Affinity-mature parathion-resistant nano antibody and application thereof

Abstract

The invention discloses an anti-parathion nano antibody with mature affinity and application of the anti-parathion nano antibody. According to the invention, mutation is carried out on the basis of an anti-parathion nano antibody Nb9 to obtain a nano antibody mutant H6 with higher affinity to parathion, and the amino acid sequence of the nano antibody mutant H6 is as shown in SEQ ID NO.1. The nano antibody can specifically recognize parathion pesticides, and compared with the nano antibody Nb9, the sensitivity is improved by 2.9 times, and the minimum detection limit is reduced by 2.6 times; meanwhile, compared with other positive mutants obtained through identification, the sensitivity is better, and the thermal stability and the tolerance to organic solvents are improved. When the fluorescent probe is further used for detecting parathion pesticide, the detected IC50 is 3.55 ng / mL, the lowest detection limit is 1.30 ng / mL, the linear range is 1.89-6.67 ng / mL, the detection result is accurate, the stability is good, and the fluorescent probe can be more widely applied to detection of parathion pesticide residues in agricultural products.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody engineering, and more specifically, relates to an affinity-matured anti-parathion nano-antibody and its application. Background Art

[0002] Parathion is a broad-spectrum, highly toxic organophosphate insecticide and acaricide with a chemical formula of C 10 H 14 NO5PS, the pure product is a light yellow liquid. Parathion has strong contact and stomach poisoning effects, has a certain fumigation effect, has no systemic effect, but has a strong penetration effect. Parathion can be used to control a variety of pests on crops such as rice, cotton and fruit trees, mainly controlling rice borers, cotton bollworms, corn borers, sorghum borers, etc. Parathion is a highly toxic insecticide that is not easily degraded in the environment. Parathion has been banned by the state. Parathion inhibits the activity of acetylcholinesterase, leading to a large accumulation of the neurotransmitter acetylcholine in the body, which in turn causes a series of neurological reactions, such as nausea, headache, weakness, chest tightness and other poisoning symptoms, and severe cases can lead to death. At present, residues are still detected in some vegetable samples, causing direct or potential serious harm to human health.

[0003] Immunoassay technology has been widely used in the field of food and environmental chemical pollution monitoring due to its advantages such as fast detection speed, simple operation, high sensitivity and low cost. At present, improving detection sensitivity has become the key research direction of immunoassay technology. In immunoassay technology, antibodies play a core role, and their performance is directly related to the sensitivity and accuracy of the detection method. Traditional antibodies are obtained through animal immunization, and their complex structure limits the application of in vitro affinity maturation. Nanobodies are a new generation of genetically engineered antibody fragments derived from heavy chain antibodies of camelids. In the process of studying nanoantibodies, they have shown excellent stability. They can maintain strong antigen binding ability under high temperature conditions and in an environment containing organic solvents. These characteristics are crucial for the application of antibodies because they make immunoassay technology based on nanoantibodies more able to meet the market's urgent demand for rapid detection.

[0004] However, there are still few nanoantibodies that can be used to detect parathion. The sensitivity, affinity and performance of existing nanoantibodies for parathion need to be improved (Zhang Yuqi. Preparation of nanoantibodies for parathion, establishment of immunoassay methods and research on molecular recognition mechanism [D]. South China Agricultural University, 2019.). In order to meet the needs of actual detection, it is particularly necessary to develop more and more sensitive nanoantibodies suitable for parathion detection, which has practical value and important significance in actual production and application. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the above-mentioned existing anti-parathion nanoantibodies and provide an affinity-matured anti-parathion nanoantibody and its application.

[0006] The first object of the present invention is to provide an anti-parathion nanobody.

[0007] The second object of the present invention is to provide a gene encoding an anti-parathion nanobody.

[0008] The third object of the present invention is to provide a recombinant expression vector.

[0009] The fourth object of the present invention is to provide a recombinant engineered bacterium or a recombinant expression cell.

[0010] A fifth object of the present invention is to provide the use of the nanobody, encoding gene, recombinant expression vector, recombinant engineered bacteria or recombinant expression cell.

[0011] The sixth object of the present invention is to provide a parathion detection product.

[0012] A seventh object of the present invention is to provide a method for detecting parathion.

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

[0014] The present invention provides an anti-parathion nano antibody, the amino acid sequence of which is shown in SEQ ID NO.1.

[0015] Based on the existing anti-parathion nanoantibody Nb9, the present invention analyzes the base sequence of the anti-parathion Nb9 nanoantibody according to the in vivo somatic hypermutation hotspots AGY and RGYW, determines the mutation hotspots, introduces mutations at the amino acid sites involved in the hotspots, constructs an antibody mutation library using site-directed saturation mutagenesis technology, and further performs affinity maturation of the anti-parathion nanoantibody through phage display and bio-panning technology, thereby obtaining a nanoantibody H6 with higher affinity for parathion than the original nanoantibody, which can specifically identify parathion pesticides, has a sensitivity increased by 2.9 times compared to the original nanoantibody Nb9, and a minimum detection limit reduced by 2.6 times. At the same time, it is also better than other positive mutation nanoantibodies identified in terms of sensitivity, and its thermal stability and tolerance to organic solvents are improved.

[0016] The present invention provides a gene encoding an anti-parathion nanobody, and the nucleotide sequence thereof is shown in SEQ ID NO.2.

[0017] The invention provides a recombinant expression vector containing a gene encoding an anti-parathion nano antibody.

[0018] The present invention provides a recombinant engineering bacterium or a recombinant expression cell, which contains the above-mentioned recombinant vector.

[0019] The present invention provides the use of the above-mentioned nano-antibody, encoding gene, recombinant expression vector, recombinant engineering bacteria or recombinant expression cell in detecting parathion.

[0020] The present invention provides the use of the above-mentioned nano-antibody, encoding gene, recombinant expression vector, recombinant engineering bacteria or recombinant expression cell in the preparation of a product for detecting parathion.

[0021] The invention provides a parathion detection product, which contains a parathion nano antibody, a coding gene, a recombinant expression vector, or a recombinant engineering bacterium or a recombinant expression cell.

[0022] The nanobody provided by the present invention is further used in an enzyme-linked immunosorbent assay method to detect parathion pesticides, which can detect parathion pesticides more sensitively and specifically. 50 The detection limit is 3.55 ng / mL, the minimum detection limit is 1.30 ng / mL, and the linear range is 1.89-6.67 ng / mL. The detection results are accurate, effective, and stable, and can be more widely used in the detection of parathion pesticide residues in agricultural products.

[0023] Therefore, the present invention provides a method for detecting parathion, which uses affinity-matured anti-parathion nanoantibodies, or the above-mentioned products for detection.

[0024] Furthermore, the method adopts enzyme-linked immunosorbent assay for detection.

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

[0026] More preferably, the method uses parathion artificial antigen as coating source to perform enzyme-linked immunosorbent assay; the parathion artificial antigen is H1-OVA, and its structural formula is:

[0027]

[0028] As a more preferred embodiment, the present invention provides a specific detection method for parathion:

[0029] S1. Prepare the coating: ELISA plate containing complete parathion pesticide antigen (H1-OVA);

[0030] S2. Add the parathion pesticide standard or the sample to be tested into the microwells of the ELISA plate, and then add the nanobody H6;

[0031] S3. Add enzyme-labeled secondary antibody and incubate;

[0032] S4. Add color developing solution and incubate;

[0033] S5. Add stop solution and measure;

[0034] 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 hole is taken as the vertical axis to establish a standard curve, and then the content of parathion pesticide in the sample to be tested is calculated according to the absorbance value of the sample to be tested.

[0035] The present invention has the following beneficial effects:

[0036] The present invention mutates the existing anti-parathion nanoantibody Nb9 to obtain a nanoantibody mutant H6 with a higher affinity for parathion than the original nanoantibody Nb9, and its amino acid sequence is shown in SEQ ID NO.1. The nanoantibody can specifically recognize parathion pesticides, and its sensitivity is 2.9 times higher than that of nanoantibody Nb9, and the minimum detection limit is reduced by 2.6 times. It is also better than other positive mutant nanoantibodies identified, and its thermal stability and tolerance to organic solvents are improved. The nanoantibody provided by the present invention is further used in an enzyme-linked immunosorbent assay method to detect parathion pesticides, and its detection IC 50 The detection limit is 3.55 ng / mL, the minimum detection limit is 1.30 ng / mL, and the linear range is 1.89-6.67 ng / mL. It has better sensitivity, accurate results and good stability for the detection of parathion, and can be more widely used in the detection of parathion pesticide residues in agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a mutation hotspot located in the CDR region of nanobody Nb9.

[0038] Figure 2 Specific amplification protocol for hotspot mutation libraries.

[0039] Figure 3 Agarose gel electrophoresis plots for hotspot mutation library construction.

[0040] Figure 4 This is the result of ic-ELISA to identify positive mutant clones.

[0041] Figure 5 This is the sequence alignment result of the mutants.

[0042] Figure 6 The figure shows the results of SDS-PAGE analysis of the purified mutant.

[0043] Figure 7 The standard curve for the detection of parathion by nanobody.

[0044] Figure 8 This is a graph showing the stability test results of the nanobody.

[0045] Fig. 9 This is a graph showing the organic tolerance test results of the nanobody. DETAILED DESCRIPTION

[0046] 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.

[0047] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0048] The amino acid sequence of the original anti-parathion Nanobody Nb9 in the embodiment is: EVQLLQSGGDSVQAGGSLRLSCVGSLYSYCISAVSWYRQAPGKEREFVSWIH RDGTTSYADSVKGRFTISQDQPKNTVYLRMNSLKPEDTAMYYCKAETLPKFG RACRNADYWGQGTQVTVSS.

[0049] Example 1 Design and construction of nanobody hotspot mutation library

[0050] Based on the parathion nanoantibody Nb9 previously studied and disclosed by the applicant team (Zhang Yuqi. Preparation of parathion nanoantibodies, establishment of immunoassay methods and study of molecular recognition mechanisms [D]. South China Agricultural University, 2019.), according to the in vivo somatic hypermutation hotspots AGY and RGYW (Y = C or T; R = A or G; W = A or T), the base sequence of the anti-parathion nanoantibody Nb9 was analyzed and the mutated amino acid sites were determined to be Ser28, Tyr29, Ser32, Lys102, Phe103, Arg105, Ala106. Based on the degenerate codon NNK (a total of 32 codon combinations, which can encode 20 amino acids), random mutations were introduced at the above amino acid sites to construct a mutation library, and the phagemid vector was constructed by overlap extension PCR and electroporated into E. coli TG1 for library construction.

[0051] The mutation hotspots located in the CDR region of the nanobody Nb9 are as follows Figure 1 As shown in Table 1, primers for introducing designed mutations into the CDR1 and CDR3 hotspot regions of Nb9 are used. A total of three rounds of PCR amplification are required to synthesize the full-length nanobody fragment. The specific amplification and construction scheme of the hotspot mutation library is as follows: Figure 2The first round of PCR amplification obtained three short DNA fragments, namely FR1-CDR1, FR2-FR3 and CDR3-FR4. The second round of PCR amplification obtained a DNA fragment of FR1-FR3. The third round of PCR amplification obtained a full-length nano antibody fragment. The PCR reaction results are shown in Figure 3 express.

[0052] Table 1 Primer sequences for hotspot mutation library

[0053]

[0054] Note: The italics in the table indicate mutation sites, where N = A / C / G / T, K = G / T.

[0055] The final obtained nanobody full-length gene fragment and the pComb3xss empty vector obtained by plasmid extraction were double-digested using restriction endonuclease Sfi I, and the Nb9 mutant fragment recovered after enzyme digestion and the pComb3xss fragment were connected by T4 DNALigase at a molar ratio of 3:1, and the constructed connection product was transformed into E.coli TG1 electrotransformation competent state. The number of colonies on the plate was counted the next day and the size of the gene library was calculated according to the dilution multiple. At the same time, 10 single clones were picked from the library capacity determination plate for DNA sequencing, the correctness and diversity of the inserted sequence were analyzed, and the diversity of the library was calculated. The clones on the culture medium were scraped off with LB culture medium, glycerol was added to adjust the concentration to 20%, and then divided into 2mL centrifuge tubes and frozen at -80°C, which is the Nb9 hot spot saturated mutant gene bacterial library resistant to parathion pesticides.

[0056] The bacterial solution after electroporation and resuscitation for 1 hour was expanded into 2×YT-Amp medium at a ratio of 1:100 and cultured at 37°C with shaking at 250 rpm until the logarithmic phase (OD 600 =0.4-0.6). Add 1 mL of helper phage M13K07, let stand in a 37°C incubator for 30 min, culture at 37°C, 250 rpm for 2 h, add kanamycin solution to a final concentration of 70 μg / mL, and culture overnight at 37°C, 250 rpm. The next day, transfer the culture solution to a 250 mL sterile centrifuge bottle and centrifuge at 4°C, 12 000 rpm for 20 min, transfer the supernatant to a new 250 mL sterile centrifuge bottle, add 1 / 5 volume of 5×PEG / NaCl solution, mix upside down, incubate on ice for 3 h, centrifuge at 4°C, 12 000 rpm for 20 min, discard the supernatant, add 1 mL of sterile PBS to resuspend the phage precipitate, and filter with a 0.22 μm filter membrane to obtain a phage display nanoantibody mutation library against parathion pesticides. Take 10 μL to measure the titer of the antibody library, and store the rest at -80°C for use.

[0057] Example 2 Bioaffinity screening and identification of anti-parathion nanobodies

[0058] 1. Screening

[0059] The complete antigen H1-OVA was prepared by coupling the hapten H1 synthesized earlier by the applicant's team with ovalbumin OVA (albumin) through the active ester method. H1-OVA was used as the coating source to perform affinity panning on the nanoantibody mutation library established in Example 1 for a total of 4 rounds. The panning scheme is shown in Table 2 below.

[0060] Table 2: Screening scheme for hotspot saturation mutation library

[0061]

[0062] The specific screening steps are:

[0063] (1) Coating: HI-OVA was used to coat the original, 3 wells were coated in each round, 100 μL per well, and another 3 wells were coated with 1 mg / mL OVA, 100 μL per well for negative panning, and coated overnight at 4°C. The next day, the liquid in the wells was shaken off, and the wells were washed twice with an automatic plate washer and patted dry. 200 μL of blocking solution was added to each well, and the blocking reaction was carried out at 37°C for 3 h. The liquid in the wells was discarded, patted dry, and placed in a 37°C oven upside down for 1 h before use.

[0064] (2) Panning: To eliminate non-specific binding of the phage library, first take 100 μL of the above-mentioned nanoantibody mutation library solution and place it in the negative panning well. After reacting at room temperature at 37°C for 1 hour, transfer it to the antigen well and incubate it at room temperature with micro-oscillation for 1 hour. Discard the solution in the well, wash the plate with 300 μL / well of sterile PBST, then wash it several times with 300 μL / well of sterile PBS and pat it dry for later use. In order to screen antibodies with higher sensitivity, drug competition elution was used in all four rounds, that is, 100 μL of gradient diluted parathion standard solution was added, incubated at 37°C with micro-oscillation for 1.5 hours, and then aspirated and collected for storage. Take 10 μL of the eluted product to calculate the titer, and the remaining eluted products were amplified and put into the next round of panning.

[0065] (3) Identification of specific phage monoclones: 96 monoclones were randomly picked from the titer measurement plates of the eluted products from the four rounds of panning, inoculated into deep-well plates containing 600 μL / well LB-Amp medium, and cultured overnight at 37°C and 150 rpm. The next day, 10 μL of the above seed culture solution was inoculated into a deep-well plate containing 1 mL / well 2×YT-Amp medium, and cultured at 37°C and 150 rpm until the logarithmic phase, which was about 3-4 hours. 10 μL IPTG (final concentration of 1 mM) was added to each well, and the expression was induced at 37°C and 150 rpm overnight. The next day, the above deep-well plate was centrifuged at 16°C and 4400 rpm for 20 minutes, the supernatant was discarded, 200 μL PBS was added to each well for re-suspending, and the plate was frozen at -80°C for 3 hours, then thawed and centrifuged. The supernatant was taken for ic-ELISA to identify positive phage clones. The identification results of positive clones are as follows: Figure 4 As shown, multiple positive phage clones were identified and named F1, G7, H5, H6, H2, D3, and B1.

[0066] 2. Identification

[0067] Subsequently, ic-ELISA was used to identify positive phage clones. The specific detection steps are as follows:

[0068] (1) Coating: Dilute the coating antigen H1-OVA to 1 μg / mL with coating solution, add 100 μL / well to a 96-well ELISA plate, and incubate at 4°C overnight.

[0069] (2) Washing and blocking: Wash twice with PBST in a plate washer and pat dry. Add 200 μL of blocking solution to each well and place in a 37°C incubator for 3 h. Remove the liquid from the well and pat dry.

[0070] (3) Sample addition: Add 50 μL / well of supernatant and 50 μL / well of PBS diluent to each well of the ELISA plate as the titer column; add 50 μL / well of supernatant and 50 μL / well of 100 ng / mL parathion to another well of the ELISA plate as the inhibition column. Incubate at 37°C for 40 min, wash 5 times with PBST, and pat dry.

[0071] (4) Adding secondary antibody: add 100 μL / well of rabbit anti-VHH-HRP (5000-fold dilution, purchased from Nanjing GenScript Biotechnology Co., Ltd.), incubate in a 37°C incubator for 30 min, wash 5 times with PBST, and pat dry.

[0072] (5) Add 100 μL / well of TMB two-component colorimetric solution and incubate at 37°C for 10 min. Add 50 μL / well of stop solution to terminate the reaction and measure the absorbance at 450 nm using a microplate reader.

[0073] According to the ic-ELISA data, the binding ability of the antibody expressed in the deep-well plate to the coated antigen was analyzed and the positive clones were sent to the sequencing company for sequencing. Then the sequences were compared and analyzed using software. Finally, multiple positive clone mutants were identified, and the comparison results of some of their sequence analysis are shown below. Figure 5 As shown, the amino acid sequence of the mutant H6 involved is shown in SEQ ID NO.1, and the nucleotide sequence encoding the nano antibody is shown in SEQ ID NO.2; the amino acid sequence of the mutant F1 is shown in SEQ ID NO.3, and the nucleotide sequence encoding the nano antibody is shown in SEQ ID NO.4; the amino acid sequence of the mutant D3 is shown in SEQ ID NO.5, and the nucleotide sequence encoding the nano antibody is shown in SEQ ID NO.6.

[0074] Example 3 Expression, purification and activity identification of mutants

[0075] 1. Expression and purification of mutants

[0076] The frozen E. coli of the mutant identified as a positive clone in the early stage was thawed, and 5 μL of the bacterial solution was drawn for streaking on the LB-Amp plate, and inverted and cultured overnight in a 37°C incubator. The next day, a single clone from the plate was picked and placed in 10 mL of LB-Amp liquid culture medium, and cultured overnight at 37°C with shaking at 250 rpm. The pComb3xss-VHH plasmid in the overnight bacteria was extracted using the plasmid mini-extraction reagent of Novezan Co., Ltd., and transformed into E. coli BL21 (DE3) by heat shock for expression. The periplasmic cavity protein was extracted by osmotic shock method, and affinity chromatography was performed through Ni-NTA filler for purification. The purified protein solution was collected, and the mutant expression and purification were identified by SDS-PAGE. The protein electrophoresis results are shown in the figure. Figure 6 As shown, each mutant was successfully expressed.

[0077] 2. Identification of mutant activity

[0078] Using H1-OVA as the coating source, an indirect competitive ELISA test was established. The specific steps are as follows:

[0079] (1) Coating: Dilute the coating source H1-OVA to 1 μg / mL with PBS, add 100 μL per well to a 96-well ELISA plate, and incubate at 4°C overnight.

[0080] (2) Blocking: The next day, wash the plate twice with an automatic plate washer and pat dry. Use 3% skim milk powder as the blocking solution, add 200 μL per well, incubate in a 37°C constant temperature incubator for 3 h, pat dry, place upside down in a 37°C oven for 2 h, and store in a 4°C refrigerator for later use.

[0081] (3) Incubation with primary antibody: Nanoantibodies Nb9, H6, F1, and D3 were diluted to working concentrations and added to the wells at a volume of 50 μL per well. 50 μL of gradient diluted drugs were added and the plates were incubated at 37°C in a constant temperature incubator for 40 min. The plates were then washed 5 times with a plate washer and patted dry.

[0082] (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 constant temperature incubator for 30 min, then remove and wash the plate 5 times with an automatic plate washer and pat dry.

[0083] (5) Color development and termination: Add 100 μL of premixed TMB two-component color development solution to each well, incubate at 37°C for 10 min, then remove from the well and add 50 μL of 10% sulfuric acid to each well to terminate the reaction.

[0084] (5) Reading: Measure the absorbance at 450 nm for curve plotting, using IC 50 As an evaluation standard for antibody sensitivity.

[0085] The sensitivity of the screened nano-antibodies to parathion pesticide was tested, and the test results are as follows: Figure 7 The IC of nanobody H6 50 It is 3.55ng / mL, which is 2.9 times higher than that of nanoantibody Nb9, the minimum detection limit is 1.30ng / mL, which is 2.6 times lower than that of nanoantibody Nb9, and the linear range is 1.89-6.67ng / mL; its effect is significantly better than nanoantibodies F1 and D3, and finally the nanoantibody H6 with higher affinity for parathion than the original nanoantibody is obtained.

[0086] 3. Specificity detection

[0087] At the same time, several other organophosphorus pesticides such as quinalphos, coumaphos and triazophos were selected as cross-drugs, and the recognition specificity of the nanoantibody was analyzed using the above IC-ELISA method to calculate the IC of each cross-drug 50 The cross-reaction rate is calculated as CR (%) = IC 50 (Parathion) / IC 50 (parathion analogue).

[0088] The results of the cross-reaction rate detection of different mutants with parathion structural analogues are shown in Table 3. The results show that the cross-reaction rate of nanoantibody H6 to coumaphos, triazophos and quinalphos is lower than that of nanoantibodies F1 and D3. The cross-reaction rates to triazophos, quinalphos and coumaphos are 8.1%, 5.4% and 11.8%, respectively. The cross-reaction rates with other structural analogues are all less than 1.0%, which has good specificity and meets the detection requirements of parathion residues.

[0089] Table 3 Specificity of mutant-based ic-ELISA method for detecting organophosphorus pesticides

[0090]

[0091] Example 4 Analysis of thermal stability and organic tolerance of mutants

[0092] 1. Thermal stability

[0093] The performance of nanoantibodies H6, F1, D3 and Nb9 was analyzed. The specific method was as follows: the nanoantibodies were diluted to the working concentration and then divided into 7 equal parts, placed in a PCR instrument, and incubated for 10 minutes in different temperature environments (4°C, 20°C, 40°C, 60°C, 75°C, 95°C). The binding ability of the nanoantibodies without heat treatment to the antigen was taken as 100% for control. After the different temperature treatment groups returned to room temperature, the ic-ELISA method was used to evaluate the binding activity of the antigen and antibody, and its thermal stability was analyzed.

[0094] The results are as follows Figure 8 As shown, the thermal stability of nanobody H6 is improved compared with Nb9, and is significantly better than that of nanobodies F1 and D3.

[0095] 2. Organic tolerance

[0096] The performance of nanoantibodies H6, F1, D3 and Nb9 was analyzed. The specific method was: using methanol solutions of different concentrations such as 10%, 20%, 30%, 40%, 50%, 60%, 70% as diluents, the nanoantibodies were diluted to the working concentration, and the antigen-antibody binding ability was determined by the ic-ELISA method. Nanoantibodies that had not been treated with organic solvents were used as controls with an antigen binding ability of 100%, and the tolerance of different nanoantibodies to methanol was evaluated.

[0097] The results are as follows Fig. 9 As shown, the organic tolerance of Nanobody H6 is improved compared with Nb9, and is significantly better than the effects of Nanobodies F1 and D3.

[0098] In summary, based on the anti-parathion nanoantibody Nb9, the present invention analyzes the base sequence of the anti-parathion Nb9 nanoantibody according to the in vivo somatic hypermutation hotspots AGY and RGYW, determines the mutation hotspots, introduces mutations at the amino acid sites involved in the hotspots, constructs an antibody mutation library using site-directed saturation mutagenesis technology, and further uses phage display and bio-panning technology to perform affinity maturation of the anti-parathion nanoantibody, thereby obtaining a nanoantibody H6 with a higher affinity for parathion than the original nanoantibody, and its amino acid sequence is shown in SEQ ID NO. 1. Nanoantibody H6 can specifically recognize parathion pesticides, and its sensitivity is 2.9 times higher than that of the original nanoantibody Nb9, and the minimum detection limit is reduced by 2.6 times. It is also better than other positive mutation nanoantibodies identified, and its thermal stability and tolerance to organic solvents are improved.

[0099] The nanobody H6 provided by the present invention is used in the enzyme-linked immunosorbent assay method for parathion pesticides, which can detect parathion pesticides more sensitively and specifically. 50 The detection limit is 3.55 ng / mL, the minimum detection limit is 1.30 ng / mL, and the linear range is 1.89-6.67 ng / mL. The detection results are accurate, effective, and stable, and can be more widely used in the detection of parathion pesticide residues in agricultural products.

[0100] 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 anti-parathion nanobody, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the anti-parathion nanobody according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that: Containing the gene according to claim 2.

4. A recombinant engineered bacterium or a recombinant expression cell, characterized in that: Containing the recombinant vector according to claim 3.

5. Use of the nanobody according to claim 1, the encoding gene according to claim 2, the recombinant expression vector according to claim 3, or the recombinant engineered bacteria or recombinant expression cells according to claim 4 in detecting parathion.

6. Use of the nanobody according to claim 1, the encoding gene according to claim 2, the recombinant expression vector according to claim 3, or the recombinant engineered bacteria or recombinant expression cells according to claim 4 in the preparation of a product for detecting parathion.

7. A parathion detection product, characterized in that: Containing the nanobody according to claim 1, the encoding gene according to claim 2, the recombinant expression vector according to claim 3, or the recombinant engineered bacteria or recombinant expression cells according to claim 4.

8. A method for detecting parathion, characterized in that: The nanobody according to claim 1 or the product according to claim 7 is used for detection.

9. The method according to claim 8, characterized in that: The method adopts enzyme-linked immunosorbent assay for detection.

10. The method according to claim 9, characterized in that: The method uses parathion artificial antigen as coating source to perform enzyme-linked immunosorbent assay; the parathion artificial antigen is H1-OVA, and its structural formula is:

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