Affinity mature anti-acrylamide derivative nano antibody mutant and application thereof

Abstract

The invention discloses an anti-acrylamide derivative nano antibody mutant with mature affinity and application of the anti-acrylamide derivative nano antibody mutant. A saturated mutation library is constructed on the basis of a wild type anti-acrylamide derivative nano-antibody, an anti-acrylamide derivative nano-antibody mutant with mature affinity and remarkably improved performance is obtained through screening by a phage display method, the IC50 of the nano-antibody mutant is 0.81 mu g / mL and is improved by 55% compared with that of the wild type nano-antibody, and the anti-acrylamide derivative nano-antibody mutant has the advantages of being high in specificity, high in sensitivity and good in stability. The lowest detection limit is 0.052 [mu] g / mL, and the linear range is 0.172-9.793 [mu] g / mL; the nanometer antibody mutant can specifically recognize an acrylamide derivative, the tolerance to an organic solvent is also remarkably improved and is nearly doubled compared with that of a wild type, and the stability is higher; when being used for an enzyme-linked immunosorbent assay method for detecting acrylamide, the enzyme-linked immunosorbent assay method is accurate in detection result and high in stability, and has important significance for detecting the content of acrylamide in food.

Description

Technical Field

[0001] The invention belongs to the field of antibody engineering, and specifically relates to an affinity-matured anti-acrylamide derivative nano antibody mutant and application thereof. Background Art

[0002] Acrylamide is a colorless, odorless, white crystal with a chemical formula of C3H5NO. Reducing sugars in food react with free amino acids at high temperatures to form acrylamide, which is widely found in baked foods 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. At the same time, excessive intake of acrylamide can lead to abnormal development of the human reproductive system and a decrease in the number and quality of sperm. Acrylamide can also affect the human nervous system, interfere with the normal communication between nerve cells, and cause imbalances in the ion concentration inside the nervous system. Therefore, it is necessary to strengthen the detection of acrylamide content in food.

[0003] At present, the detection methods for acrylamide in food at home and abroad are mainly instrumental analysis methods such as gas chromatography-mass spectrometry and spectral analysis. Although instrumental analysis methods are accurate, they are expensive and not suitable for large-scale detection on site. In recent years, immunoassay methods have been widely used in the detection of toxic and harmful substances in food due to their simplicity, speed and high sensitivity. The core reagents of immunoassay methods are antibodies. Polyclonal antibodies and monoclonal antibodies are limited in their application and development in immunoassays due to their poor stability and low yield. Nanobodies refer to the variable regions of heavy chain antibodies that naturally lack light chains in camelids. They have a small molecular weight, only 1 / 10 of the size of traditional antibodies, and are currently the smallest antigen-binding fragments. Compared with traditional antibodies, nanobodies are more stable, have excellent thermal stability and tolerance to organic solvents, and can maintain good antigen binding ability after high temperature treatment and in a certain concentration of organic solvents. Because the domain of nanoantibodies is simple, it is easy to improve the affinity of antibodies through genetic engineering.

[0004] However, there are still 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 mutant of a Nanobody 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 nanoantibody 3G, the present invention conducts homology modeling and molecular docking to determine the key recognition sites of nanoantibodies and acrylamide derivatives, constructs an antibody mutation library using site-directed saturation mutagenesis technology, and further conducts affinity maturation of the wild-type anti-acrylamide derivative nanoantibody 3G through phage display and biopanning technology, thereby obtaining a nanoantibody mutant 1C1 with a higher affinity for acrylamide derivatives than the wild-type nanoantibody 3G. The anti-acrylamide derivative nanoantibody mutant 1C1 is a wild-type nanoantibody 3G in which the 26th, 28th, 30th, 32nd, and 76th amino acids are mutated to Gly26, Arg28, Asp30, Trp32, and Pro76, respectively. The IC value of nanoantibody mutant 1C1 is 2.34, which is 1.38, which is 1.71, which is 1.91, which is 1.37, which is 1.97, which is 1.98, which is 1.99, which is 2.01, which is 2.10, which is 1.22, which is 1.99, which is 2.11 50 The detection limit of the nanoantibody mutant 1C1 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 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 recognize acrylamide derivatives, with a sensitivity of 55% higher than that of the wild-type nanoantibody. It had better tolerance to the pretreatment reagent methanol and greater stability. When used in enzyme-linked immunosorbent assay, the detection results were accurate and stable, which is of great significance for detecting the content of acrylamide in food.

[0015] Further, the amino acid sequence of the wild-type anti-acrylamide derivative nanobody 3G is shown in SEQ ID NO.3: EVQLEQSGGGPVQAGGSLRLSCTASEYTRINYCMGWVRQAPGKERERVAGFGTGGSTRYADSVKGRFTISQDNAKRTLYLEMNSLKPEDTAMYYCAARACWSNTYTYWGQGTLVTVSS.

[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, and the nucleotide sequence thereof 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 the use of the affinity-matured anti-acrylamide derivative nanobody mutant 1C1 in the preparation of a product for detecting acrylamide derivatives.

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

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

[0022] The present invention also provides the use of the recombinant expression vector or the 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 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. Prepare an ELISA plate coated with a complete antigen containing an acrylamide derivative;

[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 developing solution and incubate;

[0035] S5. Add stop solution and measure;

[0036] S6. Based on the log of drug standard concentration 10 The value is taken as the horizontal axis, and the ratio of the absorbance value of each standard concentration to the absorbance value of the zero standard well is taken as the vertical axis to establish a standard curve, 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. On the basis of the wild-type anti-acrylamide derivative nanobody 3G, the present invention adopts the site-directed saturation mutagenesis technology to construct an antibody mutation library, and obtains a nanobody mutant 1C1 with a higher affinity for quinalphos than the wild-type nanobody by panning. The amino acid sequence of the mutant is shown in SEQ ID NO.1. The nanobody can specifically recognize acrylamide derivatives, and the sensitivity is increased by 55% compared with the original wild-type nanobody (3G). The nanobody mutant 1C1 can maintain 70% of the activity at a methanol concentration of 40%, which is 1 times higher than 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, and acrylamide can be detected, and its IC50 is 0.81 μg / mL, which is 55% higher than 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 effects, 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 is the three-dimensional model of nanobody 3G;

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

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

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

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

[0045] Figure 6 It is a 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] Fig. 9 Standard curve for acrylamide detection for Nanobodies 1C1 and 3G;

[0049] Fig.10 This is the methanol tolerance curve of Nanobodies 1C1 and 3G. DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. 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 are 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 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 region, red represents CDR1 region, blue represents CDR2 region, and yellow represents CDR3 region. In order to further verify the reliability of the model, the homology model was evaluated by the amino acid dihedral angle 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 areas: core area, additional allowed area, roughly allowed area and forbidden area. 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 homology model of the nanobody 3G 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 score of Nanobody 3G is 96.154, as shown in 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. According to the docking results, the docking model with the lowest binding free energy, i.e., the highest score, was selected, such as Figure 5 shown.

[0061] By analyzing the docking results, it can be found that the interaction between nanobody 3G and XAA is mainly hydrogen bonding and hydrophobic interaction. The amino acid residue involved in antigen recognition through hydrogen bonding is Thr28 located in the CDR1 region. Threonine forms a bond of 1.567 nm with the hydrogen atom in the imino group of XAA through the oxygen atom on its side chain. Hydrogen bonds. The amino acid residues involved in antigen recognition through hydrophobic forces 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 were the key recognition sites for nanobody 3G and XAA.

[0062] Example 2 Construction of saturation mutation library

[0063] According to 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 The changes of amino acid residues within the range may have a potential impact on the binding. In this example, Glu26, Thr28, Ile30, Thr32, and Arg76 were also subjected to site-directed saturation random mutagenesis to construct a saturation mutation library of non-critical sites.

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

[0066] Table 1 Saturation mutagenesis primers

[0067]

[0068] Subsequently, the antibody fragment and pComb3Xss plasmid were double digested with restriction endonuclease SfiⅠ, and the reaction conditions were: 50℃, 4h. The digested products were purified and connected by T4 DNALigase, and the reaction conditions were: 22℃, 4h. After enzyme connection, the ligation products were electrotransformed into E.coli TG1, and the saturated mutation library capacity was calculated by dilution plating, and clones were randomly selected for sequencing to verify sequence diversity. After adding glycerol to the transformed bacterial library to a final concentration of 20%, it was divided into 1.5mL centrifuge tubes and stored at -80℃, which is the anti-acrylamide derivative nanoantibody saturated mutation library.

[0069] Take 1 mL of the mutant bacterial library and inoculate it into 200 mL of LB-Amp medium. Cultivate at 37°C, 220 rpm / min until OD 600 About 0.5. Add helper phage M13K07 at a multiplicity of infection ratio of 20:1 for static infection for 30 minutes, then add kanamycin after 1 hour at 37°C, 220rpm / min, and culture overnight. The next day, centrifuge at 12000rpm / min for 20 minutes, take the supernatant, add 1 / 5 of 20% PEG-NaCl solution, incubate on ice for 2 hours or at 4°C overnight. Subsequently, centrifuge at 12000rpm / min for 20 minutes, resuspend the phage with TBS, and obtain the anti-acrylamide derivative nanoantibody phage display library. Take 10μL to measure the titer of the antibody library, and store the rest at -80°C for 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] The acrylamide standard was derivatized with the derivatizing agent xanthanol under acidic conditions, and the derivative product after the reaction 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 scheme for panning 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-removed 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 the plate colony count, 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 refrigerator for 3 h. After thawing at room temperature, the precipitate was resuspended with 200 μL PBS buffer, and the deep-well plate was placed at 4°C and shaken 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 detection are as follows:

[0082] 1) Plate coating: Dilute the coating agent XAA-309-OVA to 1 μg / mL with the 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 the liquid in the well, add 150 μL 3% skim milk powder to each well, and block in a 37°C constant temperature water bath for 2 hours. Pat dry and set aside.

[0084] 3) Incubate with primary antibody: Add 50 μL of culture supernatant and 50 μL of PBS buffer to each well, which is the titer well. Add 50 μL of supernatant and 50 μL of 1 μg / mL acrylamide derivative solution to each well, which is the inhibition well. Incubate in a 37°C incubator for 40 minutes, wash 5 times with PBST buffer and pat dry.

[0085] 4) Incubation with secondary antibody: Add 100 μL rabbit anti-VHH-HRP secondary antibody (5000-fold dilution) to each well, incubate in a 37°C incubator for 40 min, wash 5 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, incubated in a 37°C incubator for 10 min, and then 50 μL of 10% H2SO4 was added to each well to terminate the reaction.

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

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

[0089] I(%)=(1-B / B0)×100

[0090] In the formula, 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, 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 transferred into E. coli BL21 (DE3) competent cells by chemical transformation. Single clones were randomly selected for culture 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 The value was 0.6-0.8, and the inducer IPTG was added at a final concentration of 1mM, and the expression was induced at 37°C and 220rpm for 20h. The next day, the bacteria were collected by centrifugation at 12000rpm / min. The periplasmic soluble protein was extracted by sucrose osmotic pressure method, and the soluble nanobody was obtained after purification by Ni column affinity chromatography.

[0094] Using XAA-309-OVA as the coating source, an indirect competitive ELISA test was established. 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 into the wells of the ELISA plate, and incubate at 4°C overnight.

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

[0097] 3) Sample addition: Add 50 μL / well supernatant and 50 μL / well PBS dilution to the wells of the ELISA plate as the titer column; add 50 μL / well supernatant and 50 μL of gradient dilution of XAA drug to another well of the ELISA plate as the inhibition column. Incubate in a 37°C incubator for 40 min, wash 5 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 in a 37°C incubator for 10 min, add 50 μL / well of stop solution to terminate the reaction. Measure the absorbance at 450 nm with an ELISA 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 the 26th, 28th, 30th, 32nd, and 76th amino acids 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 shown in Fig. 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 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 nanobodies not treated with organic solvents was used as a 100% control to evaluate the tolerance of different mutants to the organic solvent methanol.

[0104] The results are as follows Fig.10 As shown, 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 nanobody mutant 1C1 to methanol is significantly improved.

[0105] In summary, the present invention, based on the anti-acrylamide derivative nanoantibody 3G, performs homology modeling and molecular docking, determines the key recognition sites of the nanoantibody 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 nanoantibody through phage display and biopanning technology, and obtains a mutant nanoantibody 1C1 having a higher affinity for quinalphos than the wild-type nanoantibody. The mutant 1C1 is a wild-type nanoantibody 3G in which the 26th, 28th, 30th, 32nd, and 76th amino acids are mutated to Gly26, Arg28, Asp30, Trp32, and Pro76, respectively, and its amino acid sequence is shown in SEQ ID NO.1. The nanoantibody can specifically recognize acrylamide derivatives, and has an IC of 1.177 with the wild-type nanoantibody 3G.50 The sensitivity is 1.80 μg / mL, which is 55% higher than that of the wild type. The tolerance to organic solvents is also significantly improved, which is nearly 1 times higher than that of the wild type, and the stability is stronger. The nanobody provided by the present invention is used in the enzyme-linked immunosorbent assay method for detecting acrylamide, and acrylamide can be detected. 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 equivalent replacement methods and are included in the protection scope 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 the 26th, 28th, 30th, 32nd and 76th amino acids 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 vector according to claim 6.

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

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 as claimed in claim 1 or 2.

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