical lymph nodes of healthy adult male alpacas (purchased from the Experimental Animal Center of Dafang Bio-Nano Antibody Industry Research Institute of Jiangsu Academy of Agricultural Sciences). After the first immunization, booster immunization was performed every 14 days, for a total of five immunizations. In the last four immunizations, incomplete Freund's adjuvant was used instead of complete Freund's adjuvant. After each immunization for 7 days, venous serum was collected from the alpaca, and the antibody titer was monitored by ELISA. When the antibody titer reached 1:51200, the nanoantibody library was constructed.

[0044] 3. Construction of Nanolibrary

[0045] When the titer of alpaca serum antibody reached 1:51200, 50 mL of alpaca peripheral blood was collected, and the total RNA of peripheral blood lymphocytes was extracted using a kit, reverse transcribed into cDNA, and specific primers were designed. After two rounds of nested PCR amplification, the target bands obtained in the first round of PCR were around 1000 bp and 700 bp (such as Figure 2 The second round of PCR introduced two sticky ends, Sac I and Spe I, and finally obtained a pure VHH sequence band (as shown in Figure 3 As shown in the figure, the band size was about 470 bp. The target band was cut and recovered, and the VHH target fragment and pComb3Xss vector (purchased from Beijing Biobo Biotechnology Co., Ltd.) were double-digested with Fast Digest Sac I and Fast Digest Spe I. The VHH fragment was ligated to the pComb3Xss vector with T4 DNA ligase, and then transformed into Escherichia coli TG-1 competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.) by chemical transformation method through heat shock. The obtained single colonies were collected to obtain a nanobody library with a library capacity of 3×10 8 The positive insertion rate is 100% (e.g. Figure 4 as shown).

[0046] 4. Rescue of Listeria monocytogenes Nanobody Library

[0047] Take 1mL of the nanoantibody library and add it to 200mL of 2×YT medium containing ampicillin at a final concentration of 100μg / mL. Incubate at 37°C and 220rpm with shaking until the OD value is 0.6. Add helper phage M13K07 (purchased from Thermo Fisher Scientific, USA, catalog number: 18311019) at MOI=50:1. Let it stand at room temperature for 30min, then incubate at 37°C and 180rpm for 30min, centrifuge at 8000rpm for 10min, discard the supernatant, and use 200mL of 100μg / mL ampicillin and 75μg / mL kanamycin to precipitate the cells. The cells were resuspended in 2×YT medium, and IPTG was added at a final concentration of 0.5 mM. The cells were induced at 28°C for 16 h. The induced bacterial solution was centrifuged at 8000 rpm for 30 min. The supernatant was taken and 50 mL of high-pressure sterilized polyethylene glycol / sodium chloride (PEG / NaCl) solution was added. The cells were placed in an ice bath overnight. After the ice bath, the cells were centrifuged at 8000 rpm for 1 h. The precipitate was the recombinant phage containing the VHH sequence. The phage precipitate was resuspended in 1 mL of sterile PBS, and the phage precipitate was centrifuged at 12000 rpm for 10 min. The residual bacterial precipitate at the bottom of the centrifuge tube was discarded. The supernatant was the rescued phage.

[0048] 5. Screening of specific recombinant phage against Listeria monocytogenes

[0049] The optimal coating concentration of MurA protein was determined to be 10 μg / mL by chessboard method verification. MurA protein was coated on the ELISA plate, 100 μL per well, incubated at 37°C for 1 hour, washed five times with 0.01% phosphate buffered saline (PBST), blocked with 5% skim milk powder for 1 hour, and washed five times with 0.01% PBST, with an interval of 5 minutes each time. The rescued phage was diluted to 1×10 11pfu / mL, 200μL per well, incubate at 37℃ for 2h, then shake at 200rpm for 20min at room temperature, pour out the phages in the wells, wash the plate three times with 0.01% PBST, then wash the plate three times with 0.02% PBST, then wash the plate four times with 0.03% PBST, each time with an interval of 5min, and finally wash the plate 10 times with PBS, each time with an interval of 3min. After washing, pat the ELISA plate clean, add 150μL of glycine-HCl buffer (Glycine-HCl) with a pH of 2.2 filtered through a 0.22um filter to each well, shake at 400rpm for 20min at room temperature to elute the phages adsorbed on the ELISA plate, collect the eluate, and quickly add Tris-HCl neutralizer with a pH of 8.0 to neutralize. The neutralized recombinant phage was used to infect 3.5 mL of competent Escherichia coli TG-1 cells shaken to an OD value of 0.6, and the cells were allowed to stand at room temperature for 30 min, and then shaken at 28°C and 180 rpm for 30 min. The obtained bacterial solution was all spread on ampicillin-resistant 2×YT solid culture medium, and incubated in an incubator at 37°C overnight. The colonies were scraped to make a bacterial suspension, and the helper phage M13K07 was added again at an MOI of 50:1. The process of phage rescue and screening of specific recombinant phages was repeated for a total of 3 pannings to obtain P3-round specific recombinant phages for Listeria monocytogenes. All of them were infecting competent Escherichia coli TG-1 cells and then spread on ampicillin-resistant 2×YT solid culture medium. The obtained single colonies were tested by Phage-ELISA. The OD value obtained by ELISA was above the critical value (Cut-off), which was a positive result. The positive TG-1 strain was preserved and named TG1-pComb3Xss-MurANb4H8. The recombinant plasmid inside the TG1-pComb3Xss-MurANb4H8 strain contains a specific VHH sequence for Listeria monocytogenes MurA protein. After sequencing, the sequence was aligned and sorted using MAGA7 software, and the sequence measured was the VHH sequence against Listeria monocytogenes MurA protein. The sequencing yielded Listeria monocytogenes nanoantibody MurANb4H8, the amino acid sequence of which 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] GATGTGCAGCTGGTGGAGTCTGGGGGAGGGTTGGTTCAGAATGGGGGGTCTCTGAGACTCTCCTGTGTAGCCTCCGGAATCACCGGAAGCACATTCAGTACCCACGCGTTGGCCTGGTTCCGCCAGGCTCCAGGAAAGCAGCGTGAATGGGTCGCAGACATTAATACTGATGGTAGT ATAAGTTATGCAGAATCCGTGAGGGGCCGATTCACCATTCCAGAGACGACGCCAAGAACACGGTGTATTTGCAGATGAACAGCCTGAGAGATGAGGACACGGCCGCTATTACTGTAATGCAAGGCGCTACGGTGCGAGTTATTGGGGTCAGGGGACCCAGGTCACCGTCTCCTCA.

[0052] SEQ ID NO.3:

[0053] DVQLVESGGGLVQNGGSLRLSCVASGITGSTFSTHALAWFRQAPGKQREWVADINTDGSISYAESVRGRFTISRDDAKNTVYLQMNSLRDEDTAVYYCNARRYGASYWGQGTQVTVSS.

[0054] 6. Preparation and thiol modification of anti-Listeria monocytogenes MurA protein nanoantibody Phage-MurANb4H8 phage

[0055] Construction of GCJ-MurANb4H8 strain:

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

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

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

[0059] The resulting recombinant phage Phage-MurANb4H8 was chemically modified by coupling with cysteamine to add a thiol group. 12 PFU of phage was reacted with 1 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) for 30 min, 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 min for a total of two additions. After overnight reaction, the thiolated phage was successively in PBS and ddH 2 DHO, and finally precipitated with PEG / NaCl and washed with 1 mL ddHO. 2 O resuspend.

[0060] Example 2 Specificity 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 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 thiolated nanoantibody phage-induced colloidal gold aggregation.

[0063] Listeria monocytogenes and eight other foodborne pathogens (Salmonella Enteritidis, Escherichia coli O157:H7, Salmonella Typhimurium, Vibrio parahaemolyticus, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterococcus faecalis) were used as detection strains, and the binding ability of the recombinant phage Phage-MurANb4H8 expressing nanoantibodies to these nine foodborne pathogens was measured.

[0064] 50 μL of 10 9 CFU / mL of Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7, Salmonella typhimurium, Vibrio parahaemolyticus, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterococcus faecium were incubated with 100 μL of thiol-modified Phage-MurANb4H8 recombinant phage in a 37°C incubator for 120 min, 100 μL of 2 nM AuNPs were added to each system, and the mixture was incubated at 37°C for 100 min. 200 μL of the mixture was added to the ELISA plate, and the ELISA plate was placed in an ELISA reader. The OD value of each well was read at a wavelength of 524 / 682 nm to determine the specificity of the nanoantibody. The measurement results are shown in FIG. Figure 5 As shown. Figure 5 It can be seen that the nanoantibody Phage-MurANb4H8 has no cross-reaction to other foodborne pathogens and has a strong binding ability with Listeria monocytogenes.

[0065] Example 3 Establishment of an immunocolorimetric sensor for detecting Listeria monocytogenes based on thiolated bacteriophage-induced colloidal gold aggregation

[0066] Immunocolorimetric sensor for detection of Listeria monocytogenes:

[0067] Take 1.5mL centrifuge tubes and add 50μL of 10 diluted in gradient concentration to each tube. 3 ~10 8 CFU / mL Listeria monocytogenes, add 100 μL of thiol-modified Phage-MurANb4H8 recombinant phage to the system, place it in a 37°C incubator and incubate it for 120 min, then continue to add 100 μL of AuNPs solution to the system, incubate it at 37°C for 100 min, draw 200 μL of the reaction mixture and add it to the ELISA plate, place the ELISA plate in an ELISA reader, and detect it at wavelengths of 524 nm and 682 nm to obtain A 524 / A 682The measurement results are as follows Figure 6 As shown, a standard curve is drawn based on the obtained data. The standard curve is as shown in Figure 7 The detection limit of this method is 1.0×10 4 CFU / mL.

[0068] Example 4

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

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

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all 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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