Affinity-matured anti-acrylamide derivative nanobody mutant and its application

Abstract

The present invention discloses an affinity-matured anti-acrylamide derivative nanobody mutant and its application. The present invention constructs a saturation mutation library based on the wild-type anti-acrylamide derivative nanobody, and then screens the anti-acrylamide derivative nanobody mutant with affinity maturation and significantly improved performance through phage display method. The nanobody mutant IC 50 The detection limit of the nanoantibody was 0.81 μg / mL, which was 55% higher than that of the wild-type nanoantibody. The minimum detection limit was 0.052 μg / mL and the linear range was 0.172-9.793 μg / mL. The nanoantibody mutant could specifically recognize acrylamide derivatives and had significantly improved tolerance to organic solvents, which was nearly doubled compared to the wild-type and more stable. When used for enzyme-linked immunosorbent assay to detect acrylamide, the test results were accurate and stable, which is of great significance for detecting the content of acrylamide in food.

Description

Technical Field

[0001] The present invention belongs to the field of antibody engineering, and in particular relates to an affinity-matured anti-acrylamide derivative nanobody mutant and its application. Background Art

[0002] Acrylamide is a colorless, odorless, white crystal with the chemical formula C3H5NO. Reducing sugars in food react with free amino acids at high temperatures to form acrylamide, which is widely found in baked goods such as bread, biscuits, and coffee. Acrylamide is carcinogenic and is classified as a Class 2A carcinogen by the World Health Organization. It has potential toxic effects on the human body. Excessive intake of acrylamide can lead to abnormal development of the human reproductive system and a decrease in sperm count and quality. Acrylamide can also affect the human nervous system, interfering with normal communication between nerve cells and causing imbalances in ion concentrations within the nervous system. Therefore, it is necessary to strengthen the detection of acrylamide content in food.

[0003] Currently, the primary detection methods for acrylamide in food, both domestically and internationally, rely on instrumental methods such as gas chromatography-mass spectrometry and spectral analysis. While accurate, these methods are costly and unsuitable for large-scale, on-site testing. In recent years, immunoassays, due to their simplicity, speed, and high sensitivity, have become widely used for the detection of toxic and hazardous substances in food. The core reagents of immunoassays are antibodies. The poor stability and low yield of polyclonal and monoclonal antibodies have limited their application in immunoassays. Nanobodies are heavy chain antibody variable regions naturally found in camelids that lack light chains. Their molecular weight is small, only 1 / 10 the size of traditional antibodies, making them the smallest antigen-binding fragments currently available. Compared to traditional antibodies, nanobodies offer enhanced stability, excellent thermal stability, and tolerance to organic solvents. They can maintain good antigen-binding capacity even after high-temperature treatment and in certain organic solvent concentrations. Due to their simple structural domains, nanobodies can be easily engineered to enhance their affinity.

[0004] However, there are currently few highly sensitive nanoantibodies that can be used to detect acrylamide. The sensitivity of existing wild-type anti-acrylamide derivative nanoantibodies cannot meet the needs of actual detection. They have poor tolerance to methanol, a sample pretreatment reagent, which affects the antibody activity in actual detection. Their organic solvent tolerance and affinity need to be improved. Therefore, in order to meet the needs of actual detection, it is necessary to develop more antibodies that are more sensitive, have better affinity and stability and can be used for the detection of acrylamide, which is of great significance for actual production applications. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing anti-acrylamide derivative nanobodies and provide an affinity-matured anti-acrylamide derivative nanobody and its application.

[0006] The first object of the present invention is to provide an affinity-matured anti-acrylamide derivative nanobody mutant.

[0007] The second object of the present invention is to provide a gene encoding a Nanobody mutant against acrylamide derivatives.

[0008] The third aspect of the present invention is to provide the application of affinity-matured anti-acrylamide derivative nanobody mutants.

[0009] The fourth aspect of the present invention is to provide a recombinant expression vector.

[0010] The fifth aspect of the present invention is to provide a recombinant expression cell.

[0011] The sixth aspect of the present invention is to provide a method for detecting acrylamide.

[0012] The seventh aspect of the present invention is to provide a kit for detecting acrylamide derivatives.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0014] Based on the wild-type anti-acrylamide derivative nanobody 3G, the present invention conducts homology modeling and molecular docking to determine the key recognition sites of the nanobody and acrylamide derivatives, constructs an antibody mutation library using site-directed saturation mutagenesis technology, and further performs affinity maturation of the wild-type anti-acrylamide derivative nanobody 3G through phage display and biopanning technology, thereby obtaining a nanobody mutant 1C1 having a higher affinity for acrylamide derivatives than the wild-type nanobody 3G. The anti-acrylamide derivative nanobody mutant 1C1 is a wild-type nanobody 3G in which amino acids 26, 28, 30, 32, and 76 are mutated to Gly26, Arg28, Asp30, Trp32, and Pro76, respectively. The IC of the nanobody mutant 1C1 is 0. 50 The lowest detection limit was 0.052 μg / mL, and the linear range was 0.172-9.793 μg / mL. The nanobody mutant 1C1 could maintain 70% activity at a methanol concentration of 40%, which was 1 times higher than that of the wild type. It could specifically identify acrylamide derivatives, and its sensitivity was 55% higher than that of the wild type nanobody. It had better tolerance to methanol, a pretreatment reagent, and was more stable. When used in enzyme-linked immunosorbent assay, the test results were accurate and stable, which is of great significance for detecting the content of acrylamide in food.

[0015] Furthermore, the amino acid sequence of the wild-type anti-acrylamide derivative Nanobody 3G is shown in SEQ ID NO.3: EVQLEQSGGGPVQAGGSLRLSCTASEYTRINYCMGWVRQAPGKERERVAGFGTGGSTRYADSVKGRFTISQDNAKRTLYLEMNSLKPEDTAMYYC AARACWSNTYTYWGQGTLVTVSS.

[0016] Furthermore, the amino acid sequence of the anti-acrylamide derivative nanobody mutant 1C1 is shown in SEQ ID NO.1: EVQLEQSGGGPVQAGGSLRLSCTAS G Y R R D N W CMGWVRQAP GKERERVAGFGTGGSTRYADSVKGRFTISQDNAK P TLYLEMNSLKPEDTAMY YCAARACWSNTYTYWGQGTLVTVSS.

[0017] The present invention provides a gene encoding a nanobody mutant 1C1 against acrylamide derivatives, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0018] The present invention provides the use of the affinity-matured anti-acrylamide derivative nanobody mutant 1C1 in detecting acrylamide derivatives.

[0019] The present invention provides use of the affinity-matured anti-acrylamide derivative nanobody mutant 1C1 in preparing a product for detecting acrylamide derivatives.

[0020] The present invention also provides a recombinant expression vector containing a gene encoding a nanobody mutant 1C1 against acrylamide derivatives.

[0021] The present invention also provides a recombinant expression cell containing the above recombinant vector.

[0022] The present invention also provides the use of the recombinant expression vector or recombinant expression cell in acrylamide detection or in the preparation of an acrylamide detection kit.

[0023] The present invention also provides a method for detecting acrylamide, which uses affinity-matured anti-acrylamide derivative nanobody mutant 1C1 for detection.

[0024] Furthermore, enzyme-linked immunosorbent assay (ELISA) was used for detection.

[0025] Preferably, an indirect enzyme-linked immunosorbent assay is used.

[0026] More preferably, the affinity-matured anti-acrylamide derivative nanobody mutant 1C1 is used and the acrylamide derivative artificial antigen is used as the coating source for enzyme-linked immunosorbent assay.

[0027] Preferably, the acrylamide derivative artificial antigen is the full antigen XAA-309-OVA, which is prepared by coupling the hapten XAA-309 with ovalbumin OVA (albumin) through the active ester method.

[0028] Furthermore, the structural formula of hapten XAA-309 is:

[0029]

[0030] As the most preferred embodiment, the specific detection method is:

[0031] S1. Preparation of ELISA plates coated with complete antigens containing acrylamide derivatives;

[0032] S2. Derivatize the acrylamide standard with an excess of a derivatizing agent, add the derivative standard or the sample to be tested into the microwells of the ELISA plate, and then add the nanobody

[0033] S3. Add enzyme-labeled secondary antibody and incubate;

[0034] S4. Add color development solution and incubate;

[0035] S5. Add stop solution and measure;

[0036] S6. Using the log of drug standard concentration 10 The value is the horizontal axis, and the ratio of the absorbance value of each standard concentration to the absorbance value of the zero standard well is the vertical axis. A standard curve is established, and then the content of acrylamide in the sample to be tested is calculated according to the absorbance value of the sample to be tested.

[0037] The present invention also provides a kit for detecting acrylamide derivatives, comprising affinity-matured anti-acrylamide derivative nanobody mutants.

[0038] The present invention has the following beneficial effects:

[0039] The present invention provides an affinity-matured anti-acrylamide derivative nanobody mutant. Based on the wild-type anti-acrylamide derivative nanobody 3G, the present invention uses site-directed saturation mutagenesis technology to construct an antibody mutation library. A nanobody mutant 1C1 with a higher affinity for quinalphos than the wild-type nanobody is obtained by panning. The amino acid sequence of the mutant is shown in SEQ ID NO.1. The nanobody can specifically recognize acrylamide derivatives. Compared with the original wild-type nanobody (3G), the sensitivity is increased by 55%. The nanobody mutant 1C1 can maintain 70% of its activity at a 40% methanol concentration, which is 1 times higher than that of the wild type and has stronger stability. The nanobody provided by the present invention is used in an enzyme-linked immunosorbent assay method for detecting acrylamide. It can detect acrylamide with an IC50 of 0.81 μg / mL, which is 55% higher than that of the wild type. The minimum detection limit is 0.052 μg / mL and the linear range is 0.172-9.793 μg / mL. The method provided by the present invention has accurate detection results, good effect and high stability, and can be more widely used in the detection of acrylamide content in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the three-dimensional model of nanobody 3G;

[0041] Figure 2 Ramachandran plot of the homology modeling model for Nanobody 3G;

[0042] Figure 3 is the ERRAT map of the homology model of nanobody 3G;

[0043] Figure 4 This is the Verify3D map of the Nanobody 3G homology model;

[0044] Figure 5 The three-dimensional model (A) and two-dimensional diagram (B) of the interaction between nanobody 3G and acrylamide derivatives;

[0045] Figure 6 This is the saturation mutagenesis scheme for Nanobody 3G;

[0046] Figure 7 Identify positive mutant clones for ic-ELISA;

[0047] Figure 8 This is the standard curve of each mutant after phage display screening;

[0048] Figure 9 Standard curve for acrylamide detection of Nanobodies 1C1 and 3G;

[0049] Figure 10 Methanol tolerance curves of Nanobodies 1C1 and 3G. DETAILED DESCRIPTION

[0050] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0051] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0052] Example 1 Homology modeling and molecular docking of anti-acrylamide derivative nanobodies

[0053] 1. Homology modeling and model evaluation of anti-acrylamide derivative nanobodies

[0054] The amino acid sequence of the anti-acrylamide derivative nanobody 3G (SEQ ID NO. 3: EVQLEQ SGGGPVQAGGSLRLSCTASEYTRINYCMGWVRQAPGKERERVAGFGTGGST RYADSVKGRFTISQDNAKRTLYLEMNSLKPEDTAMYYCAARACWSNTYTY WGQGTLVTVSS) was uploaded to the AlphaFold online server. After screening, a model with the highest score was finally obtained. The three-dimensional model of nanobody 3G is shown in FIG. Figure 1 As shown, green represents FR regions, red represents CDR1 regions, blue represents CDR2 regions, and yellow represents CDR3 regions. To further verify the reliability of the model, the homology model was evaluated using the amino acid dihedral angles of the Ramachandran plot and the ERRAT and Verify 3D indicators.

[0055] Among them, the Pull-type diagram can be used to analyze whether the skeleton structure and conformation of the model meet the stability requirements of the structure. The results show the partitioning of the amino acids in the model, which is mainly divided into four regions: core region, additional allowed region, roughly allowed region and forbidden region. Figure 2 As shown, 99% of the amino acid residues fall in the core region and 100% of the amino acids are located in favorable positions. According to the Pull-type diagram scoring criteria (greater than 90% of the amino acids fall in the core region), the Nanobody 3G homology model can be considered reliable.

[0056] The ERRAT index evaluates the three-dimensional structure of the model through crystallography, indicating the number of non-bonded interactions (side chains) formed between pairs of different atomic types within a range of 0.35 nm. A theoretical score greater than 85 points indicates that the model is reliable, while the homology model of Nanobody 3G scored 96.154. Figure 3 As shown, it meets the requirements.

[0057] The Verify 3D module is used to score the compatibility of the model's three-dimensional structure with any amino acid sequence. The results are as follows: Figure 4 As shown, 96.61% of the amino acid residues in the homology model of Nanobody 3G have a 3D-1D score ≥ 0.1, which exceeds the 80% qualification line, indicating that the model is reliable.

[0058] In summary, the structural model finally obtained in this embodiment is reliable and can be used for subsequent research.

[0059] 2. Molecular docking of nanobody 3G and acrylamide derivative XAA

[0060] After the model evaluation confirmed its rationality, the three-dimensional structures of nanobody 3G and acrylamide derivative XAA were input into Lead IT software for molecular docking. After adding missing atoms, specifying protonation states and optimizing hydrogen bond parameters, the protein and small molecule were docked. Based on the docking results, the docking model with the lowest binding free energy, i.e., the highest score, was selected. Figure 5 shown.

[0061] By analyzing the docking results, it can be found that the interaction forces between nanobody 3G and XAA are mainly hydrogen bonds and hydrophobic interactions. The amino acid residue that participates in antigen recognition through hydrogen bonding is Thr28 located in the CDR1 region. Threonine forms a bond with the hydrogen atom in the imino group of XAA through the oxygen atom on its side chain. The amino acid residues involved in antigen recognition through hydrophobic interactions are Glu26, Tyr27, Thr28, Arg29, Ile30, Asn31, and Tyr32 in the CDR1 region, and Asn73 and Arg76 in the FR3 region. Alanine scanning was used to verify the key amino acid sites, and it was found that Thr27, Arg29, Asn31, and Asn73 are the key recognition sites for nanobody 3G and XAA.

[0062] Example 2 Construction of saturation mutation library

[0063] Based on the above molecular docking and alanine scanning results, site-directed saturation random mutagenesis was performed on the key amino acid sites Thr27, Arg29, Asn31, and Asn73 to construct a saturation mutation library.

[0064] At the same time, due to the antigen-antibody binding site Changes in amino acid residues within the range may have a potential impact on binding. In this example, site-directed saturation random mutagenesis was also performed on Glu26, Thr28, Ile30, Thr32, and Arg76 to construct a saturation mutation library of non-critical sites.

[0065] For the above 9 mutation points, two saturation mutation libraries were constructed respectively. Site-directed saturation mutagenesis was performed using overlap extension PCR and three rounds of amplification to obtain the complete antibody fragment. The mutation scheme is as follows: Figure 6 The primer sequences used are shown in Table 1 below. After the reaction, a small amount of PCR product was taken for agarose gel electrophoresis to verify whether the bands were correct and single.

[0066] Table 1 Saturation mutagenesis primers

[0067]

[0068] Subsequently, the antibody fragment and pComb3Xss plasmid were double-digested with the restriction endonuclease Sfi I at 50°C for 4 hours. The digested products were purified and ligated using T4 DNA Ligase at 22°C for 4 hours. After enzymatic ligation, the ligated products were electroporated into E. coli TG1. The saturation mutation library capacity was calculated by dilution plating, and randomly selected clones were sequenced to verify sequence diversity. Glycerol was added to the transformed library to a final concentration of 20%, aliquoted into 1.5 mL centrifuge tubes, and stored frozen at -80°C. This constituted the anti-acrylamide derivative nanobody saturation mutation library.

[0069] Take 1 mL of the mutant bacterial library and inoculate it into 200 mL of LB-Amp medium. Incubate at 37°C and 220 rpm / min until the OD 600 About 0.5. Add helper phage M13K07 at a multiplicity of infection of 20:1 and infect statically for 30 minutes. Then, add kanamycin after 1 hour at 37°C and 220 rpm / min and culture overnight. The next day, centrifuge at 12,000 rpm / min for 20 minutes, remove the supernatant, add 1 / 5 of 20% PEG-NaCl solution, and incubate on ice for 2 hours or at 4°C overnight. Subsequently, centrifuge at 12,000 rpm / min for 20 minutes and resuspend the phage in TBS to obtain the anti-acrylamide derivative nanobody phage display library. Pipette 10 μL to determine the titer of the antibody library, and store the rest at -80°C for later use.

[0070] Example 3 Screening and identification of anti-acrylamide derivative nanobody mutants

[0071] The complete antigen XAA-309-OVA was prepared by coupling the hapten XAA-309 (structural formula shown in Formula 1) synthesized in the early stage of the research laboratory of the present invention with ovalbumin OVA (albumin) through the active ester method.

[0072]

[0073] Acrylamide standards were derivatized using the derivatizing agent xanthanol under acidic conditions. The derivative product was appropriately diluted and used as an inhibitory drug for IC-ELISA detection. The structural formula of the acrylamide derivative is shown in Formula 2 below:

[0074]

[0075] The panning scheme for the two saturation mutation libraries constructed in Example 2 above is as follows:

[0076] Table 2: Site saturation mutation library screening scheme

[0077]

[0078] (1) Coating: Dilute the coating agent to 1 μg / mL with PBS. Coat 3 background-free wells (hemocyanin, bovine serum albumin, and chicken ovalbumin, 1 mg / mL, 100 μL / well) and the coating agent wells (100 μL / well) in each round and incubate overnight at 37°C. The next day, wash the plate twice with PBST buffer, add 150 μL of blocking solution to each well, and incubate at 37°C for 3 hours. Discard the blocking solution, dry at 37°C for 1 hour, and store in a refrigerator at 4°C for later use.

[0079] (2) Screening: Take 100 μL of the nanoantibody library and add it to the background-removed well and incubate at 37°C for 1 hour. Then transfer the liquid to the original coated well and incubate at 37°C for 1 hour. Discard the liquid in the original coated well, wash 5 times with PBST buffer and 15 times with PBS buffer. Use competitive elution, add 100 μL of gradient diluted acrylamide derivative solution, and incubate at 37°C for 1 hour. Take 10 μL of the eluted product to calculate the titer by counting the colonies on the plate, and the remaining eluted product is used for the next round of screening after auxiliary phage rescue amplification.

[0080] (3) Selection and identification of specific phage clones: 96 phage clones were randomly selected and inoculated into a deep-well plate containing 500 μL / well LB medium (Amp), and cultured at 37°C, 180 rpm, and shaken overnight. 20 μL of overnight bacteria were inoculated into a deep-well plate containing 1 mL / well medium (Amp), and cultured at 37°C, 180 rpm, and shaken for 3 h. A final concentration of 1 mM IPTG was added to each well, and cultured at 28°C, 180 rpm, and shaken overnight. The next day, centrifuged at 4500 rpm for 20 min, discarded the supernatant, and placed in a -80°C ultra-low temperature freezer for 3 h. After thawing at room temperature, the precipitate was resuspended in 200 μL PBS buffer, and the deep-well plate was shaken at 4°C for 1 h. Centrifuged at 4500 rpm for 20 min, and the supernatant was taken for ic-ELISA detection.

[0081] The specific steps of ic-ELISA testing are as follows:

[0082] 1) Coating: Dilute the coating agent XAA-309-OVA to 1 μg / mL with coating solution. Add 100 μL of the diluted coating agent to each well and incubate in a 37°C constant temperature water bath for 12-14 hours.

[0083] 2) Blocking: The next day, wash each well twice with PBST buffer solution, pat dry, add 150 μL of 3% skim milk powder to each well, and block in a 37°C water bath for 2 h. Pat dry and set aside.

[0084] 3) Incubation with primary antibody: Add 50 μL of culture supernatant and 50 μL of PBS buffer to each well (titer wells). Add 50 μL of supernatant and 50 μL of a 1 μg / mL acrylamide derivative solution to each well (inhibition wells). Incubate at 37°C for 40 min, then wash five times with PBST buffer and pat dry.

[0085] 4) Incubation with secondary antibody: Add 100 μL of rabbit anti-VHH-HRP secondary antibody (5000-fold dilution) to each well, incubate in a 37°C incubator for 40 min, wash five times with PBST buffer, and pat dry.

[0086] 5) Color development and termination: 100 μL of TMB two-component color development solution was added to each well, and the cells were incubated in a 37°C incubator for 10 min. 50 μL of 10% H 2 SO 4 was then added to each well to terminate the reaction.

[0087] 6) Reading: Read the absorbance at 450 nm using a microplate reader.

[0088] According to the ic-ELISA test results, the inhibition rate (I) was calculated using the following formula:

[0089] I(%)=(1-B / B0)×100

[0090] Where B0 is the absorbance value corresponding to the titer well, and B is the absorbance value corresponding to the inhibition well;

[0091] The results of ic-ELISA identification of positive mutant clones are as follows Figure 7 As shown in the figure, a total of 6 mutants (2A1, 1C1, 2H6, 1A1, 2E1, 2D1) with inhibition rates exceeding 60% were obtained. Subsequently, the different mutants were sent to a sequencing company for gene sequencing and comparative analysis of the amino acid sequences.

[0092] Example 4 Soluble expression and activity identification of mutants

[0093] The pComb3Xss-VHH plasmids of the six mutants obtained in Example 3 were extracted using a plasmid extraction kit and transformed into E. coli BL21 (DE3) competent cells by chemical transformation. Single clones were randomly selected and cultured in LB medium. Subsequently, the bacterial solution was inoculated into a large volume of LB medium at a ratio of 1:100 until the OD 600 When the pH value was 0.6-0.8, IPTG was added to a final concentration of 1 mM and expression was induced at 37°C, 220 rpm for 20 hours. The next day, the cells were harvested by centrifugation at 12,000 rpm / min. Periplasmic soluble proteins were extracted by sucrose osmotic pressure and purified by Ni column affinity chromatography to obtain soluble nanobodies.

[0094] An indirect competitive ELISA assay was established using XAA-309-OVA as the coating agent. The specific steps are as follows:

[0095] 1) Coating: Dilute the coating antigen XAA-309-OVA to 1 μg / mL with coating solution, add 100 μL / well to the wells of the ELISA plate, and incubate at 4°C overnight.

[0096] 2) Washing and Blocking: Wash twice with PBST on a plate washer, pat dry, add 150 μL / well of blocking solution, block in a 37°C incubator for 3 h, and then remove the liquid from the wells and pat dry.

[0097] 3) Sample loading: Add 50 μL / well of supernatant and 50 μL / well of PBS dilution to each well of the ELISA plate as the titer column. Add 50 μL / well of supernatant and 50 μL of serially diluted XAA to another well of the ELISA plate as the inhibition column. Incubate at 37°C for 40 min. Wash five times with PBST and pat dry.

[0098] 4) Add enzyme-labeled secondary antibody: Add 100 μL / well of rabbit anti-VHH-HRP (5000-fold dilution), incubate in a 37°C incubator for 30 min, wash 5 times with PBST, and pat dry.

[0099] 5) Color development and termination: Add 100 μL / well of TMB two-component color development solution, incubate at 37°C for 10 min, and terminate the reaction by adding 50 μL / well of stop solution. Measure the absorbance at 450 nm using a microplate reader.

[0100] The sensitivity of the 6 Nanobody mutants screened in Example 3 was tested, and the results were as follows: Figure 8As shown, mutant 1C1 has the best effect. The anti-acrylamide derivative nanobody mutant 1C1 is a wild-type nanobody 3G in which amino acids 26, 28, 30, 32, and 76 are mutated to Gly26, Arg28, Asp30, Trp32, and Pro76, respectively. Its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the nanobody mutant 1C1 is shown in SEQ ID NO.2.

[0101] The standard curves of nanobody 1C1 and nanobody 3G for detecting acrylamide are as follows Figure 9 As shown, the IC of Nanobody 1C1 is shown 50 It was 0.81 μg / mL, which was 55% higher than that of the wild-type nanobody 3G. The minimum detection limit of nanobody 1C1 was 0.052 μg / mL, and the linear range was 0.172-9.793 μg / mL. Finally, the affinity-matured anti-acrylamide derivative nanobody 1C1 was obtained.

[0102] Example 5 Verification of organic solvent tolerance of mutants

[0103] The nanobody mutant 1C1 and the wild-type nanobody 3G were diluted to working concentrations with different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70%) of methanol as a buffer solution, and the antigen-antibody binding ability was determined by ic-ELISA. The detection method was the same as in Example 2. The antigen binding ability of the nanobody not treated with organic solvent was used as a 100% control to evaluate the tolerance of different mutants to the organic solvent methanol.

[0104] The results are as follows Figure 10 As shown, the mutant 1C1 can still maintain more than 70% binding activity at a methanol concentration of 40%. Compared with the wild-type nanobody 3G, the tolerance of the nanobody mutant 1C1 to methanol is significantly improved.

[0105] In summary, the present invention, based on the anti-acrylamide derivative nanobody 3G, performs homology modeling and molecular docking to determine the key recognition sites of the nanobody and the acrylamide derivative XAA, constructs an antibody mutation library using site-directed saturation mutagenesis technology, and further performs affinity maturation of the anti-acrylamide derivative nanobody through phage display and biopanning technology, obtaining a mutant nanobody 1C1 with a higher affinity for quinalphos than the wild-type nanobody. Mutant 1C1 is a wild-type nanobody 3G in which amino acids 26, 28, 30, 32, and 76 are mutated to Gly26, Arg28, Asp30, Trp32, and Pro76, respectively. Its amino acid sequence is shown in SEQ ID NO.1. The nanobody can specifically recognize acrylamide derivatives and has an IC of 100% with that of the wild-type nanobody 3G.50 The sensitivity is 1.80 μg / mL, which is 55% higher than that of wild type. The tolerance to organic solvents is also significantly improved, which is nearly 1 times higher than that of wild type. The stability is stronger. The nanobody provided by the present invention is used in the enzyme-linked immunosorbent assay method for detecting acrylamide, which can detect acrylamide. Its IC 50 The detection limit of acrylamide in agricultural products was 0.81 μg / mL, which was 55% higher than that of the wild type. The minimum detection limit was 0.052 μg / mL and the linear range was 0.172-9.793 μg / mL. The detection results of this method were accurate, effective and stable, and it can be more widely used in the detection of acrylamide content in agricultural products.

[0106] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An affinity-matured anti-acrylamide derivative nanobody mutant 1C1, characterized in that: The anti-acrylamide derivative nanobody mutant 1C1 is a wild-type nanobody 3G in which amino acids 26, 28, 30, 32, and 76 are mutated to Gly26, Arg28, Asp30, Trp32, and Pro76, respectively; the amino acid sequence of the wild-type anti-acrylamide derivative nanobody is shown in SEQ ID NO.

3.

2. The Nanobody mutant 1C1 according to claim 1, characterized in that The amino acid sequence of the anti-acrylamide derivative nanobody mutant 1C1 is shown in SEQ ID NO.

1.

3. A gene encoding an anti-acrylamide derivative nanobody mutant 1C1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. Use of the affinity-matured anti-acrylamide derivative Nanobody mutant 1C1 according to claim 1 or 2 in detecting acrylamide.

5. Use of the affinity-matured anti-acrylamide derivative Nanobody mutant 1C1 according to claim 1 or 2 in the preparation of a product for detecting acrylamide.

6. A recombinant expression vector, characterized in that: Containing the gene according to claim 3.

7. A recombinant expression cell, characterized in that Containing the recombinant expression vector according to claim 6.

8. A method for detecting acrylamide, characterized in that: The detection is performed using the affinity-matured anti-acrylamide derivative nanobody mutant 1C1 according to claim 1 or 2.

9. The method according to claim 8, characterized in that Enzyme-linked immunosorbent assay was used for detection.

10. A kit for detecting acrylamide derivatives, characterized in that: Comprising the affinity matured anti-acrylamide derivative nanobody mutant 1C1 according to claim 1 or 2.

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