A nano antibody against Listeria monocytogenes and its application

Abstract

The present invention discloses a nano antibody for Listeria monocytogenes and its application, and relates to the field of biotechnology. The amino acid sequence of the nano antibody is shown in SEQ ID NO.3. The present invention uses the p60 protein of Listeria monocytogenes expressed in prokaryotes as an immunogen to immunize alpacas, extracts total RNA of peripheral blood lymphocytes, amplifies VHH fragments by reverse transcription and two rounds of nested PCR, digests the VHH fragments with pComb3Xss vectors through enzyme digestion, connects and transforms into TG1 competent cells, and successfully screens out a nano antibody LMNb2E10 against Listeria monocytogenes p60 protein through three rounds of panning using phage display technology. The nano antibody can specifically identify Listeria monocytogenes, and the double-antibody sandwich ELISA method established by the nano antibody 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 in particular to a nano antibody of Listeria monocytogenes and application thereof. Background Art

[0002] Listeria monocytogenes (LM), also known as Listeria monocytogenes, is a Gram-positive small rod, a facultative anaerobic bacterium, and the causative pathogen of listeriosis. LM mainly infects the intestines. The bacterium is widely distributed in nature, salt-tolerant, and can reproduce under low temperature conditions. It mainly uses food as a vector and is one of the most deadly foodborne pathogens. Pregnant women, newborns, the elderly, and immunocompromised patients are susceptible to this disease, and the main clinical manifestations include symptoms of miscarriage, sepsis, meningoencephalitis, and febrile gastroenteritis.

[0003] Listeriosis outbreaks were first documented in 1981 and have been linked to the consumption of a variety of contaminated foods. Listeria can be found in rotting vegetables, sewage, sludge, produce, and certain types of food. Human infection is primarily due to the consumption of contaminated foods such as fresh cheese, cooked and frozen poultry products, pork products, meat products, hot dogs, smoked fish, vegetables, seafood, salads, and ready-to-eat (RTE) foods stored at refrigerated temperatures. The development of robust, sensitive, and reproducible methods for the detection of Listeria monocytogenes is particularly necessary.

[0004] The p60 protein is an extracellular protein secreted by LM, encoded by the iap gene. It was first discovered in 1989 and is an extracellular protein necessary for invading host cells. Since the p60 protein may be an auxiliary protein for LM to infect mammalian cells, it is also called an infection auxiliary protein. There is a typical signal peptide sequence at the N-terminus of the gene sequence encoding the p60 protein, which indicates that the p60 protein is a secretory protein with peptidoglycan hydrolase activity required for normal cell division. The secretion amount is high when LM infects the host, which helps to enhance the toxicity of LM during the infection process. The p60 protein can be secreted in all isolated Listeria, which shows that the p60 protein of Listeria monocytogenes can be used in traditional immunological tests to detect Listeria monocytogenes in food.

[0005] At present, the detection methods for Listeria monocytogenes involve traditional plate counts, immunological tests, molecular biological tests, electrochemical tests, biosensors, microfluidic systems, etc., showing different sensitivities and specificities. At present, my country generally uses the plate count method as the gold standard for detection, but due to its long detection cycle, poor timeliness, cumbersome steps, and the need for repeated bacterial growth, the best detection time is often missed, causing losses to production practice. The application of immunological detection methods often relies on antigen-antibody binding, which simplifies the operation and greatly shortens the detection time. Compared with traditional molecular biological tests such as PCR, immunological methods have higher specificity, reduce the probability of false positives, have good repeatability, and are easy to standardize. The new detection method established based on this has currently shown good detection performance.

[0006] In camelids such as alpacas and dromedaries, as well as cartilaginous fish such as sharks and rays, there is a type of heavy-chain antibody (HCAbs) that naturally lacks light chains. The variable region of its heavy chain is called a nanobody (Nbs), also known as a single-domain antibody (VHH). The molecular weight of a nanobody is only 15kDa. Compared with traditional IgG antibodies, nanobodies have the advantages of easy expression, easy modification, good stability, strong specificity, and high affinity. Most importantly, nanobodies can recognize hidden antigen epitopes that conventional antibodies cannot recognize. The extended CDR1 region and longer CDR3 region of nanobodies give them prominent antigenic determinants that can penetrate deep into the gaps of folded proteins. This structural change allows nanobodies to bind to the surface and cracks of antigens, giving them better recognition of antigens.

[0007] Immunoassays implemented by nanobodies can be used to detect microorganisms including bacteria and viruses. The use of nanobodies to detect pathogenic microorganisms in food has gradually matured, and nanobodies will surely become a powerful tool for immunological detection in the future. More importantly, the application of nanobodies will promote the development of rapid, sensitive and specific detection methods, which can show higher accuracy and stability than existing immunological detection methods, and are conducive to simplifying operations and shortening detection time. It is expected to replace the current ELISA detection method based on conventional antibodies.

[0008] However, the current immunological detection methods for Listeria mainly use traditional monoclonal antibodies and polyclonal antibodies, but traditional antibodies have large molecular weight, low stability, high cost, and production and preparation require immunization of a large number of experimental animals, which is not in line with animal welfare. The application of existing detection still cannot meet the requirements of production practice. Therefore, it is particularly necessary to develop new antibodies with high stability, strong specificity, high affinity and simple preparation process. Summary of the invention

[0009] The purpose of the present invention is to provide a nano antibody for Listeria monocytogenes and its application to solve the problems existing in the above-mentioned prior art. The nano antibody can specifically identify Listeria monocytogenes, and the double antibody sandwich ELISA method established by using the nano antibody 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 nano antibody against Listeria monocytogenes, and the amino acid sequence is shown in SEQ ID NO.3.

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

[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 the use of the above-mentioned nano-antibody in the preparation of an ELISA kit for detecting Listeria monocytogenes.

[0018] Furthermore, the ELISA kit is a double antibody sandwich ELISA kit.

[0019] The present invention also provides a double-antibody sandwich ELISA kit, comprising the above-mentioned nano-antibody.

[0020] Furthermore, the double antibody sandwich ELISA kit also includes an enzyme-labeled plate, a detection antibody, an enzyme-labeled secondary antibody, a washing solution, a color developing solution and / or a stop solution;

[0021] The nano antibody is coated in the ELISA plate.

[0022] The present invention discloses the following technical effects:

[0023] The present invention uses the p60 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 them into TG1 competent cells, and uses phage display technology to successfully screen out a nano antibody LMNb2E10 against the p60 protein of Listeria monocytogenes after three rounds of panning. Based on this, a prokaryotic recombinant expression vector pET28a-LMNb2E10 is constructed, and is transformed into Escherichia coli BL21 competent cells by heat shock method. After IPTG induction, the recombinant protein is affinity purified, and the recombinant protein is verified by Western blot to be the nano antibody LMNb2E10 that specifically recognizes the p60 protein of Listeria monocytogenes, which has high specificity, strong stability and good sensitivity. The double antibody sandwich ELISA method established based on the nano antibody LMNb2E10 has a detection limit of 1.0×10 4 CFU / mL, which has the advantage of high sensitivity.

[0024] The present invention uses nanoantibody LMNb2E10 to prepare a sandwich ELISA kit, which is used to detect Listeria monocytogenes, and the detection limit can reach 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

[0025] 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.

[0026] Figure 1 This is a Western Blot protein electrophoresis identification diagram of the purified p60 protein; where M: Marker; 1: purified p60 protein;

[0027] Figure 2 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;

[0028] Figure 3 The following is the restriction enzyme electrophoresis identification diagram of the vector and VHH sequence; where M: Marker; 1: pComb3XXS without restriction enzyme digestion; 2: pComb3XSS with double restriction enzyme digestion; 3: VHH sequence with double restriction enzyme digestion;

[0029] Figure 4 This is the electrophoresis identification diagram of the insertion positive rate of the nanobody library; where M: Marker; 1-48: PCR products of 48 randomly selected monoclonal colonies;

[0030] Figure 5 This is a Western Blot protein electrophoresis identification diagram of the nanobody of Listeria monocytogenes; wherein, M: Marker; 1: nanobody LMNb2E10;

[0031] Figure 6 The specific ELISA test results of the nanoantibody for Listeria monocytogenes;

[0032] Figure 7 This is the standard curve diagram of the nanoantibody against Listeria monocytogenes for detecting Listeria monocytogenes. DETAILED DESCRIPTION

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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 those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example 1

[0039] 1. Preparation of Antigen

[0040] The p60 protein unique to Listeria monocytogenes was selected as the immunogen. The strain expressing the p60 protein was the BL21-pET28a-iap prokaryotic expression strain pre-constructed in this experiment. Kanamycin was added at a final concentration of 100 μg / mL, and the culture was cultured at 220 r / min in a 37°C shaker until the OD value was 0.5. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM, and the culture was induced at 30°C 180 r / min for 6 hours. The bacterial precipitate was collected and resuspended in a binding solution without urea. After ultrasonic disruption, the supernatant was taken and purified by nickel column to obtain the purified p60 protein. The electrophoresis Western Blot identification of the purified p60 protein is shown in Figure 1 .

[0041] The construction method of the BL21-pET28a-iap prokaryotic expression strain is as follows: PCR amplification of the gene iap encoding the p60 protein (the nucleotide sequence is shown in SEQ ID NO.1), introduction of two restriction sites, Nde I and Xho I, directionally cloning it into the pET28a (+) vector (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.), and transformation into the BL21 expression strain, and successfully constructing the BL21-pET28a-iap prokaryotic expression strain.

[0042] Formula of binding solution without urea: Na 3 PO 4 12H 2 O 7.603 g / L, NaCl 29.22 g / L and imidazole 2.722 g / L.

[0043] SEQ ID NO.1:

[0044]

[0045] 2. Alpaca Immunity

[0046] 1 mL of purified p60 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 1:25600, the nanoantibody library was constructed.

[0047] 3. Construction of Nanolibrary

[0048] When the titer of alpaca serum antibody reached 1:25600, 75 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, two sticky ends of Sac I and Spe I were introduced, and finally a pure VHH sequence band was obtained (such as Figure 2 The target band was cut and recovered, and the VHH target fragment and pComb3Xss vector (purchased from Beijing Bio-Bowei Biotechnology Co., Ltd.) were double-digested with Fast Digest Sac I and Fast Digest Spe I (as shown in the table). Figure 3 As shown in Figure 2), the VHH fragment was ligated to the pComb3Xss vector using T4 DNA ligase and then transformed into Escherichia coli TG-1 competent cells by heat shock 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).

[0049] 4. Rescue of Listeria monocytogenes Nanobody Library

[0050] Take 1 mL of the nanoantibody library and add it to 200 mL of 2×YT medium, then add ampicillin with a final concentration of 100 μg / mL, and culture at 37°C, 220 rpm until the OD value is 0.6, add helper phage M13K07 (purchased from Thermo Fisher Scientific, catalog number: 18311019) at MOI=50:1, let it stand for 30 min, and incubate at 37°C Culture at 180rpm for 30min, centrifuge at 8000rpm for 10min, discard the supernatant, resuspend the bacterial pellet with 200mL of 2×YT medium, pre-add 100μg / mL ampicillin and 75μg / mL kanamycin to the medium, then add IPTG with a final concentration of 1mM, induce at 28℃ for 16h, centrifuge the induced bacterial solution at 8000rpm for 30min, take the supernatant, add 1 / 5 of the total volume of sterile polyethylene glycol / sodium chloride (PEG / NaCl) solution, ice bath for more than 4h, centrifuge at 12000rpm for 1h after the ice bath, the precipitate is the recombinant phage containing the VHH sequence, resuspend the phage precipitate with sterile PBS, centrifuge at 12000rpm for 10min, discard the residual bacterial pellet at the bottom of the centrifuge tube, and the supernatant is the rescued phage.

[0051] 5. Screening of specific recombinant phage against Listeria monocytogenes

[0052] The optimal coating concentration of p60 protein was determined to be 10 μg / mL by chessboard method verification. The p60 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), 5 minutes apart each time, and tapped clean. The plate was blocked with 5% skim milk powder for 1 hour, washed five times with 0.01% PBST, 5 minutes apart each time, and tapped clean for later use. The rescued phage was diluted to 1×10 11pfu / mL, 200μL per well, incubate at 37℃ for 2h, shake at 25℃, 200rpm for 20min, wash the plate three times with 0.01% PBST, wash the plate three times with 0.02% PBST, wash the plate three 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 pH 2.2 glycine-HCl buffer (Glycine-HCl) to each well, shake at 37℃, 400rpm for 20min, elute the phage adsorbed on the ELISA plate, collect the eluate, and add pH 8.0 Tris-HCl neutralizer to neutralize it, so that the final concentration is adjusted to about pH 7.0. The neutralized recombinant phage was infected with competent Escherichia coli TG-1, allowed to stand for 30 minutes, and shaken at 28°C and 180 rpm for 30 minutes. The resulting bacterial solution was resuspended in 500 μL of 2×YT medium and all of it was spread on ampicillin-resistant 2×YT solid medium. The culture was inverted in a 37°C incubator overnight, and the colonies were scraped to make a bacterial suspension. The helper phage M13K07 was added again, and the process of phage rescue and screening of specific recombinant phages was repeated. A total of 4 pannings were performed to obtain a specific recombinant phage for Listeria monocytogenes. After infecting competent Escherichia coli TG-1 cells, it was spread on ampicillin-resistant 2×YT solid medium. The resulting single colony was tested by Phage-ELISA. The OD value obtained by ELISA was above the Cut-off, which was a positive result. The positive TG-1 strain was preserved and named LMNb2E10. The recombinant plasmid inside the LMNb2E10 strain contains a specific VHH sequence for Listeria monocytogenes. After sequencing, the sequence was aligned and sorted using MAGA7 software, and the sequence measured was the VHH sequence against Listeria monocytogenes. The sequencing yielded the Listeria monocytogenes nanoantibody LMNb2E10, whose nucleotide sequence is shown in SEQ ID NO.2 and whose amino acid sequence is shown in SEQ ID NO.3.

[0053] SEQ ID NO.2:

[0054] GATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTTAGACTCTCCCGTGCAGCCTCTGGAAGCACCTTCAGACTATATGCGATGGGCTGGTACCGCCAGGCTCCAGGAAAGCAGCATGAGTTGGTCGCAGGTATTACGAGTGGAGGTGCAACAAACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGATCATGTTGTATCTGCAAATGAACAACCTAAAACCCGAAGACACGGCCGTCTATTACTGTGCGACCGTGACTTGTACGAGTTGGACCCAGCACATGGGGGAATATGACGTCTCCGGCCAGGGGACCCAGGTCACCGTCTCCTCA。

[0055] SEQ ID NO.3:

[0056] DVQLVESGGGLVQAGGSLRLSRAASGSTFRLYAMGWYRQAPGKQHELVAGITSGGATNYADSVKGRFTISRDNAKIMLYLQMNNLKPEDTAVYYCATVTCTSWTQHMGEYDVSGQGTQVTVSS。

[0057] 6. Prokaryotic expression and purification of the nanobody LMNb2E10 against Listeria monocytogenes

[0058] According to the sequencing results of the LMNb2E10 positive strain, specific primers were designed, and two sticky ends, Nde I and Xho I, were introduced for PCR amplification. The prokaryotic expression vector pET28a (+) (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) and the LMNb2E10 fragment were double-digested with Fast Digest Nde I enzyme and Fast Digest Xho I enzyme, and then the LMNb2E10 fragment was ligated to pET28a (+) using T4 DNA ligase to construct the recombinant expression plasmid pET28a-LMNb2E10 (nucleotide sequence shown in SEQ ID NO.4). The recombinant expression plasmid pET28a-LMNb2E10 was transformed into Escherichia coli BL21 competent cells by heat shock method to construct the recombinant expression strain BL21-pET28a-LMNb2E10. The recombinant expression strain BL21-pET28a-LMNb2E10 was cultured to an OD value of 0.8, and IPTG with a final concentration of 1 mM was added. The mixture was induced at 25°C for 20 h, and centrifuged at 8000 rpm for 10 min. The bacterial pellet was washed 3 times with sterile PBS, and resuspended with 15 mL PBS for the last time. The pellet was ultrasonically precipitated at 4°C. The ultrasonic program was ultrasonic time of 3 s, interval time of 3 s, power of 60%, and ultrasonic time of 20 min. The ultrasonic supernatant was purified by nickel column. 5× loading buffer was added to the purified protein eluate, and the sample was fully boiled and then run Western Blot. The obtained 15 kDa protein band was the purified nanoantibody LMNb2E10 against Listeria monocytogenes p60 protein (such as Figure 5 as shown).

[0059] Example 2 Specificity verification of nanobody LMNb2E10

[0060] The 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.

[0061] A double antibody sandwich ELISA detection method was established using nanobody LMNb2E10 as the capture antibody and anti-LM polyclonal antibody (purchased from LifeSpan BioSciences, USA, product number: LS-C71735-1) as the detection antibody.

[0062] 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 faecium) were used as detection strains to measure the binding ability of the nanoantibody LMNb2E10 to these nine foodborne pathogens.

[0063] The nanoantibody LMNb2E10 at 10 μg / mL was used as the coating source, 100 μL per well was coated on the ELISA plate, placed in a 37°C incubator for 1 hour, washed 5 times with 0.01% PBST buffer, 5 minutes each time; 100 μL of 5% skim milk powder was added to each well, blocked in a 37°C incubator for 1 hour, washed 5 times with 0.01% PBST buffer, 5 minutes each time; 100 μL of 10 8 CFU / mL inactivated Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7, Salmonella typhimurium, Vibrio parahaemolyticus, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterococcus faecium were incubated at 37°C for 1 hour and washed 5 times with 0.01% PBST buffer; 100 μL of anti-LM polyclonal antibody was added to each well, incubated at 37°C for 1 hour, and washed 5 times with 0.01% PBST buffer; 100 μL of HRP-labeled goat anti-rabbit antibody (purchased from Beijing Solebow Technology Co., Ltd.) was added to each well, incubated at 37°C for 1 hour, and washed 5 times with 0.01% PBST buffer; 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate colorimetric solution was added to each well and incubated in the dark for 15 minutes; 50 μL of 2 mol / L H 2 SO 4 The reaction was terminated by adding the solution, and the ELISA plate was placed in an ELISA reader. The OD value of each well was read at 450 nm to determine the specificity of the nanoantibody. The results were as follows: Figure 6 As shown. Figure 6 It can be seen that the nanoantibody LMNb2E10 has no cross-reaction to other foodborne pathogens and has a strong binding ability with Listeria monocytogenes.

[0064] Example 3 Establishment of Nanoantibody Sandwich ELISA Method Based on Nanoantibody LMNb2E10

[0065] Double antibody sandwich ELISA method for detecting Listeria monocytogenes:

[0066] 10 μg / mL LMNb2E10 was used as the coating source, 100 μL per well was coated on the ELISA plate, placed in a 37°C incubator for 1 hour, washed 5 times with 0.01% PBST buffer, 5 minutes each time; 200 μL of 5% skim milk powder was added to each well, blocked in a 37°C incubator for 1 hour, washed 5 times with 0.01% PBST buffer, 5 minutes each time; 100 μL of gradient dilutions of 10 3 ~10 8 CFU / mL inactivated Listeria monocytogenes was incubated at 37℃ for 1h and washed 5 times with 0.01% PBST buffer; 100μL anti-LM polyclonal antibody was added to each well, incubated at 37℃ for 1h, and washed 5 times with 0.01% PBST buffer; 100μL HRP-labeled goat anti-rabbit antibody was added to each well, incubated at 37℃ for 1h, and washed 5 times with 0.01% PBST buffer; 100μL TMB substrate colorimetric solution was added to each well and incubated in the dark for 15min; 50μL 2mol / L H 2 SO 4 The reaction was terminated by adding 1% ethanol and 0.5% ethanol. The ELISA plate was placed in an ELISA reader and the OD value of each well was read at 450 nm. The standard curve was drawn using the logistic fitting curve model. Figure 7 As shown. Figure 7 It can be concluded that the detection limit of this method is 1.0×10 4 CFU / mL.

[0067] Example 4

[0068] A double nano-antibody sandwich ELISA detection kit for Listeria monocytogenes, comprising the following components:

[0069] ELISA plate coated with nanobody LMNb2E10 (capture antibody);

[0070] Detection antibody: polyclonal antibody against LM;

[0071] Enzyme-labeled secondary antibody: Anti-phage M13 antibody with HRP label;

[0072] Washing solution: 0.01% PBST buffer;

[0073] Color development solution: TMB substrate color development solution;

[0074] Stop solution: 2mol / LH 2 SO 4 Solution.

[0075] 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, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

2. A gene encoding a nanobody according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

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

4. A recombinant host cell, characterized in that Comprising the recombinant expression vector according to claim 3.

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

6. Use of the nanobody as claimed in claim 1 in the preparation of an ELISA kit for detecting Listeria monocytogenes.

7. The use according to claim 6, characterized in that: The ELISA kit is a double antibody sandwich ELISA kit.

8. A double antibody sandwich ELISA kit, characterized in that: Comprising the Nanobody of claim 1.

9. The double antibody sandwich ELISA kit according to claim 8, characterized in that: The double antibody sandwich ELISA kit also includes an enzyme-labeled plate, a detection antibody, an enzyme-labeled secondary antibody, a washing solution, a color developing solution and / or a stop solution; The nano antibody is coated in the ELISA plate.

Images