Affinity-matured anti-parathion nanobody and its application

Abstract

The present invention discloses an affinity-matured anti-parathion nanobody and its application. The present invention performs mutation on the anti-parathion nanobody Nb9 to obtain a nanobody mutant H6 with higher affinity for parathion, the amino acid sequence of which is shown in SEQ ID NO.1. The nanobody can specifically recognize parathion pesticides, and its sensitivity is 2.9 times higher than that of nanobody Nb9, and the minimum detection limit is reduced by 2.6 times; it is also better than the sensitivity of other positive mutants identified, and its thermal stability and tolerance to organic solvents are improved. It is further used 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. The detection results are accurate and stable, and it can be more widely used in the detection of parathion pesticide residues in agricultural products.

Description

Technical Field

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

[0002] Parathion is a broad-spectrum, highly toxic organophosphate insecticide and acaricide with the chemical formula C 10 H 14 NO5PS, the pure product is a light yellow liquid. Parathion has strong contact and stomach poisoning effects, a certain fumigation effect, no systemic effect, but 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 stem 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 in severe cases, death. Residues are still detected in some vegetable samples, posing a direct or potential serious threat 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. Currently, improving detection sensitivity has become a key research direction in immunoassay technology. Antibodies play a core role in immunoassay technology, 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. During the research process of nanobodies, they have demonstrated remarkable stability and can maintain strong antigen-binding ability under high temperature conditions and in environments containing organic solvents. These characteristics are crucial for the application of antibodies, as they make nanoantibody-based immunoassay technology more able to meet the urgent market demand for rapid detection.

[0004] However, there are currently few nanoantibodies that can be used to detect parathion, and the sensitivity, affinity and performance of existing nanoantibodies to parathion need to be improved (Zhang Yuqi. Preparation of nanoantibodies to parathion, establishment of immunoassay methods and research on molecular recognition mechanisms [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 applications of the nanobody, encoding gene, recombinant expression vector, recombinant engineered bacteria or recombinant expression cells.

[0011] A 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 nanobody, 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 nanoantibody Nb9 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 biopanning technology, thereby obtaining a nanoantibody H6 with higher affinity for parathion than the original nanoantibody, which can specifically recognize parathion pesticides. Compared with the original nanoantibody Nb9, the sensitivity is increased by 2.9 times, and the minimum detection limit is reduced by 2.6 times. At the same time, it is also better than other positive mutation nanoantibodies identified, and its thermal stability and tolerance to organic solvents are improved.

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

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

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

[0019] The present invention provides the use of the above-mentioned nanobody, encoding gene, recombinant expression vector, recombinant engineered 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 engineered 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 engineered 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 the coating source for 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 coated plate containing the complete parathion pesticide antigen (H1-OVA).

[0030] S2. Add the parathion pesticide standard or the test sample to 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. 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 to establish a standard curve, and then calculate the content of parathion pesticide in the sample to be tested 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. Its amino acid sequence is shown in SEQ ID NO.1. The nanoantibody can specifically recognize parathion pesticides. Compared with the nanoantibody Nb9, the sensitivity is increased by 2.9 times and the minimum detection limit is reduced by 2.6 times. It is also more sensitive than other positive mutant nanoantibodies identified. Its thermal stability and tolerance to organic solvents are also improved. The nanoantibody provided by the present invention is further used in an enzyme-linked immunosorbent assay method to detect parathion pesticides. The IC 50 The minimum 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 the nanobody Nb9.

[0038] Figure 2 Develop a specific amplification protocol for hotspot mutation libraries.

[0039] Figure 3 Agarose gel electrophoresis diagram of hotspot mutation library construction.

[0040] Figure 4 The figure shows the results of ic-ELISA identification of 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] Figure 9 This is a graph showing the organic tolerance test results of nanobodies. DETAILED DESCRIPTION

[0046] 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.

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

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

[0049] Example 1 Design and Construction of Nanobody Hotspot Mutation Library

[0050] Based on the previously published parathion nanoantibody Nb9 (Zhang Yuqi. Preparation of parathion nanoantibodies, establishment of immunoassay methods, and study of molecular recognition mechanisms [D]. South China Agricultural University, 2019), the base sequence of the anti-parathion nanoantibody Nb9 was analyzed based on the in vivo somatic hypermutation hotspots AGY and RGYW (Y = C or T; R = A or G; W = A or T). The mutated amino acid sites were identified as Ser28, Tyr29, Ser32, Lys102, Phe103, Arg105, and Ala106. Based on the degenerate codon NNK (a total of 32 codon combinations, encoding 20 amino acids), random mutations were introduced at the above amino acid sites to construct a mutation library. A 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 2As shown. The 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 the full-length nanoantibody fragment. The PCR reaction results are shown as follows. Figure 3 express.

[0052] Table 1 Primer sequences for hotspot mutation library

[0053]

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

[0055] The resulting full-length nanobody gene fragment and the pComb3xss empty vector obtained from the plasmid extraction were double-digested using the restriction endonuclease Sfi I. The Nb9 mutant fragment recovered from the digestion and the pComb3xss fragment were ligated using T4 DNA Ligase at a molar ratio of 3:1. The resulting ligation product was transformed into E. coli TG1 electrocompetent cells. The next day, the number of colonies on the plates was counted, and the size of the gene library was calculated based on the dilution factor. Ten single clones were selected from the library capacity measurement plates for DNA sequencing to analyze the correctness and diversity of the inserted sequences and calculate the diversity of the library. Colonies on the culture medium were scraped off with LB medium, adjusted to 20% glycerol, and aliquoted into 2 mL centrifuge tubes. The cells were then frozen at -80°C to create the parathion-resistant Nb9 hotspot saturation mutant gene library.

[0056] The bacterial suspension after electroporation and recovery 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). 1 mL of helper phage M13K07 was added, and the culture was allowed to stand in a 37°C incubator for 30 min. The culture was then incubated at 37°C, 250 rpm, for 2 h. Kanamycin solution was added to a final concentration of 70 μg / mL, and the culture was shaken at 37°C, 250 rpm, and incubated overnight. The next day, the culture was transferred to a 250 mL sterile centrifuge bottle and centrifuged at 4°C, 12,000 rpm, for 20 min. The supernatant was transferred to a new 250 mL sterile centrifuge bottle, and 1 / 5 volume of 5× PEG / NaCl solution was added. The culture was mixed by inversion and incubated on ice for 3 h. The precipitate was centrifuged at 4°C, 12,000 rpm, and 20 min. The supernatant was discarded, and the phage pellet was resuspended in 1 mL of sterile PBS. The precipitate was filtered through a 0.22 μm filter to obtain a phage-displayed nanoantibody mutant library against parathion pesticides. 10 μL of the library was aspirated to determine the titer of the antibody library, and the remainder was stored at -80°C for later 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 team with ovalbumin OVA (albumin) through the active ester method. H1-OVA was used as the coating source to perform affinity panning on the nanobody 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 plan for hotspot saturation mutation library

[0061]

[0062] The specific screening steps are:

[0063] (1) Coating: 3 wells were coated with HI-OVA in each round, 100 μL per well. 3 more wells were coated with 1 mg / mL OVA, 100 μL per well for negative panning. Coating was carried out at 4°C overnight. The next day, the liquid in the wells was discarded, 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 continued at 37°C for 3 h. The liquid in the wells was discarded, and the wells were patted dry. The wells were then placed in an oven at 37°C, inverted, and placed for 1 h before use.

[0064] (2) Panning: To eliminate the 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 for 1 hour at 37°C, transfer it to the antigen well and incubate it at room temperature with micro-shaking 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-shaking for 1.5 hours, and then aspirated and collected for storage. Take 10 μL of eluted product to calculate the titer, and the remaining eluted product was amplified and put into the next round of panning.

[0065] (3) Identification of specific phage monoclones: 96 monoclones were randomly picked from the titer determination plate of the eluted products after 4 rounds of panning, inoculated into a deep-well plate containing 600 μL / well LB-Amp medium, and cultured at 37°C and 150 rpm overnight. 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 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, frozen at -80°C for 3 hours, thawed and centrifuged, and the supernatant was taken for ic-ELISA identification of positive phage clones. The results of positive clone identification 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, positive phage clones were identified using IC-ELISA. 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 on 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. Discard the liquid in the well and pat dry.

[0070] (3) Sample loading: 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 five 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, 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 data of ic-ELISA, 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 nanobody 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 nanobody 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 nanobody is shown in SEQ ID NO.6.

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

[0075] 1. Mutant expression and purification

[0076] The frozen E. coli mutants identified as positive clones were thawed, and 5 μL of the bacterial solution was used to streak the LB-Amp plate and inverted in a 37°C incubator overnight. The next day, single clones from the plate were picked and placed in 10 mL of LB-Amp liquid medium, shaken and cultured at 37°C and 250 rpm overnight. The pComb3xss-VHH plasmid in the overnight bacteria was extracted using the plasmid mini-extraction reagent from Novezan Co., Ltd. and transformed into E. coli BL21 (DE3) by heat shock for expression. The periplasmic protein was extracted by osmotic shock and purified by affinity chromatography using Ni-NTA filler. The purified protein solution was collected and the mutant expression and purification status were identified by SDS-PAGE. The protein electrophoresis results are shown in Figure 2. Figure 6 As shown, each mutant was successfully expressed.

[0077] 2. Identification of mutant activity

[0078] An indirect competitive ELISA assay was established using H1-OVA as the coating agent. The specific steps are as follows:

[0079] (1) Coating: Dilute the coating agent 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 using an automatic plate washer and pat dry. Add 200 μL of 3% skim milk powder to each well as a blocking solution and incubate in a 37°C constant temperature incubator for 3 h. Pat dry, then invert and bake in a 37°C oven for 2 h. Store in a 4°C refrigerator until ready for use.

[0081] (3) Incubation with primary antibodies: Dilute the nanoantibodies Nb9, H6, F1, and D3 to the working concentrations and add 50 μL of each well to the wells. Add 50 μL of the serially diluted drugs and incubate in a 37°C constant temperature incubator for 40 min. Wash the plate 5 times with a plate washer and pat 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, remove from the well, wash the plate 5 times with an automatic plate washer, and pat dry.

[0083] (5) Color development and termination: Add 100 μL of pre-mixed TMB two-component color development solution to each well, incubate at 37°C for 10 min, then remove the solution 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 plotting the curve, using IC 50 As an evaluation standard for antibody sensitivity.

[0085] The sensitivity of the screened nanoantibodies to parathion pesticide was tested, and the test results were as follows: Figure 7 The IC of nanobody H6 50 The minimum detection limit was 3.55 ng / mL, which was 2.9 times higher than that of nanoantibody Nb9. The minimum detection limit was 1.30 ng / mL, which was reduced by 2.6 times. The linear range was 1.89-6.67 ng / mL. Its effect was significantly better than that of nanoantibodies F1 and D3, and finally the nanoantibody H6 with higher affinity for parathion than the original nanoantibody was 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 cross-reaction rate test results 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 the 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 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 nanoantibody without heat treatment to the antigen was used 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 nanobody 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%, and 70% as diluents, the nanoantibodies were diluted to the working concentration, and the antigen-antibody binding ability was determined by ic-ELISA method. Nanoantibodies that were not 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 Figure 9 As shown, the organic tolerance of Nanobody H6 was improved compared with Nb9, and was significantly better than that of Nanobody 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 performs affinity maturation of the anti-parathion nanoantibody through phage display and biopanning technology, obtaining a nanoantibody H6 with a higher affinity for parathion than the original nanoantibody, the amino acid sequence of which is shown in SEQ ID NO. 1. Nanoantibody H6 can specifically recognize parathion pesticides, with a sensitivity 2.9 times higher than that of the original nanoantibody Nb9, and a minimum detection limit reduced by 2.6 times. It is also more sensitive 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 considered as equivalent replacement methods and are included in the scope of protection 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 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 cell 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 cell 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 the coating source to perform enzyme-linked immunosorbent assay; the parathion artificial antigen is H1-OVA, and its structural formula is:

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