A nano-antibody-based electrochemical detection method for porcine epidemic diarrhea virus and an antibody preparation method

Abstract

The invention discloses an electrochemical detection method for porcine epidemic diarrhea virus based on nano antibodies and an antibody preparation method, which relates to the field of immunoassay technology. The electrochemical detection method comprises the following steps: dropping MUA solution on the electrode surface, placing it in water-saturated air, and incubating at room temperature for 20 hours; rinsing the electrode with ethanol and air-drying; placing the prepared electrode in a mixed solution containing NHS solution and EDC solution, activating for 60 minutes, and then washing the electrode with PBS solution; adding a nano antibody solution with a concentration of 7.4 μg / μL on the electrode surface, placing it at room temperature for 3 hours, and then washing the electrode with PBS solution; adding 0.1% BSA solution on the electrode surface, blocking for 1.5 hours, and washing the electrode with PBS solution and air-drying after blocking; assembling the modified electrode sheet, adding antigen solution, and performing electrochemical detection after reacting at room temperature for 15 minutes. The invention combines nano antibodies with electrochemical immunosensors to establish an electrochemical detection method for porcine epidemic diarrhea virus based on nano antibody technology.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a porcine epidemic diarrhea virus electrochemical detection method based on nanoantibodies and an antibody preparation method. Background Art

[0002] Porcine epidemic diarrhea (PED) is one of the diseases that causes large-scale diarrhea in pigs. The disease is caused by the invasion of porcine epidemic diarrhea virus (PEDV) in pigs. The main characteristics of the disease are diarrhea. In severe cases, watery diarrhea, depression and gradual weight loss may occur. Porcine epidemic diarrhea virus (PEDV) has the characteristics of high pathogenicity and strong mutation ability. Therefore, it is very necessary to find effective measures to quickly detect PEDV. In past studies, immunoassays were considered an important detection method. However, conventional antibodies involved in immunoassays are difficult to recombinantly express and are easily affected by the environment. Compared with classical antibodies, nanobodies (Nanobodies, Nb) are not only easy to express in prokaryotes or eukaryotes, but also can exist stably under extreme conditions, and can be used as a substitute for classical antibodies.

[0003] Biosensors have the advantages of portability (because they are miniature), simplicity, automation, cost-effectiveness, high stability, and short detection time. In addition, biosensors can provide real-time responses, so they are suitable for point-of-care detection and have been introduced to measure quantitative signals from probe-bound analytes. Currently, many electrochemical immunosensors have been developed, such as voltammetric immunosensors, amperometric immunosensors, barrier immunosensors, ECL immunosensors, etc., and various strategies have been proposed to detect biomarkers, so biosensors have been widely used in various fields, including clinical diagnosis, agriculture, food industry, environmental monitoring, and quality control.

[0004] Since antibodies are often used as recognition molecules for specific binding to target analytes, the present application combines nanoantibodies with electrochemical immunosensors to establish a method for rapid detection of PEDV, providing a new detection method for the detection of porcine epidemic diarrhea virus. Summary of the invention

[0005] The technical solution of the present invention to solve the above technical problems is to provide a method for electrochemical detection of porcine epidemic diarrhea virus based on nanoantibodies, comprising the following steps: Preparation of modified electrode: MUA solution (11-mercaptoundecanoic acid) was added dropwise on the electrode surface, placed in water-saturated air, and incubated at room temperature for 20 h; the electrode was rinsed with ethanol and air-dried; Electrode activation: The prepared electrode was placed in a mixture of NHS solution (a solution containing N-hydroxysuccinimide) and EDC solution (a solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) for 60 min, and then the electrode was washed with PBS solution (phosphate buffered saline); Nanoantibody labeling: Add a nanoantibody solution with a concentration of 7.4 μg / μL to the electrode surface, leave it at room temperature for 3 h, and then wash the electrode with PBS solution; Blocking treatment: add 0.1% BSA solution (containing bovine serum albumin (BSA)) to the electrode surface and block for 1.5 hours. After blocking, wash the electrode with PBS solution and air dry. Electrode assembly and detection: Assemble the modified electrode sheets, add antigen solution, react at room temperature for 15 minutes, and then perform electrochemical detection; Among them, the electrochemical detection parameter settings are: detection voltage is 4μA or 8μA, standing time is 1s, starting potential is -0.5V, ending potential is 0.5V, step potential is 0.001V, modulation amplitude is 0.025V, and frequency is 100Hz.

[0006] Furthermore, in the step of preparing the modified electrode, the concentration of the MUA solution is 1 mM, the drop volume is 50 μL, and the culture conditions are water-saturated air, room temperature, and 20 hours.

[0007] Furthermore, in the electrode activation step, the concentration of the NHS solution is 20 mM, and the concentration of the EDC solution is 10 mM.

[0008] Furthermore, in the nano-antibody labeling step, the volume of the nano-antibody solution added is 30 μL.

[0009] Furthermore, in the blocking step, the added volume of the BSA solution is 50 μL.

[0010] Furthermore, in the electrode assembly and detection steps, the solvent of the antigen solution is a mixed solution of 10 mM potassium ferrocyanide and 10 mM potassium ferrocyanide, and the added volume of the antigen solution is 28 μL.

[0011] In order to solve the above technical problems, the present invention also proposes a method for preparing a PEDV-S nanobody, which is used to prepare the nanobody as described above, comprising the following steps: pCold-II vector (Escherichia coli cold shock expression vector) was used as the expression vector; EcoRⅠ and KpnI were selected as restriction sites; According to the gene sequence of the target nanobody, the gene primers were designed using Primer5 software; An EcoRI endonuclease site was added to the 5' end of the upstream primer, and a KpnI endonuclease site was added to the 5' end of the downstream primer. The technical solution of the present invention combines nanoantibodies with electrochemical immunosensors. The PEDV-S nanoantibody is used as a recognition molecule for specifically binding to a target analyte, and an electrochemical detection method for porcine epidemic diarrhea virus based on nanoantibody technology is established. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0013] Figure 1 This is a flow chart of the steps of the electrochemical detection method for porcine epidemic diarrhea virus based on nanoantibodies according to the present invention; Figure 2 This is the PCR verification diagram of the recombinant plasmid of the present invention; Figure 3 This is a double enzyme digestion verification diagram of the recombinant plasmid of the present invention; Figure 4 This is the SDS-PAGE identification result diagram of the present invention; Figure 5 This is a diagram of the Western Blot identification results of the present invention; Figure 6 This is the SDS-PAGE identification diagram of the purified protein of the present invention; Figure 7 This is a graph showing the binding force of the nanobody of the present invention; Figure 8 This is a graph showing the binding potency of two nanobodies of the present invention; Fig. 9 This is a graph showing the specificity of the nanoantibody detected by ELISA of the present invention; Fig.10 For electrochemical detection of the present invention; Fig.11 This is a test diagram of the electrochemical detection repeatability of the present invention; Fig.12 Schematic diagram of the vector construction of the present invention. DETAILED DESCRIPTION

[0014] The present invention proposes a porcine epidemic diarrhea virus electrochemical detection method based on nanoantibodies and an antibody preparation method, aiming to design a PEDV-S nanoantibody for use in the porcine epidemic diarrhea virus electrochemical detection method.

[0015] The electrochemical detection method for porcine epidemic diarrhea virus based on nanoantibodies and the antibody preparation method proposed in the present invention are described below in specific embodiments: Main reagents and materials: Four strains of PEDV-S nanoantibodies were preserved by the applicant's laboratory; pCold-II plasmid was preserved by the applicant's experiment; E.coli BL21 (DE3) competent cells were purchased from Beijing Solebow Technology Co., Ltd.; IPTG and nickel column were purchased from Sangon Biotech (Shanghai) Co., Ltd.; BCA protein quantitative determination kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; TMB colorimetric solution and stop solution were purchased from Beijing Solebow Technology Co., Ltd.

[0016] Example 1: A method for electrochemical detection of porcine epidemic diarrhea virus based on nanoantibodies, comprising the following steps: Preparation of modified electrode: drop MUA solution on the electrode surface, place in water-saturated air, and incubate at room temperature for 20 h; rinse the electrode with ethanol and air dry; Electrode activation: The prepared electrode was placed in a mixture of NHS solution and EDC solution for 60 min, and then the electrode was washed with PBS solution; Nanobody labeling: Add a nanobody solution (PEDV-S nanobody prepared in Example 3) with a concentration of 7.4 μg / μL to the electrode surface, leave it at room temperature for 3 h, and then wash the electrode with PBS solution; Blocking treatment: add 0.1% BSA solution to the electrode surface and block for 1.5 hours. After blocking, wash the electrode with PBS solution and air dry. Electrode assembly and detection: Assemble the modified electrode sheets, add antigen solution, react at room temperature for 15 minutes, and then perform electrochemical detection; Among them, the electrochemical detection parameter settings are: detection voltage is 4μA or 8μA, standing time is 1s, starting potential is -0.5V, ending potential is 0.5V, step potential is 0.001V, modulation amplitude is 0.025V, and frequency is 100Hz.

[0017] Furthermore, in the step of preparing the modified electrode, the concentration of the MUA solution is 1 mM, the drop volume is 50 μL, and the culture conditions are water-saturated air, room temperature, and 20 hours.

[0018] Furthermore, in the electrode activation step, the concentration of the NHS solution is 20 mM, and the concentration of the EDC solution is 10 mM.

[0019] Furthermore, in the nano-antibody labeling step, the volume of the nano-antibody solution added is 30 μL.

[0020] Furthermore, in the blocking step, the added volume of the BSA solution is 50 μL.

[0021] Furthermore, in the electrode assembly and detection steps, the solvent of the antigen solution is a mixed solution of 10 mM potassium ferrocyanide and 10 mM potassium ferrocyanide, and the added volume of the antigen solution is 28 μL.

[0022] Specifically, 1mM MUA solution was used to modify the electrode, 50μL of solution was added to the electrode, and it was placed in water-saturated air and incubated at room temperature for 20h. The next day, the electrode was rinsed with ethanol and air-dried; the electrode was placed in a solution containing 20mM NHS solution and 10mM EDC solution (25μL each), activated for 60min, and washed with PBS solution after 1h; 30μL (concentration of 7.4μg / μL) of nanoantibodies were added to the electrode holes for labeling, and after being placed at room temperature for 3h, the electrode was washed with PBS and then blocked with 50μL of 0.1% BSA solution for 1.5h. After blocking, the electrode was washed with PBS and air-dried. Assemble the electrode sheet, add 28ul antigen (solvent: a mixed solution of 10mM potassium ferrocyanide and 10mM potassium ferrocyanide), and test on the machine after 15min at room temperature. The detection parameters are: voltage: 4uA or 8uA; standing time: 1s; starting potential: -0.5V; ending potential: 0.5V; step potential: 0.001V; modulation amplitude: 0.025V; frequency: 100Hz.

[0023] The marked electrode pieces were tested on the machine. The results were as follows: Fig.10 As shown, A is the negative control; B is the antigen diluted 1:100; C is the antigen diluted 1:10000. It can be seen that the current peak values ​​are different for different antigen concentrations, and they meet the inverse proportional growth condition, that is, the lower the antigen concentration, the larger the peak value. Therefore, in order to ensure the reliability of this experiment, a repeated test was carried out, and the results are shown as follows Fig.11 As shown, A is the negative control; B is the antigen diluted 1:100; C is the antigen diluted 1:10000. The conclusion is consistent with the previous one, proving that the electrochemical detection method was successfully established in this application. Example 2: A method for preparing a PEDV-S nanobody, such as Fig.12 As shown, the following steps are included: pCold-II vector was used as the expression vector; EcoRⅠ and KpnI were selected as restriction sites; According to the gene sequence of the target nanobody, the gene primers were designed using Primer5 software; An EcoRⅠ nuclease site was added to the 5' end of the upstream primer, and a KpnI nuclease site was added to the 5' end of the downstream primer; and the pCold-PEDV plasmid was constructed. Example 3: A method for preparing a PEDV-S nanobody, comprising the following steps: pCold-II vector was used as the expression vector; EcoRⅠ and KpnI were selected as restriction sites; According to the gene sequences of the four nanobodies, gene primers were designed using Primer5 software; The EcoRⅠ endonuclease site was added to the 5' end of the upstream primer, and the KpnI endonuclease site was added to the 5' end of the downstream primer; the pCold-PEDV plasmids were constructed, namely pCold-PEDV-13; pCold-PEDV-41; pCold-PEDV-61; and pCold-PEDV-86. All primers in this method were synthesized by GenScript Technology Co., Ltd.

[0024] The nucleotide sequence of pCold-PEDV-13 is: CTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGACACTCTCCTGTGCCGTCTCTGGATACGCCTATATGCCATACTACATGGCCTGGTACCGCCAGGCCCCAGGGAGTGAACGCGAATTGGTCTCAAATATTATCAGTAATGGAGGCACATATTATGCAGACTCCGTGA AGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGTTCTGAGGACACGGCCATGTATTATTGTAAAGCAGTTGTTTCACAGGACGGTTGGATCCAAAGGGTGCCTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGC.

[0025] The amino acid sequence of pCold-PEDV-13 is: LQESGGGSVQAGGSLTLSCAVSGYAYMPYYMAWYRQAPGSERELVSNIISNGGTYYADSVKGRFTISQDNAKNTVYLQMNSLSSEDTAMYYCKAVVSQDGLDPKGAYWGQGTQVTVSSAA。

[0026] The nucleotide sequence of pCold-PEDV-41 is as follows: CTGCAGGAGTCTGGGGGAGGCTCGGTGCAGACTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGCCACACCTACACTATCTGTGGTATGGCCTGGTACCGCCAGGCTCCAGGGAAGGGGCGCGAGTTGGTCTCAAATATTTTACGTGGCGGTGATTCATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGATAACGCCAAGAATACGGTATATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCCACTTATTACTGTAAAGCAGCGCGCTTAGATCGTGGTAGTTGTGTCTGGCATAACGACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGC。

[0027] The amino acid sequence of pCold-PEDV-41 is as follows: LQESGGGSVQTGGSLRLSCAASGHTYTICGMAWYRQAPGKGRELVSNILRGGDSYYADSVKGRFTISQDNAKNTVYLQMNSLRPEDTATYYCKAARLDRGSCVWHNDWGQGTQVTVSSAA。

[0028] The nucleotide sequence of pCold-PEDV-61 is as follows: CTGCAGGAGTCTGGGGGAGGCTCGGTCCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGATACACCTCCATCACCTACAGTAGATGTGCCATGAGCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGGAGGTTTCAACTATACTTCGTGGTGGTGCCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCGTCTCCCAAGACAAAGCCAAGAATACGATGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTAAGACACGCGGAGGTGATTACTGCGAGCAGAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGC。

[0029] The amino acid sequence of pCold-PEDV-61 is as follows: LQESGGGSVQAGGSLRLSCVASGYTSITYSRCAMSWYRQAPGKEREEVSTILRGGATNYADSVKGRFTVSQDKAKNTMYLQMNSLKPEDTAVYYCKTRGGDYCEQNYWGQGTQVTVSSAA。

[0030] The nucleotide sequence of pCold-PEDV-86 is as follows: ctgcagGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTGGCCTCTGGATTCACCTTCAGTAGGTTTCTCATGAAATGGGTCCGCCAAATTCCAGGGAAGGGACTCGAGTGGGTCGCAAGTATTGGCCGGAGTGATGATTACACATCGTATGGAGACTCCGTGGAGGGCCGATTCACCATCTCCAGAGACAACGACAAGAACACGCTGTATCTGCAATTGAACAGTCTGAAAACTGAGGACACGGCCATATATTACTGTGAAATAGGGAACTCTGTCGATGGTACTAGACCTCTTTCGGGGCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAgcggccgc。

[0031] The amino acid sequence of pCold-PEDV-86 is: LQESGGGLVQPGGSLRLSCVASGFTFSRFLMKWVRQIPGKGLEWVASIGRSDDYTSYGDSVEGRFTISRDNDKNTLYLQLNSLKTEDTAIYYCEIGNSVDGTRPLSGRGQGTQVTVSSAA. 1. Verify the pCold-PEDV recombinant vector: The extracted positive plasmid was verified by PCR, and a 529 bp target band appeared. Figure 2 ; Double enzyme digestion verification, 4378bp and 381bp target bands appeared respectively, such as Figure 4 The positive plasmids were extracted and sent to GenScript for sequencing. The alignment rate was 100%, which was consistent with expectations, indicating that the pCold-PEDV recombinant vector was successfully constructed.

[0032] 2. Induced expression and purification of recombinant protein: The four correctly sequenced pCold-PEDV plasmids were transformed into BL21 (DE3) expression competent cells, 200 μL of the bacteria were spread on LB / AMP plates, and placed in a 37°C constant temperature incubator for overnight culture. 100 μL of the above culture was inoculated into 10 mL of LB / AMP medium, and cultured in a 37°C 220 r / min constant temperature shaker until the logarithmic phase, 1 mL of the culture was taken, centrifuged at 12000g at 4°C for 2 min, and the bacteria were collected.

[0033] The remaining 9 mL of culture was taken out of the incubator, and 9 μL of IPTG stock solution (final concentration of 1 mmol / L) was added immediately and 30 min later. The culture was induced at 200 r / min and 15 °C for 24 h.

[0034] After induction, the induced bacteria were centrifuged at 12000 g for 10 min at 4°C to collect the bacteria, add 1 mL PBS to resuspend the bacteria, and centrifuge again to collect the bacteria. After repeating twice, the bacteria were resuspended with 500 μL PBS and ultrasonically lysed at a power of 180 W, a working time of 10 s, a rest time of 10 s, and ultrasonic lysis for 20 min. The supernatant and precipitate were collected after high-speed centrifugation of the lysate. Samples were kept for each step of the operation and analyzed by SDS-PAGE. Figure 4 As shown in the figure, the results showed that compared with the induced and uninduced pCold-PEDV, the induced pCold-PEDV bacteria had an obvious protein band near 14KD, which was consistent with the expected size of the protein. In order to further confirm whether the protein was the target protein, a Western Blot experiment was performed, as shown in the figure. Figure 5As shown, the results showed that the His monoclonal antibody specifically bound to the protein at this location, indicating that the protein was correctly expressed and existed in the form of inclusion bodies. The recombinant proteins of PEDV-S-13 and PEDV-S-41 were induced to express in large quantities, and the Nb2 recombinant protein with the His tag in the supernatant was purified using a Ni-NTA affinity chromatography column. Figure 6 As shown, 50 mM imidazole can elute the target protein well with good purity.

[0035] 3. Binding detection and titer analysis: PEDV virus was diluted to 100 μg / mL with PBS buffer, and 100 μL (10 μg) was added to each well of a 96-well ELISA plate. An equal volume of PSB was added directly to the well without antigen control, and the plate was placed at 4°C for overnight coating. The plate was taken out the next day, the coating solution was discarded, 200 μL of 2.5% skim milk powder was added to each well, and the plate was blocked in a 25°C incubator for 1 h. The blocking solution was discarded, and the recombinant nanoantibody was taken, diluted 1:1 with 2.5% skim milk powder, and added to the ELISA plate. 100 μL was added to each well and incubated in a 25°C incubator for 2 h. After discarding the nano-antibodies, add 100 μL of His-tagged enzyme-labeled antibody, wash the ELISA plate 4 times with TBST, add 100 μL of TMB colorimetric substrate to each well, and color in a 25°C incubator in the dark for 30 minutes. Finally, add 50 μL of ELISA stop solution to terminate the reaction, and use an ultra-micro spectrophotometer to measure the OD450 of each well. The OD value indirectly reflects the binding strength. Figure 7 As shown, the results show that the capabilities of S-13 and S-41 are similar.

[0036] At the same time, the titer analysis test is carried out, and the screened nano antibodies are diluted in multiples, and the two are diluted in multiples and then tested by ELISA method, such as Figure 8 As shown, it can be seen that the binding force of S-41 is better than that of S-13.

[0037] 4. Specificity detection: PEDV virus and laboratory-stored PDCOV, PCV, TGEV, and PRRSV viruses were diluted to 100 μg / mL with PBS buffer, and 100 μL (10 μg) was added to each well of a 96-well ELISA plate. An equal volume of PBS was added directly to the well without antigen control, and the plate was placed at 4°C for overnight coating. The plate was taken out the next day, the coating solution was discarded, and 200 μL of 2.5% skim milk powder was added to each well, and the plate was blocked in a 25°C incubator for 1 hour. The blocking solution was discarded, and the recombinant nanoantibody was taken, diluted 1:1 with 2.5% skim milk powder, and added to the ELISA plate, and 100 μL was added to each well, and the plate was incubated in a 25°C incubator for 2 hours. After discarding the nanoantibodies, add 100 μL of His-tagged enzyme-labeled antibody, wash the ELISA plate 4 times with TBST, add 100 μL of TMB colorimetric substrate to each well, and color develop in a 25°C incubator in the dark for 30 min. Finally, add 50 μL of ELISA stop solution to terminate the reaction. Fig. 9 As shown, it is specific for PEDV only. The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for electrochemical detection of porcine epidemic diarrhea virus based on nanoantibodies, characterized in that: The following steps are involved: Preparation of modified electrode: MUA solution was added dropwise on the electrode surface, placed in water-saturated air, and incubated at room temperature for 20 h; Rinse the electrodes with ethanol and air dry; Electrode activation: The prepared electrode was placed in a mixture of NHS solution and EDC solution for 60 min, and then the electrode was washed with PBS solution; Nanoantibody labeling: Add a nanoantibody solution with a concentration of 7.4 μg / μL to the electrode surface, leave it at room temperature for 3 h, and then wash the electrode with PBS solution; Blocking treatment: add 0.1% BSA solution to the electrode surface and block for 1.5 hours. After blocking, wash the electrode with PBS solution and air dry. Electrode assembly and detection: Assemble the modified electrode sheets, add antigen solution, react at room temperature for 15 minutes, and then perform electrochemical detection; Among them, the electrochemical detection parameter settings are: detection voltage is 4μA or 8μA, standing time is 1s, starting potential is -0.5V, ending potential is 0.5V, step potential is 0.001V, modulation amplitude is 0.025V, and frequency is 100Hz.

2. The electrochemical detection method for porcine epidemic diarrhea virus based on nanoantibodies according to claim 1, characterized in that: In the step of preparing the modified electrode, the concentration of the MUA solution is 1 mM, the drop volume is 50 μL, and the culture conditions are water-saturated air, room temperature, and 20 hours.

3. The electrochemical detection method for porcine epidemic diarrhea virus based on nanoantibodies according to claim 1, characterized in that: In the electrode activation step, the concentration of the NHS solution is 20 mM, and the concentration of the EDC solution is 10 mM.

4. The electrochemical detection method for porcine epidemic diarrhea virus based on nanoantibodies according to claim 1, characterized in that: In the nanobody labeling step, the volume of the nanobody solution added is 30 μL.

5. The electrochemical detection method for porcine epidemic diarrhea virus based on nanoantibodies according to claim 1, characterized in that: In the blocking step, the added volume of the BSA solution is 50 μL.

6. The electrochemical detection method for porcine epidemic diarrhea virus based on nanoantibodies according to claim 1, characterized in that: In the electrode assembly and detection steps, the solvent of the antigen solution is a mixed solution of 10 mM potassium ferrocyanide and 10 mM potassium ferrocyanide, and the added volume of the antigen solution is 28 μL.

7. A method for preparing a PEDV-S nanobody, for preparing the nanobody as claimed in claim 1, characterized in that: The following steps are involved: pCold-II vector was used as the expression vector; EcoRⅠ and KpnI were selected as restriction sites; According to the gene sequence of the target nanobody, the gene primers were designed using Primer5 software; An EcoRI endonuclease site was added to the 5' end of the upstream primer, and a KpnI endonuclease site was added to the 5' end of the downstream primer.

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