AU2020103284A4 - Nanobody for carbofuran pesticide and preparation method and use thereof - Google Patents

Abstract

The present invention discloses a nanobody of a carbofuran pesticide and a preparation method and use thereof. An amino acid sequence of the nanobody is shown in SEQ ID NO.1. The gene encoding the nanobody has a nucleotide sequence shown in SEQ ID NO.2. A nanobody specific to a carbofuran pesticide is obtained in the present invention. The nanobody is capable of detection of the carbofuran pesticide, and a detection result is accurate, with good effect and good stability, and it has a good detection effect under the conditions of high temperature and organic solvent. The nanobody can not only be widely used in the detection of carbofuran pesticide residues in an agricultural product, but also can be used as a precursor, which can be modified by random or site-directed mutagenesis technology to obtain mutations with better properties (affinity, specificity, stability, etc.). It is used for development and further use in food, medicine, agriculture and other fields, and has great application promotion value.

Description

NANOBODY FOR CARBOFURAN PESTICIDE AND PREPARATION METHOD AND USE THEREOF

TECHNICAL FIELD The present invention relates to the field of biotechnology, and in particular, to a nanobody for carbofuran pesticide and a preparation method and use thereof.

BACKGROUND Carbamate compounds are used as insecticides, acaricides, herbicides and bactericides on pesticides, and have formed a large category of pesticides, with many varieties, good efficacy and low toxicity. Carbamate pesticides are a main cause of acute poisoning of pesticides, and are also key tested species for pesticide residues in vegetables at present. Carbofuran, trade name furadan, is a broad-spectrum carbamate insecticide, acaricide and nematicide, and can be used for a variety of crops to control more than 300 pests and nematodes in the soil and on the ground. It has a function of shortening the growth period of crops, promoting growth and development of crops, and thus effectively increasing crop yields. It is widely used in pest control of vegetables, fruits, food crops and etc. However, carbofuran is highly toxic to humans and animals, and it is hard to be degraded and easily causes environmental pollution. China has stipulated a maximum residue limit of carbofuran in food, but there are still many illegal use, and thus it is necessary to strengthen the detection of carbofuran pesticide residues. At present, the analysis of carbofuran residue generally uses gas chromatography (GC for short), high performance liquid chromatography (HPLC for short) and chromatography-mass spectrometry technique. These methods are sensitive and accurate, and can simultaneously determine multiple drugs, but sample pre-processing is complicated, tedious and time-consuming. In addition, expensive equipment and professional operators are required, the detection cost is high, and it is difficult to meet the needs of on-site, batch, and rapid detection of samples. Therefore, it is of great significance to develop a simple and fast analytical method for on-site monitoring of pesticide residues. However, although an antibody-based immunoassay method has advantages of fast, sensitive, and high-throughput, the antibodies are often poorly stable and easy to be inactivated under extreme conditions. Therefore, there is currently a lack of a stable anti-carbofuran antibody.

SUMMARY OF THE INVENTION A purpose of the present invention is to overcome the shortcomings of the prior art, to provide a nanobody for carbofuran pesticide and a preparation method and use thereof. A first purpose of the present invention is to provide a nanobody specifically recognizing carbofuran. A second purpose of the present invention is to provide a gene encoding the nanobody specifically recognizing carbofuran. A third purpose of the present invention is to provide a recombinant vector. A fourth purpose of the present invention is to provide a recombinant cell. A fifth purpose of the present invention is to provide use of one or more of the nanobody, the gene, the recombinant vector and the recombinant cell in detecting carbofuran or preparing a detection kit for carbofuran. A sixth purpose of the present invention is to provide a preparation method for the nanobody. A seventh purpose of the present invention is to provide a detection method for carbofuran. An eighth purpose of the present invention is to provide a kit for detecting carbofuran. In order to achieve the above purposes, the present invention is realized by the following technical solutions. Nanobody has good stability and high affinity, which overcomes the shortcomings of small molecule functional antibodies, and at the same time, it also has advantages of small molecular weight, weak immunogenicity, strong tissue penetration and etc. that monoclonal antibodies and polyclonal antibodies do not have. In the present invention, by phage display technology, a nanobody that can specifically bind to a target molecule (carbofuran complete antigen) is screened from a camel-derived immune single-domain heavy-chain antibody library, and a method of a rapid, sensitive and stable detection of carbofuran residues in vegetables and tea is established. Therefore, the present invention claims the following content. A nanobody specifically recognizing carbofuran, and an amino acid sequence thereof being shown in SEQ ID NO.1. A gene encoding the nanobody specifically recognizing carbofuran, and a nucleotide sequence of the nanobody is shown in SEQ ID NO.2. A recombinant vector, which is a vector containing the gene. Preferably, the vector is an expression vector. Preferably, the expression vector is pComb3xss. A recombinant cell, which is a cell carrying the recombinant vector, or a cell capable of expressing the nanobody. Preferably, the cell is Escherichiacoli BL21(DE3). Use of one or more of the nanobody, the gene, the recombinant vector and the recombinant cell in detecting carbofuran or preparing a detection kit for carbofuran. A preparation method for the nanobody including the following steps: SI, cloning an encoding gene of the nanobody to an expression vector to obtain a recombinant vector; S2, transforming the recombinant vector into a recipient cell to obtain a recombinant cell; S3, cultivatiing the recombinant cell and inducing expression of the nanobody; and S4, separating and purifying the nanobody. Preferably, in the step S3, the recombinant cell is cultured to a log phase with an OD6 0 0 value of 0.6-0.8, and the nanobody is induced to express. Preferably, in the step S3, 0.1 mM to 1ImM IPTG is used to induce expression at 28°C to 37°C for 12 to 16 hours. More preferably, in the step S3, 1 mM IPTG is used to induce expression at 37°C for 12 hours. Preferably, in the step S4, protein in a periplasmic cavity is extracted by a sucrose osmotic pressure method, and the soluble nanobody in the periplasmic cavity is obtained after one-step Ni column purification. A detection method for carbofuran utilizing the nanobody. Preferably, an indirect ELISA method was developed for detection. A carbofuran complete antigen by coupling a carbofuran hapten shown in formula (I) with a carrier protein is used as a coating antigen, and the nanobody is used as a detection antibody, 0

O NH '- COOH

(I). Preferably, the carrier protein is ovalbumin. More preferably, the detection method includes the following steps: coating the coating antigen in microplate; adding carbofuran standards or test samples, then adding the detection antibody; adding enzyme-labeled secondary antibody, and incubating; adding substrate solution, and incubating; adding stopping solution and conduction of measurement; operating the log10 of concentrations of the carbofuran standards as the abscissa, and ratio of an absorbance value of each concentration of the carbofuran standards to an absorbance value of zero standard well as the ordinate to establish a standard curve, and then calculating a content of carbofuran in the test samples according to the absorbance values of the test samples. Furthermore preferably, using a carbofuran complete antigen by coupling the carbofuran hapten shown in formula (I) with ovalbumin as a coating antigen, optimally diluting of the coating antigen, adding into the microplate with 100 tL per well, and incubating overnight at 37°C; washing the plate with a microplate washer twice, draining the liquid from the well by shaking, adding 120 pL blocking solution, incubating at 37 0C for 2 hours, draining the liquid from the well by shaking,placing the plate upside down, drying at 37°C for 1 hour; adding 50 tL the carbofuran standard or the test sample into each well, then adding 50ptL nanobody, incubating at 37°C for 1 hour, washing the plate with the microplate washer for 5 times, draining the liquid from the well by shaking; adding 100pL anti-HA-HRP antibody (a dilution of 20000 times) into each well, incubating at 37°C for 40 minutes, washing the plate with the microplate washer for 5 times, draining the liquid from the well by shaking; adding 100 pL TMB substrate solution into each well, incubating at 37°C for 10 minutes; then adding 50 pL 10% H 2 SO4 to stop the substrate reaction; measuring absorbance value at 450nm with an instrument; analyzing the log10 of concentrations of carbofuran as the abscissa, and ratio B/B 0 of the absorbance value as the ordinate, and fitting the standard curve with a four-parameter fitting module of Origin 9.0. A kit for detection of carbofuran contains the nanobody. Preferably, an indirect ELISA method is developed for detection. The kit further contains a carbofuran complete antigen by coupling a carbofuran hapten shown in formula (I) with a carrier protein as a coating antigen, and the nanobody is used as a detection antibody, 0

O NH' COOH N0

(I). Preferably, the carrier protein is ovalbumin. Preferably, it further contains an enzyme-labeled secondary antibody, a substrate solution and a stopping solution. Compared with the prior art, the present invention has the following beneficial effects. A nanobody specific to carbofuran pesticide is obtained in the present invention. The nanobody is capable of detecting the carbofuran pesticide, and a detection result is accurate, with good effect and good stability, and it has a good detection effect under the conditions of high temperature and organic solvent. The nanobody can not only be widely used in the detection of carbofuran pesticide residues in an agricultural product, but also can be used as a precursor, which can be modified by random or site-directed mutagenesis technology to obtain mutations with better properties (affinity, specificity, stability, etc.). It is used for development and further use in food, medicine, agriculture and other fields, and has great application promotion value.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a detection result of phage clones by indirect competitive ELISA. FIG. 2 shows a standard curve for detection of carbofuran pesticide. FIG. 3 shows a binding capacity of the nanobody for 5 minutes at high temperature. FIG. 4 shows a binding activity of the nanobody at 95°C. FIG. 5 shows a binding ability of the antibody to an antigen with methanol as a diluent. FIG. 6 shows a binding ability of the antibody to an antigen with acetonitrile as a diluent.

DETAILED DESCRIPTION The present invention is further described in detail below in combination with the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention, and are not used to limit the scope of the present invention. The test methods used in the following embodiments are conventional methods, unless otherwise specified; and the materials and reagents used are commercially available reagents and materials, unless otherwise specified.

Embodiment 1 Construction of immuned library of anti-carbofuran pesticide nanobody I. Experimental method 1. Preparation of complete antigens BFNB-OVA and BFNB-KLH Carbofuran pesticide hapten BFNB was coupled with albumin (OVA,) and keyhole limpet heocyanin (KLH), respectively to prepare complete antigens BFNB-OVA and BFNB-KLH. The chemical formula of the carbofuran pesticide hapten BFNB was:

O J NH' " COOH NO

2. Immunization of bactrian camel 500 pg of BFNB-KLH was taken to be emulsified with an equal volume of Freund's complete adjuvant, and multi-point immunization injections were performed on the neck of the bactrian camel subcutaneously. Booster immunization was performed every 2 weeks, each time 500 pg of BFNB-KLH emulsified with an equal volume of Freund's incomplete adjuvant and then immunization was performed, and blood was collected from veins one week after each immunization. Indirect competitive ELISA method was used to determine serum titers. 3. RNA extraction and cDNA synthesis A blood sample with the best serum inhibition was taken for lymphocyte separation and RNA extraction. The RNA extraction was performed according to the Trizol reagent method of Invitrogen. RNA was used as a template, the instructions of TARAKA's first-strand reverse transcription kit was followed to synthesize a first strand of cDNA. 4. Acquisition of gene segments of nanobody The amplification was performed by PCR technology with the use of Taq Mix DNA polymerase to obtain the gene encoding the variable region of the camel heavy chain antibody (primers are shown in Table 1 below). Table 1 Primer sequence for amplification of VHH gene: Primer Sequence P1 5'-CATGCCATGACTCGCGGCCCAGGCGGCCGTCCTGGCTGCTCTTCTA CAAGG-3' P2 5'-CATGCCATGACTCGCGGCCGGCCTGGCCCGTGCATTCTGGTTCAGG TTTTGGTTGTGG-3' P3 5'-CATGCCATGACTCGCGGCCGGCCTGGCCCTTGCATACTTCATTCGTT CC-3' QI 5'-GTCCTGGCTGCTCTTCTACAAGG-3' Q2 5'-GGTACGTGCTGTTGAACTGTTCC-3'

Q3 5'-ACTGGCCCAGGCGGCCGAGGTGCAGCTGSWGSAKTCKG-3' Q4 5'-ACTGGCCGGCCTGGCCTGAGGAGACGGTGACCWGGGTC-3'

One-step PCR and two-step PCR were performed on the first strand of cDNA obtained in the previous step, respectively. (1) One-step PCR used primers P1 and P2 and primers P1 and P3 to amplify the target fragments respectively, and the reaction conditions were: 94°C for 5 minutes, 94°C for 30 seconds, 61°C for 30 seconds, 72°C for 1 minute, 30 cycles, extended at 72°C for 10 minutes. PCR products were respectively subjected to agarose gel electrophoresis, and fragments of about 500 bp were recovered, and the target fragments were recovered through a DNA gel recovery kit, and combined to obtain nanobody gene fragments, which were quantified and stored at -20°C for further use. (2) Among two-step PCR amplification, the first step of PCR used primers Q Iand Q2 to amplify, and the reaction conditions were: 94°C for 5 minutes, 94°C for 30 seconds, 55°C for seconds, 72°C for 1 minute, 30 cycles, extended at 72°C for 10 minutes. The PCR products of the first step were subjected to agarose gel electrophoresis, fragments of about 500 bp were recovered, and the target fragments were recovered by the DNA gel recovery kit. The recovered target fragments were used as a template, primers Q3 and Q4 of the second step were used for amplification, and the PCR reaction conditions were the same as in the first step. Nanobody gene fragments were obtained by further gel cutting and recovery, which were quantified and stored at -20°C for later use. 5. Preparation ofnanobody gene bank Phagemid vector pComb3xss and the two nanobody gene fragments obtained in the previous step were subjected to sfiI double digestion. The pComb3xss vector and the two nanobody gene fragments were obtained through gel recovery and PCR purification. Then pComb3xss and the target fragment were mixed at a molar ratio of 1:3, and T4 ligase was used for ligation reaction overnight at 16°C. The above ligation product was precipitated and recovered by PCR Clean Up Kit, and the products were dissolved in 35 pl of sterile water. The ligation products in the one-step method and the two-step method were mixed at 1:1, and were electrotransformed into competent cells E. coli ER2738 with a total of 13 times, the transformed bacteria solution was cultured on a shaker at 200 rpm and 37°C for 1 hour to recover and grow. The transformed bacteria solution was diluted in a gradient to spread on the LB culture plate containing ampicillin and tetracycline for overnight culture at 37°C. On the next day, the single clone spread on the plate were randomly picked up and sent to the company for sequencing to identify the diversity of the antibody library. The capacity was calculated from the number and diversity of clones. The cells were scraped with the culture medium, and sterile glycerol with a final concentration of 20% was added, and stored at -80°C, which was the nanobody gene library for carbofuran pesticide. 6. Preparation of nanobody phage library 1 mL of the cryopreserved nanobody library was taken and inoculated into 200 mL of LB (Amp, Tet) medium, and the shaking at 37°C and 250 rpm was performed to reach a log phase (OD 6 0 0=0.6 to 0.8). 1 mL of helper phage (1x1012 pfu/mL) was added, and further cultivation was performed at 37°C for 30 minutes, shaking cultivation was performed at 250 rpm for 2 hours, then kanamycin solution (helper phage resistance) was added with a final concentration of 70 tg/mL, shaking cultivation was performed at 250 rpm overnight. The culture medium was centrifuged at 4°C and 12000 rpm for 20 minutes, the supernatant was transferred to a new centrifuge tube, 50 mL of 5xPEG/NaCl was added and mixed w, and incubation was performed on ice for 4 hours. Centrifugation was performed at 4°C and 12000 rpm for 15 minutes, the supernatant was abandoned, 1 mL of TBS was added to resuspend the phage precipitate, filtration was performed with 0.22 pm membrane, 20% sterilized glycerin was added, and the nanobody phage library was stored at -20°C .

Embodiment 2 Construction of anti-carbofuran pesticide nanobody immune library I. Experimental method 1. BFNB-OVA was used as a coating antigen, each well of the microplate contained 100 pL, and incubation was performed in a 37°C water bath overnight. The coating concentration gradient was 10 ptg/mL, 5 pg/mL, 1 pg/mL, 0.2 ptg/mL. After 12 hours of coating, the plate was washed twice with PBST, the wells of 10 pg/ml and 1 pg/ml were added with 120 pl of1% BSA, the wells of 5 pg/ml and 0.2 pg/ml were added with 120 pl of 1% fish gelatin protein, the plate was blocked for 3 hours, and dried at 37°C for further use. 2. 100 pl of phage library (about 1011 pfu) was added into 2% BSA, OVA and KLH carrier proteins respectively, incubation was performed at 37°C for 1 hour, and the antibodies that non-specifically adsorb the carrier proteins were removed and then the unbinding phages were transferred into the wells of coating antigen, shaking was performed at room temperature for 1 hour, unbound phages were discarded, and the plate was washed with PBST for 5 times, 10 times, 15 times, 15 times in four rounds. The phage antibody adsorbed in the wells was eluted by 100pL of eluent (2 tg/mL, 1 tg/mL, 0.5 pg/mL, 0.1 ptg/mL of carbofuran solution). 10 pL eluted phages were used for titer determination, and the remaining eluted phages were used for the next round of screening after the amplification of helper phage rescue. A total of 4 rounds of screening (see Table 2) were carried out. The third and fourth rounds of phage single clone were randomly selected for indirect competitive ELISA. Table 2 Screening strategy table: Round Coated antigen Input Output Recovery rate Enrichment (pg/mL) (cfu) (cfu) 1 10 101 105 10~6

2 5 1012 107 10- 100 3 1 102 107 10- 1 4 0.2 1012 105 10~6 -100

Recovery = output phage / input phage Enrichment = after round / previous round II. Experimental result The results of indirect competitive ELISA for the third and fourth rounds of phage single clone are shown in FIG. 1, and nine nanobodies with different sequences were obtained: Nb309 (393bp), Nb316 (393bp), Nb328 (375bp), Nb391 (393bp), Nb393 (372bp), Nb415 (372bp), Nb438 (372bp), Nb480 (384bp), Nb489 (366bp). After the performance analysis, Nb316 has the best inhibition rate, and the amino acid sequence thereof is shown in SEQ ID NO.1; and the nucleotide sequence thereof is shown in SEQ ID NO.2.

Embodiment 3 Preparation of nanobody of carbofuran pesticide and Establishment of indirect competitive ELISA method I. Experimental method 1. The Nb316-pComb3xss plasmid was extracted by an extraction kit, and then transformated into the competent E. coli BL21(DE3) by chemical transformation method. A single clone was tpicked up for PCR identification and sequencing, and the inserted fragments were determined as target fragments. The BL21(DE3) bacterial colony containing the nanobody target fragments was cultured to a log phase with an OD6 0 0 value of 0.6-0.8, A final concentration of 1mM IPTG was added to induce expression at 37°C for 12 hours. The next day, centrifugation was performed to obtain the bacteria. Then the periplasmic cavity protein was extracted by the sucrose osmotic pressure method, and the soluble nanobody Nb316 in the periplasmic cavity was obtained after one-step Ni column purification. 2. BFNB-OVA was used as the coating antigen, diluted to a working concentration of 1 pg/mL, and added with 100 tL per well to a 96-well microplate, , and incubation was performed overnight at 37C. The next day, the plate was washed twice with the plate washer, the liquid was drained from the well by shaking, 120pL of a blocking solution was added, and incubation was performed at 37°C for 2 hours, the liquid was drained from the well by shaking and the plate was placed upside down at 37°C oven for 1 hour, and then was taken out for further use. Each well was added with 50 tL of gradiently diluted drug and 50pL of nanobody, and three replicates was performed for each concentration. Incubation was performed at 37°C for 1 hour, the plate was washed with the plate washer for 5 times, the liquid was drained from the well by shaking, each well was added with 100 tL of anti-HA-HRP antibody (a dilution of 20000 times), incubation was performed at 37°C for 40 minutes, the plate was washed with the plate washer for 5 times, and the liquid was drained from the well by shaking. Each well was added with 100 tL of TMB subatrate solution, incubation was performed at 37°C for 10 minutes, 50 tL of 10% H2SO 4 was then added to stop the reaction, The absorbance value at 450 nm was measured with an instrument. Concentration of carbofuran was taken as the abscissa, ratio of the absorbance value B/B 0 was taken as the ordinate, and a standard curve was fitted with a four-parameter fitting module of Origin 9.0. II. Experimental result The results show that the standard curve of the indirect competitive ELISA (as shown in FIG. 2) for the detection of carbofuran based on nanobody Nb2 has an IC5 0 of 7.27±0.88 ng/mL, and a linear range of 1.44 ng/mL to 30.40 ng/mL.

Embodiment 4 Detection of various carbamate pesticides by indirect competitive ELISA I. Experimental method 1. BFNB-OVA was used as the coating antigen, Nb316 was the detection antibody, and the indirect competitive ELISA method was used to evaluate the specificity and sensitivity of carbamate pesticides. The specific steps were as follows: each well was added with 50pL of gradient diluted drug and 50pL of nanobody, and three replicates was performed for each concentration. Incubation was performed at 37°C for 1 hour, the plate was washed with the plate washer for times, the liquid was drained from the well by shaking, each well was added with 100 tL of anti-HA-HRP antibody (a dilution of 20000 times), incubation was performed at 37°C for minutes, the plate was washed with the plate washer for 5 times, and the liquid was drained from the well by shaking. Each well was added with 100 tL of TMB substrate solution, incubation was performed at 37°C for 10 minutes, and then 50 tL of 10% H2SO 4 was added to stop the reaction, The absorbance value at 450 nm was measured with an enzyme-labeled instrument. Concentration of carbofuran was taken as the abscissa, ratio of the absorbance value B/B 0was taken as the ordinate, and a standard curve was fitted with a four-parameter fitting module of Origin 9.0 to obtain respective IC5 0 value. The following equation was used to calculate the cross reactivity of each drug and anti-carbofuran nanobody:

CR (%)=100x IC5 0 (carbofuran ) / IC5 0 (carbofuran analogue). 2. The results show that the cross reactivity of nanobody Nb316 with benfuracarb, fenobucarb, carbosulfan, 3-hydroxycarbofuran and isoprocarb was 5.05%, 3.52%, 2.56%, 1.97% and 0.35%, respectively, and the cross reactivity of the other 7 pesticides was less than 0.1% (see Table 3). Table 3 The sensitivity and specificity of pesticides detected by ic-ELISA with Nb316: Compound name IC5 0 Cross reactivity (%) Carbofuran 7.27 100 Benfuracarb 142.51 5.05 Fenobucarb 204.6 3.52 Carbosulfan 280.93 2.56 3-Hydroxycarbofuran 366.05 1.97 Isoprocarb 1351.82 0.53 Carbaryl > 2000 < 0.1 Aldicarb > 2000 < 0.1 Methomyl > 2000 < 0.1 Pirimicarb > 2000 < 0.1 Mesurol > 2000 < 0.1 Tsumacide > 2000 < 0.1

Embodiment 5 Thermal stability analysis of nanobody of carbofuran pesticide I. Experimental method 1. The nanobody Nb316 was diluted to the working concentration, divided into seven equal parts which were placed in water baths at 20°C, 35°C, 50°C, 65°C, 80°C and 95°C for minutes, respectively. After the antibody returned to room temperature, the binding ability of the antibody to the antigen was measured by ic-ELISA, the binding ability of the unheated antibody to the antigen was regarded as 100%, and the stability of the nanobody at different temperatures was evaluated. 2. The nanobody Nb316 was diluted to the working concentration, divided into seven equal parts which were placed in a water bath at 95°C, and were heated for 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, and 60 minutes, respectively. After the antibody returned to room temperature, the binding ability of the antibody to the antigen was measured by ic-ELISA of Embodiment 4, the binding ability of the unheated antibody to the antigen was regarded as 100%, and the stability of nanobody at high temperature with time was evaluated. II. Experimental result The results show that nanobody Nb 316 can still maintain ~100% of the activity at 95°C for 5 minutes. Therefore, the nanobody still has strong binding ability at high temperatures (FIG. 3). At 95°C, from 0 minute to 60 minutes, the nanobody always maintains about 100% of the binding activity (FIG. 4). Embodiment 6 Thermal stability analysis of nanobody of carbofuran pesticide I. Experimental method Different concentrations (10%, 20%, 40%, 60% and 80%) of methanol and acetonitrile were used as diluents, the nanobody Nb316 was diluted to the same working concentration, ic-ELISA of Embodiment 4 was used to determine the binding ability of the antibody to the antigen, the binding ability of the antibody without dilution with the organic solvent diluent to the antigen was regarded as 100%, and the tolerance of the nanobody to different organic solvents and different concentrations of the same organic solvent was evaluated. II. Experimental result The results show that when the concentration of methanol is 25%, the nanobody Nb316 can still maintain about 100% of the binding activity, and when the concentration of methanol is 50%, the binding activity is still 50% (FIG. 5). In the acetonitrile environment, the binding activity of the nanobody will not decrease when the concentration of acetonitrile is lower than 20%, and the nanobody still has 50% of the binding activity when the concentration of acetonitrile is 30% (FIG. 6). Therefore, the nanobody has excellent resistance to organic solvents, and will not be affected by the residual organic solvents during the pretreatment process of actual sample detection.

Claims (10)

What is claimed is:

1. A nanobody specifically recognizing carbofuran, characterized in that, an amino acid sequence thereof is shown in SEQ ID NO.1.

2. A gene encoding a nanobody specifically recognizing carbofuran, characterized in that, a nucleotide sequence thereof is shown in SEQ ID NO.2.

3. A recombinant vector, characterized in that, the recombinant vector is a vector connected with the gene according to claim 2.

4. A recombinant cell, characterized in that, the recombinant cell is a cell carrying the recombinant vector according to claim 3, or a cell capable of expressing the nanobody according to claim 1.

5. Use of one or more of the nanobody according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3 and the recombinant cell according to claim 4 in detecting carbofuran or preparing a detection kit for carbofuran.

6. A preparation method for the nanobody according to claim 1, characterized in that, the preparation method comprises the following steps: S1, cloning an encoding gene of the nanobody according to claim 1 to an expression vector to obtain a recombinant vector; S2, transforming the recombinant vector into a recipient cell to obtain a recombinant cell; S3, cultivating the recombinant cell and inducing expression of the nanobody; and S4, separating and purifying the nanobody.

7. A detection method for carbofuran, characterized in that, the detection method utilizes the nanobody according to claim 1.

8. The detection method according to claim 7, characterized in that, an indirect ELISA method for detection was developed, a carbofuran complete antigen obtained by coupling a carbofuran hapten shown in formula (I) with a carrier protein is used as a coating antigen, and the nanobody according to claim 1 is used as a detection antibody, 0

OK NH" COOH

(I).

9. A kit for detecting carbofuran, characterized in that, the kit contains the nanobody according to claim 1.

10. The kit according to claim 9, characterized in that, an indirect ELISA method for detection was developed, the kit further contains a carbofuran complete antigen obtained by coupling a carbofuran hapten shown in formula (I) with a carrier protein as a coating antigen, and the nanobody according to claim 1 is used as a detection antibody, 0

0 NH" - COOH

(I).