A nanobody against s1 protein of porcine epidemic diarrhea virus

Abstract

The application provides a kind of anti-PEDV's S1 protein nanobody, the amino acid sequence of the provided nanobody is SEQ ID NO:8, can effectively combine porcine epidemic diarrhea virus S1 protein, simultaneously has stable PEDV virus neutralization activity.The application provides a kind of nanobody capable of specific recognition and neutralization of porcine epidemic diarrhea virus, the nanobody screened has high specific recognition and binding capacity to porcine epidemic diarrhea virus, and has the application prospect of preparing the prevention and treatment or detection reagent of porcine epidemic diarrhea virus.

Description

Technical Field

[0001] This invention belongs to the field of nanobody preparation technology, specifically relating to a nanobody against the S1 protein of porcine epidemic diarrhea virus (PEDV). Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease in pigs caused by porcine epidemic diarrhea virus (PEDV), characterized by watery diarrhea, vomiting, and dehydration. PEDV, belonging to the genus *Alpha-coronavirus* of the family Coronaviridae, was first isolated in the UK in 1971. PEDV can infect pigs of all ages, but suckling piglets with underdeveloped immune systems are more susceptible, and the mortality rate after infection can reach 100%. Currently, there are no specific drugs against PEDV; vaccination, enhanced husbandry practices, and biosecurity measures are key to controlling porcine epidemic diarrhea.

[0003] PEDV is an enveloped, single-stranded, positive-sense RNA virus, approximately 28 kb in length, encoding four structural proteins: spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The PEDV S protein is a glycoprotein located on the viral surface, playing a crucial role in host cell invasion, viral virulence, and the induction of neutralizing antibodies. Based on function, the S protein can be divided into S1 and S2. The S1 domain facilitates receptor recognition, while the S2 domain anchors to the viral membrane and triggers membrane fusion; therefore, the S protein is a major virulence gene of PEDV. The S1 protein contains the virus's main neutralizing epitope, capable of stimulating the body to produce neutralizing antibodies, making the S1 protein a candidate for vaccine and diagnostic reagent development.

[0004] In the 1990s, scientists discovered unique antibodies in camels and sharks that naturally lacked the constant region 1 (CH1) of both the light and heavy chains, naming them heavy chain antibodies (HCAbs). Subsequent research revealed that the variable domain of the heavy chain (VHH) is the main functional domain for HCAbs to bind antigens. Cloning this domain resulted in VHHs, the smallest antibody with complete antibody function. With a small molecular weight of approximately 15 kDa and an ellipsoidal crystal structure (2.5 nm in diameter and 4 nm in length), they are also known as nanobodies (Nb). These are currently the smallest functional antibody fragments, capable of independently recognizing antigens. They possess characteristics such as small molecular weight, structural stability, high tolerance to strong acids and bases, high affinity, strong tissue penetration, and the ability to recognize antigen gap epitopes, allowing for further modification and expression. Based on these advantages, nanobodies show broad application prospects in antibody drug development, disease diagnosis, drug residue analysis, and environmental monitoring.

[0005] The small size endows nanobodies with dual pharmacokinetic characteristics. On the one hand, the small size of nanobodies allows for rapid penetration into tissues; on the other hand, the molecular weight of nanobodies is much lower than the renal cutoff value for glomerular filtration (60 kDa), and rapid renal clearance can easily reduce drug efficacy. This necessitates that nanobody drugs remain in the tissue for a sufficiently long time to be absorbed and delivered to the target organ to exert their therapeutic effect. In the actual development of nanobody drugs, pharmacokinetic characteristics can be improved by extending the drug half-life or using drug delivery and controlled-release systems. Multivalent antibodies are polymers of monovalent antibodies that recognize the same or different epitopes, possessing higher antigen affinity activity and significantly extended half-life in vivo, thus having a wider range of functions and applications. Due to their simple structure, nanobodies can be modified using molecular biology methods, easily producing multivalent or multispecific antibodies, and are easily expressed in large quantities through prokaryotic systems, significantly reducing production costs. Summary of the Invention

[0006] This invention provides a nanobody targeting the S1 protein of porcine epidemic diarrhea virus (PEDV). The provided nanobody can effectively bind to the S1 protein of PEDV while exhibiting stable PEDV neutralizing activity. It provides an effective biomolecular product for the prevention and control of porcine epidemic diarrhea.

[0007] This invention first provides a nanobody against the S1 protein of porcine epidemic diarrhea virus. The nanobody comprises three complementarity-determining regions, CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1, CDR2, and CDR3 are as follows:

[0008] CDR1 sequence: GMIFSANAEG (SEQ ID NO:1)

[0009] CDR2 sequence: AQTSEGSTKYADSV (SEQ ID NO:2)

[0010] CDR3 sequence: ALVMATISPEYNEDY (SEQ ID NO: 3);

[0011] The nanobody provided by this invention has a constant region comprising FR1, FR2, FR3, and FR4, and its amino acid sequence information is as follows:

[0012] FR1: QAGLQESFGYLFQSAGMLFLGCANS (SEQ ID NO: 4), FR2: WGRAFPGKMRELNA (SEQ ID NO: 5),

[0013] FR3: KFRYTIGRDNEKNTGYLQMGSLKPADTSVYMCQ (SEQ ID NO: 6),

[0014] FR4: WFQKTQVCVSD (SEQ ID NO:7).

[0015] The nanobody described herein has the following specific amino acid sequence:

[0016] QAGLQESFGYLFQSAGMLFLGCANSGMIFSANAEGWGRAFPGKMRELNAAQTSEGSTKYADSVKFRYTIGRDNEKNTGYLQMGSLKPADTSVYMCQALVMATISPEYNEDYWFQKTQVCVSD(SEQ ID NO:8);

[0017] The present invention also provides a nucleic acid fragment for encoding the above-mentioned nanobody, one specific sequence of which is as follows:

[0018] CAGGCCGGCCTGCAGGAGAGCTTCGGCTACCTGTTCCAGAGCGCCGGCATGCTGTTCCTGGGCTGCCAACAGCGGCATGATCTTCAGCGCCAACGCCGAGGGCTGGGGCAGGGCCTTCCCCGGCAAGATGAGGGAGCTGAACGCCGCCCAGACCAGCGAGGGCAGCACCAAGTACGCCGACAG CGTGAAGTTCAGGTACACCATCGGCAGGGACAACGAGAAGAACACCGGCTACCTGCAGATGGGCAGCCTGAAGCCCGCCGACACCAGCGTGTACATGTGCCAGGCCCTGGTGATGGCCACCATCAGCCCCGAGTACAACGAGGACTACTGGTTCCAGAAGACCCAGGTGTGCGTGAGCGAC(SEQ IDNO:9).

[0019] In another aspect, the present invention provides a recombinant expression vector for recombinantly expressing the above-mentioned nanobody;

[0020] The present invention also provides host cells containing the above-described recombinant expression vector.

[0021] As a specific example, the host cell is a competent BL21(DE3) cell;

[0022] The present invention also provides the use of the nanobody in the preparation of products for the prevention or treatment of porcine epidemic diarrhea virus.

[0023] This invention provides a nanobody capable of specifically recognizing and neutralizing porcine epidemic diarrhea virus (PEDV). The screened nanobody exhibits highly specific recognition and binding ability to PEDV, and has the potential for application in the preparation of preventive, therapeutic, or diagnostic reagents for PEDV. Attached Figure Description

[0024] Figure 1 The graph shows the serum titer of anti-S1 protein in alpacas.

[0025] Figure 2 This is an electrophoresis image of the VHH gene amplified by nested PCR in the first round.

[0026] Figure 3 This is an electrophoresis image of the VHH gene amplified by nested PCR in the second round.

[0027] Figure 4 This is a photograph of the volume of a nanobody phage library.

[0028] Figure 5 Photographs showing the positive rate of nanobody phage libraries.

[0029] Figure 6 This is a sequence diversity comparison diagram of a nanobody phage library.

[0030] Figure 7 This is an SDS-PAGE image of purified monovalent nanobodies.

[0031] Figure 8 This is a diagram illustrating the specificity identification of nanobodies.

[0032] Figure 9 The figure shows the results of the nanobody neutralization experiment.

[0033] Figure 10 This image shows the antiviral activity of monovalent nanobodies as verified by indirect immunofluorescence.

[0034] Figure 11 This is a graph showing the titer of viral progeny after treatment with monovalent nanobodies.

[0035] Figure 12 The graph shows the qRT-PCR assay results for the antiviral efficacy of monovalent nanobodies. Detailed Implementation

[0036] This invention involves immunizing alpacas with recombinant porcine epidemic diarrhea virus (PEDV) S1 protein expressed in vitro. Peripheral blood was collected from the alpacas after immunization, and total RNA was extracted and reverse transcribed into cDNA. The gene fragment encoding the variable region (VHH) of the alpaca heavy chain antibody was amplified using nested PCR. The VHH gene was ligated into a vector and transformed into competent cells to construct a phage-displaying nanobody library. Positive phages were screened and identified, and the target nanobody was obtained after sequencing and sequence alignment analysis.

[0037] The vocabulary used in this invention specification is described as follows:

[0038] Nanobodies: A type of naturally occurring antibody that lacks a light chain and contains only a heavy chain (heavy chain antibody). The variable region of this type of antibody is approximately 12-15 kDa, capable of recognizing and binding antigens with extremely high affinity; it is the smallest active antigen-binding fragment. Nanobodies are characterized by their small molecular weight, high affinity, high stability, and water solubility.

[0039] Nanobodies consist of a complementarity-determining region (CDR) and a constant region (also called a framework region) (FR). The CDRs include CDR1, CDR2, and CDR3, while the FRs include FR1, FR2, FR3, and FR4. The amino acid sequences of the constant regions are more conserved than those of the CDRs. However, due to amino acid sequence variations in the CDRs, different nanobodies exhibit different antibody titers.

[0040] The present invention will now be described in detail with reference to the embodiments.

[0041] Example 1: Construction of a PEDV S1 protein-specific nanobody phage library

[0042] 1. Alpaca Immunization

[0043] Each time, 2 mL of PEDV S12 recombinant protein at a concentration of 1.6 mg / mL was emulsified with an equal volume of 201 adjuvant and immunized in one adult male alpaca. Immunization was repeated every two weeks thereafter, for a total of five immunizations. One week after the last immunization, 20 mL of peripheral blood was collected for constructing a phage display library. The PEDV S1 antibody titer in alpaca serum was detected using indirect ELISA. The results are as follows: Figure 1 As shown, the titer reached 1:256000, indicating a good immunizing effect.

[0044] 2. Isolation of peripheral blood lymphocytes and extraction of RNA

[0045] The collected anticoagulated blood was diluted with culture medium and then isolated using an alpaca peripheral blood lymphocyte separation kit. The isolated cells were counted using a hemocytometer, and RNA was extracted directly using a total RNA extraction kit.

[0046] 3. Amplification of antibody variable region genes

[0047] First, first-strand cDNA was synthesized using reverse transcriptase with RNA as a template. Then, the VHH gene was amplified using nested PCR. The heavy chain variable regions of all immunoglobulins, including the VH region of the heavy chain and the VHH region of heavy chain antibodies, were amplified from the cDNA. Following the kit instructions, the approximately 700bp target fragment was recovered by agarose gel electrophoresis. The results are shown below. Figure 2 As shown. The second round of PCR was performed using this product as a template. After 1% agarose gel electrophoresis, a fragment of approximately 400 bp was excised and purified using a gel extraction kit to recover the target fragment VHH. The results are shown below. Figure 3 As shown.

[0048] The sequence information of the primers for the two rounds of nested PCR amplification is as follows:

[0049] VHH1-F:5′-GATGGTGGCTGCTCTTCTACTTCA-3′,

[0050] VHH1-R:5′-TGCGTGCTGTAATTGTTCC-3′;

[0051] VHH2-F:5′-GGTGGAGCTCCATCAGTCTGGGGGAGR-3′,

[0052] VHH2-R: 5′-CTAGTGCGGCCGCTGAGGAGACGGTGACCTGG GT-3′.

[0053] 4. Construction of phage display vectors

[0054] The VHH target gene fragment and the pCANTAB5E phage vector were digested with Pst I and Not I restriction endonucleases, respectively. After agarose gel electrophoresis, the fragments were purified using a gel extraction kit. The double-digested VHH target gene fragment was ligated to the pCANTAB5E phage vector using T4 DNA ligase.

[0055] 5. Harvest of phage antibody library

[0056] The ligation product was transferred into TG1 competent cells via electroporation at 2.5 kV for 5 ms. After electroporation, the cells were cultured at 37°C with shaking at 200 rpm for 1 h. 100 μL of the culture was collected for library identification, and the remaining culture was plated on LB / AMP-GLU plates and incubated overnight at 37°C. The next day, the bacterial colony was collected using a cell scraper, mixed with 1 / 3 volume of 50% glycerol, aliquoted, and stored at -80°C.

[0057] 6. Quality evaluation of phage libraries

[0058] Take 100 μL of the culture from the previous step, serially dilute it, and plate it on LB / AMP-GLU ampicillin plates. After overnight incubation, count the colonies on each dilution plate. Calculate the antibody library capacity based on the colony count and dilution factor. The results are as follows: Figure 4 As shown, the reservoir capacity is 6.4 × 10⁻⁶. 13 Twenty single colonies were randomly selected and cultured overnight in 1 mL LB medium. The positivity rate of the bacterial culture was identified by PCR using pCANTAB 5E-F and pCANTAB 5E-R primers. The results are as follows: Figure 5 As shown, the positive rate reached 96%. The positive clones were sent to Shanghai Bioengineering Technology Co., Ltd. for sequencing. The sequencing results were compared using MegAlign software to identify library diversity. The results are as follows: Figure 6As shown, the constructed library exhibits high diversity.

[0059] The sequence information of the positive identification sequencing primers is as follows:

[0060] pCANTAB5E-F:5′-AATACGCCAATGGCCTCTCG-3′,

[0061] pCANTAB5E-R: 5′-CTACTGCGGCAGCTGAGTAGACGGTGACCTGGGT-3′.

[0062] Example 2: Screening for specific nanobodies targeting the S1 protein

[0063] 1. Rescue of phage antibody libraries

[0064] Take 500 μL of the primary library and add it to 100 mL of 2×YT-A medium. Incubate at 37°C and 250 rpm until the OD600 reaches approximately 0.6. Add helper phage M13KO7 with an MOI of 20:1 and incubate at 37°C for 1 h. Centrifuge at 4000 rpm, discard the supernatant, and resuspend the precipitate in 200 mL of 2×YT-AK medium. Incubate overnight at 37°C and 220 rpm. Centrifuge again, collect the supernatant, add 40 mL of PEG / NaCl, and incubate at 4°C on ice for 4 h. Centrifuge at 8000 rpm for 20 min, discard the supernatant, and resuspend the phage precipitate in 2 mL of PBS solution for later use.

[0065] 2. Solid-phase panning of recombinant phages

[0066] PEDV S1 protein was coated onto the bottom of an ELISA plate, with PBS used as a control. After overnight coating at 4°C, the uncoated protein was washed off, and the plate was blocked with 1% BSA. After blocking, the blocking buffer was washed away, and the rescued library was added. After incubation, unbound phages were washed away. Triethylamine was used for phage elution, followed by infection, propagation, and titer determination. At least three rounds of washing were performed, and the enrichment was increased to greater than 1 × 10⁻⁶. 3 Then the selection process is considered complete.

[0067] 3. Identification of positive nanobody clones

[0068] Single colonies were picked from the plates diluted and spread after the third round of screening and inoculated into 1 mL of 2×TY-AG culture medium. OD 600When the concentration reaches 0.6, IPTG is added for induction. The culture is collected and subjected to three freeze-thaw cycles at -80℃. The supernatant is collected by centrifugation, which is the crude extract of soluble recombinant nanobody. PEDV S1 protein is coated onto a 96-well microplate (100 μL / well) and incubated overnight at 4℃. A negative control well is also included. After washing, the plate is blocked with 1% BSA. The crude extracted nanobody is added to each well, incubated, and unbound nanobody is washed away. Primary antibody is added, incubated, and unbound primary antibody is washed away. Enzyme-labeled secondary antibody is added, incubated, and unbound secondary antibody is washed away. TMB chromogenic solution is added, incubated, and stop solution is added. The OD450 value is measured using a microplate reader. Figure 7 The image shows the ELISA identification results of the nanobody positive clone.

[0069] 4. Sequencing analysis of specific nanobodies

[0070] Clones with positive ELISA results were selected and sent to the company for sequencing. The sequencing results were then compared using MegAlign software.

[0071] The nucleotide sequence of one type of nanobody is as follows:

[0072] CAGGCCGGCCTGCAGGAGAGCTTCGGCTACCTGTTCCAGAGCGCCGGCATGCTGTTCCTGGGCTGCCAACAGCGGCATGATCTTCAGCGCCAACGCCGAGGGCTGGGGCAGGGCCTTCCCCGGCAAGATGAGGGAGCTGAACGCCGCCCAGACCAGCGAGGGCAGCACCAAGTACGCCGAC AGCGTGAAGTTCAGGTACACCATCGGCAGGGACAACGAGAAGAACACCGGCTACCTGCAGATGGGCAGCCTGAAGCCCGCCGACACCAGCGTGTACATGTGCCAGGCCCTGGTGATGGCCACCATCAGCCCCGAGTACAACGAGGACTACTGGTTCCAGAAGACCCAGGTGTGCGTGAGCGAC.

[0073] The amino acid sequence of the encoded nanobody is as follows:

[0074] QAGLQESFGYLFQSAGMLFLGCANSGMIFSANAEGWGRAFPGKMRELNAAQTSEGSTKYADSVKFRYTIGRDNEKNTGYLQMGSLKPADTSVYMCQALVMATISPEYNEDYWFQKTQVCVSD.

[0075] The amino acid sequence from position 1 to 25 is FR1, the amino acid sequence from position 26 to 35 is CDR1, the amino acid sequence from position 36 to 49 is FR2, the amino acid sequence from position 50 to 63 is CDR2, the amino acid sequence from position 64 to 96 is FR3, the amino acid sequence from position 97 to 111 is CDR3, and the amino acid sequence from position 112 to 122 is FR4.

[0076] Example 3: Expression and purification of S1 protein-specific nanobodies

[0077] Induced expression of nanobodies

[0078] The pET-21a-Nb recombinant plasmid was synthesized by a biotechnology company and transformed into competent BL21(DE3) cells. Positive clones were identified by culture PCR. Positive clones were inoculated into culture medium, and after the OD600 reached approximately 0.6, 0.3 mM IPTG was added for induction. The induced bacterial culture was collected, centrifuged, resuspended in PBS, and then sonicated. The supernatant and precipitate were collected after centrifugation and analyzed by SDS-PAGE. The nanobody was found to express inclusion bodies.

[0079] 1. Purification of recombinant nanobody proteins

[0080] (1) Equilibration of Ni-NTA column. Add 5 mL of Ni Resin to the empty column to treat the column, then add the sample to be purified and incubate overnight at 4°C.

[0081] (2) Collect the flow solution, then use the prepared Buffer A to elute the impurities and collect the liquid. Elute the target protein with 1 mL of Buffer B each time, for a total of 5 elutions. Process the column after elution for future use.

[0082] (3) Take 100 μL of each of the collected liquid samples and perform SDS-PAGE analysis. The results are as follows: Figure 7 As shown, the purification effect is good.

[0083] 2. Refolding of recombinant nanobody proteins

[0084] The denaturation and purification buffer for the target protein contained 8M urea, so the urea concentration in the buffer needed to be gradually reduced (6M, 4M, 2M, 0M) to achieve renaturation of the target protein. The denatured and purified recombinant protein was added to a dialysis bag, and dialysis was started at 4°C with 6M urea dialysate. The dialysate was changed every 12 hours, and finally, the protein was dialyzed twice in PBS.

[0085] Example 4: Verification of the specificity and antiviral effect of nanobodies against PEDV

[0086] 1. Validation of nanobody specificity

[0087] The virus was coated onto an ELISA plate at a concentration of 10 μg / mL. CSFV, PRV, and PCV2 were also coated as controls, and the plate was incubated overnight. After washing, 1% BSA was added for blocking, followed by the addition of nanobodies. The plate was incubated at 37°C for 2 hours, then washed and rabbit anti-His antibody was added. After further washing, HRP-labeled anti-rabbit monoclonal antibody was added. After TMB color development, the OD was recorded. 450 Value. Result as follows Figure 8 As shown, the screened nanobodies do not bind to other viruses and exhibit good specificity.

[0088] 2. Neutralization experiment of nanobody

[0089] 100 μL of nanobody was serially diluted twofold. The diluted Nb was then mixed with an equal volume of 1000 TCID50. 50 Mix PEDV / mL and incubate at 37°C for 1 hour. Use PEDV-negative serum as a negative control. After 1 hour, add the PEDV-Nb mixture to a 96-well plate and incubate at 37°C for 1 hour. Change the medium and add trypsin treated with 5 μg / mL TPCK, and continue culturing at 37°C. After 36 hours, fix the cells with 4% paraformaldehyde, block with 0.1% Triton-100 and 5% skim milk, and then add rabbit polyclonal antibody against PEDV S1 protein and HRP-labeled goat anti-rabbit secondary antibody sequentially. After TMB staining, measure OD. 450 Value. Result Figure 9 The results showed that a Nb concentration of 50 μM could completely neutralize the virus.

[0090] 3. Indirect immunofluorescence assay to verify neutralization effect

[0091] Cells were cultured to a density of 80-90%, and diluted Nb was then mixed with an equal volume of 1000 TCID50. 50 Mix with / mL PEDV, incubate at 37℃ for 1 h, add pure culture medium as a negative control, and incubate for 36 h. Wash three times with DPBS, then add 4% paraformaldehyde, 1% BSA solution and 0.1% Triton X-100 sequentially, incubating for 30 min each time. Subsequently, add rabbit polyclonal antibody against S1 protein and FITC-labeled goat anti-rabbit antibody sequentially, and incubate at room temperature for 1 h. Wash, add DAPI staining solution and stain for 10 min, then observe fluorescence under a fluorescence microscope. Results are as follows. Figure 10 As shown, N has good antiviral activity, and the effect varies in a concentration-dependent manner.

[0092] 4. TCID 50 qRT-PCR for detecting viral infection

[0093] Vero is 1×10 6Nbs were seeded at a density of 1000 nucleotides / mL into 96-well plates for approximately 24 hours. The Nbs were then diluted to different concentrations (5, 10, or 20 μM) with DMEM and treated with 1000 TCID50 solution. 50 Cells were treated with 1 / mLPEDV. After incubation at 37°C and 5% CO2 for 2 hours, the incubation medium was removed, and the cells were washed three times with PBS. Fresh DMEM containing 5 μg / mL TPCK was added. Cells were cultured for another 36 hours, and the cell supernatant was collected for progeny virus titration. Cells were then collected for real-time quantitative PCR analysis. The results are as follows: Figure 11 and Figure 12 As shown, the viral progeny titer and N gene expression level were significantly reduced after treatment with nanobodies, consistent with the IFA results.

[0094] In summary, the nanobodies provided by this invention can efficiently bind to the S1 protein of porcine epidemic diarrhea virus. The nanobodies exhibit high stability against extreme pH, high temperatures, and protein degradation. Based on these unique biological properties, they have broad application potential in the prevention or treatment of porcine epidemic diarrhea virus diseases.

Claims

1. A nanobody against the S1 protein of porcine epidemic diarrhea virus, characterized in that, The nanobody comprises three complementarity-determining regions CDR1, CDR2, and CDR3 and four constant regions FR1, FR2, FR3, and FR4, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 are SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

2. The nanobody as described in claim 1, characterized in that, The amino acid sequences of the constant regions FR1, FR2, FR3 and FR4 are SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively.

3. The nanobody as described in claim 1, characterized in that, The amino acid sequence of the nanobody is SEQ ID NO:

8.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecular segment is used to encode the nanobody of claim 1.

5. The nucleic acid molecule as described in claim 4, characterized in that, The sequence of the nucleic acid molecule is SEQ ID NO:

9.

6. A recombinant expression vector, characterized in that, The recombinant expression vector is used for recombinant expression of the nanobody of claim 1.

7. A host cell, characterized in that, The host cell carries the recombinant expression vector of claim 6.

8. The host cell as described in claim 7, characterized in that, The host cell is a competent BL21(DE3) cell.

9. The use of the nanobody of claim 1 in the preparation of articles for the prevention or treatment of porcine epidemic diarrhea virus.

10. A product for the prevention or treatment of porcine epidemic diarrhea virus, characterized in that, The product contains the nanobody as described in claim 1.

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