Nanobodies against pedv n protein and uses thereof

Abstract

The application relates to the technical field of nanobody, and specifically provides a nanobody against PEDV N protein and application thereof. The nanobody comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein the CDR1 is selected from the amino acid sequence shown in SEQ ID NO. 1; the CDR2 is selected from the amino acid sequence shown in SEQ ID NO. 2; and the CDR3 is selected from the amino acid sequence shown in SEQ ID NO. 3. The nanobody provided by the application can specifically recognize the PEDV N protein, and the antibody titer is greater than 512K and the sensitivity is high.

Description

Technical Field

[0001] This application relates to the field of nanobody technology, specifically providing a nanobody against PEDV N protein and its application. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDV). Clinical symptoms in affected pigs include watery diarrhea, vomiting, and weight loss. Pigs of all ages can be affected, but newborn piglets are more susceptible, with a morbidity rate as high as 100% and a mortality rate of (90-100)%. Currently, there are no specific drugs against PEDV; vaccination, improved husbandry practices, and biosecurity measures are key to controlling PED.

[0003] PEDV is a single-stranded positive-sense RNA virus with a genome size of approximately 28,000 nt. It has seven open reading frames (ORFs) encoding three non-structural proteins (ORF1a, ORF1b, and ORF3) and four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). These structural proteins play crucial roles in PEDV adhesion, invasion, replication, transcription, assembly, and immune evasion. The N protein, an RNA-binding protein located inside the viral particle, has a molecular weight of (55-58) kDa. The N protein and viral genomic RNA form a helical structure that constitutes the viral nucleocapsid. The N protein also binds to the cell membrane, providing a basis for viral RNA replication, transcription, and assembly. Furthermore, the N protein plays an important role in inducing cellular and humoral immunity; studies have confirmed that the N protein is an effective immune adjuvant.

[0004] Nanobodies were discovered by Professor Hamers and his team at the Free University of Brussels, Belgium. A naturally occurring heavy chain antibody (HcAbs) lacking both the light chain and the constant region of the first heavy chain exists in camels; its antigen-binding domain consists of only a single variable region (VHH) of the heavy chain. Subsequent genomic studies revealed that the DNA encoding the constant region of the first heavy chain is spliced ​​out during mRNA processing, thus generating this type of heavy chain antibody lacking both the light chain and the constant region of the first heavy chain. The VHH is currently the smallest known naturally occurring antibody fragment with antigen recognition function, approximately 15 kDa. Due to its nanoscale size (approximately 2.5 nm in diameter and 4 nm in height), it is also known as a nanobody. Nanobodies possess advantages such as excellent affinity, organic solvent tolerance, and stability, and have gradually become a low-cost, high-performance emerging detection antibody. However, compared to nanobodies against human viruses, research on nanobodies against animal viruses is relatively scarce. Summary of the Invention

[0005] In view of this, this application provides a nanobody against PEDV N protein and its use.

[0006] In a first aspect, this application provides a nanobody against PEDV N protein, the nanobody comprising three complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0007] The CDR1 is selected from the amino acid sequence shown in SEQ ID NO.1;

[0008] The CDR2 is selected from the amino acid sequence shown in SEQ ID NO.2;

[0009] The CDR3 is selected from the amino acid sequence shown in SEQ ID NO.3.

[0010] In some alternative embodiments, the nanobody comprises four framework regions FR1, FR2, FR3, and FR4, wherein:

[0011] The FR1 includes the amino acid sequence shown in SEQ ID NO.4, or an amino acid sequence containing one or more (e.g., 1, 2, 3, 4 or 5) amino acids substituted, deleted or inserted, or any combination thereof relative to SEQ ID NO.4, or is composed of the amino acid sequence shown in SEQ ID NO.4;

[0012] And / or, the FR2 comprises an amino acid sequence as shown in SEQ ID NO.5, or an amino acid sequence containing one or more (e.g., 1, 2, 3, 4 or 5, etc.) amino acids substituted, deleted or inserted or any combination thereof relative to SEQ ID NO.5, or composed of an amino acid sequence as shown in SEQ ID NO.5;

[0013] And / or, the FR3 comprises an amino acid sequence as shown in SEQ ID NO. 6, or an amino acid sequence containing one or more (e.g., 1, 2, 3, 4 or 5, etc.) amino acids substituted, deleted or inserted or any combination thereof relative to SEQ ID NO. 6, or composed of an amino acid sequence as shown in SEQ ID NO. 6;

[0014] And / or, the FR4 comprises an amino acid sequence as shown in SEQ ID NO.7, or an amino acid sequence containing one or more (e.g., 1, 2, 3, 4 or 5, etc.) amino acids substituted, deleted or inserted, or any combination thereof relative to SEQ ID NO.7, or is composed of an amino acid sequence as shown in SEQ ID NO.7.

[0015] In some alternative embodiments, the nanobody comprises an amino acid sequence as shown in SEQ ID NO. 8, or an amino acid sequence containing one or more amino acid substitutions, deletions, or insertions, or any combination thereof, relative to SEQ ID NO. 8 and other than SEQ ID NO. 1, 2, 3, or composed of an amino acid sequence as shown in SEQ ID NO. 8.

[0016] The nanobody of this application includes a complementarity determinant region (CDR) and spaced framework regions (FR), with the domains arranged in the pattern FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; wherein substitution, deletion or insertion of one or more amino acids or any combination thereof occurs in the framework regions FR1, FR2, FR3, and FR4.

[0017] In some specific embodiments, the FR1 consists of an amino acid sequence as shown in SEQ ID NO.4.

[0018] In some specific embodiments, the FR2 consists of an amino acid sequence as shown in SEQ ID NO.5.

[0019] In some specific embodiments, the FR3 consists of an amino acid sequence as shown in SEQ ID NO. 6.

[0020] In some specific embodiments, the FR4 consists of an amino acid sequence as shown in SEQ ID NO.7.

[0021] In some alternative embodiments, the nanobody comprises or is composed of the amino acid sequence shown in SEQ ID NO. 8. In some specific embodiments, the nanobody comprises the amino acid sequence shown in SEQ ID NO. 8. In other specific embodiments, the nanobody is composed of the amino acid sequence shown in SEQ ID NO. 8.

[0022] In a second aspect of this application, this application provides a nucleic acid molecule encoding the nanobody described in the first aspect of this application.

[0023] Based on the amino acid sequence of the nanobody described in the first aspect of this application, those skilled in the art can obtain the nucleic acid molecule encoding the nanobody described in the first aspect of this application. Due to the degeneracy of codons, the nucleotide sequence of a nucleic acid molecule is not unique, and all nucleic acid molecules capable of encoding the nanobody described in the first aspect of this application are within the scope of protection of this application.

[0024] In some specific embodiments, the nucleic acid molecule includes nucleotide sequence I encoding CDR1, nucleotide sequence II encoding CDR2, and nucleotide sequence III encoding CDR3, wherein:

[0025] The nucleotide sequence I is selected from the nucleotide sequence shown in SEQ ID NO. 9;

[0026] The nucleotide sequence II is selected from the nucleotide sequence shown in SEQ ID NO. 10;

[0027] Nucleotide sequence III is selected from the nucleotide sequence shown in SEQ ID NO.11.

[0028] In some alternative embodiments, the nucleic acid molecule further includes nucleotide sequence IV encoding the FR1, nucleotide sequence V encoding the FR1, nucleotide sequence VI encoding the FR1, and nucleotide sequence VII encoding the FR1, wherein:

[0029] The nucleotide sequence IV includes the nucleotide sequence shown in SEQ ID NO. 12, or a nucleotide sequence containing one or more nucleotide substitutions, deletions or insertions relative to SEQ ID NO. 12, or any combination thereof, or is composed of the nucleotide sequence shown in SEQ ID NO. 12;

[0030] And / or, the nucleotide sequence V comprises a nucleotide sequence as shown in SEQ ID NO. 13, or a nucleotide sequence containing one or more nucleotide substitutions, deletions or insertions relative to SEQ ID NO. 13, or any combination thereof, or is composed of a nucleotide sequence as shown in SEQ ID NO. 13;

[0031] And / or, the nucleotide sequence VI comprises the nucleotide sequence shown in SEQ ID NO. 14, or a nucleotide sequence containing one or more nucleotide substitutions, deletions or insertions or any combination thereof relative to SEQ ID NO. 14, or is composed of the nucleotide sequence shown in SEQ ID NO. 14;

[0032] And / or, the nucleotide sequence VII comprises a nucleotide sequence as shown in SEQ ID NO. 15, or a nucleotide sequence containing one or more substitutions, deletions or insertions of nucleotides relative to SEQ ID NO. 15, or any combination thereof, or is composed of a nucleotide sequence as shown in SEQ ID NO. 15.

[0033] In some alternative embodiments, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO. 16, or a nucleotide sequence containing one or more substitutions, deletions or insertions of nucleotides or any combination thereof relative to SEQ ID NO. 16 and other than SEQ ID NO. 9, 10, 11, or composed of a nucleotide sequence as shown in SEQ ID NO. 16.

[0034] In some specific embodiments, the nucleotide sequence IV consists of a nucleotide sequence as shown in SEQ ID NO.12.

[0035] In some specific embodiments, the nucleotide sequence V consists of a nucleotide sequence as shown in SEQ ID NO.13.

[0036] In some specific embodiments, the nucleotide sequence VI consists of a nucleotide sequence as shown in SEQ ID NO.14.

[0037] In some specific embodiments, the nucleotide sequence VII consists of a nucleotide sequence as shown in SEQ ID NO.15.

[0038] In some alternative embodiments, the nucleic acid molecule comprises or is composed of the nucleotide sequence shown in SEQ ID NO. 16. In some specific embodiments, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO. 16. In other specific embodiments, the nucleic acid molecule is composed of the nucleotide sequence shown in SEQ ID NO. 16.

[0039] In a third aspect, this application provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect of this application.

[0040] In some alternative embodiments, the recombinant expression vector includes, but is not limited to, plasmid vectors, phage vectors, viral vectors, artificial chromosome vectors, etc.

[0041] In a fourth aspect of this application, this application provides a host cell comprising the nucleic acid molecule described in the second aspect of this application and / or the recombinant expression vector described in the third aspect of this application, or the host cell being transformed or transfected by the nucleic acid molecule described in the second aspect of this application and / or the recombinant expression vector described in the third aspect of this application.

[0042] In some alternative implementations, the host cell includes, but is not limited to, microbial cells, insect cells, or other animal cells.

[0043] In some alternative embodiments, the host cell is selected from prokaryotic or eukaryotic cells. In some specific embodiments, the host cell is selected from prokaryotic cells (e.g., competent BL21(DE3) cells). In other specific embodiments, the host cell is selected from competent BL21(DE3) cells.

[0044] In a fifth aspect of this application, this application provides the use of the nanobody described in the first aspect of this application, and / or the nucleic acid molecule described in the second aspect of this application, and / or the recombinant expression vector described in the third aspect of this application, and / or the host cell described in the fourth aspect of this application in the preparation of products for detecting PEDV antibodies, and / or the preparation of products for preventing diseases or conditions caused by PEDV, and / or the preparation of products for treating diseases or conditions caused by PEDV.

[0045] In a sixth aspect of this application, this application provides a pharmaceutical composition comprising the nanobody described in the first aspect of this application, and / or the nucleic acid molecule described in the second aspect of this application, and / or the recombinant expression vector described in the third aspect of this application, and / or the host cell described in the fourth aspect of this application.

[0046] In a seventh aspect of this application, this application provides a vaccine composition comprising the nanobody described in the first aspect of this application, and / or the nucleic acid molecule described in the second aspect of this application, and / or the recombinant expression vector described in the third aspect of this application, and / or the host cell described in the fourth aspect of this application.

[0047] In an eighth aspect of this application, this application provides a detection reagent or kit for detecting PEDV antibodies, said detection reagent or kit comprising the nanobody described in the first aspect of this application.

[0048] In some alternative implementations, the detection kit is selected from enzyme-linked immunosorbent assay (ELISA) kits, fluorescence immunoassay kits, or chemiluminescence immunoassay kits.

[0049] This application has the following beneficial effects:

[0050] First, the nanobody of this application can specifically recognize PEDV N protein, and the antibody titer is >512K, which is highly sensitive.

[0051] Secondly, since the nanobody of this application can specifically recognize PEDV N protein, it is expected that the nanobody of this application can be used to prepare products for detecting PEDV antibodies, products for preventing diseases or conditions caused by PEDV, and products for treating diseases or conditions caused by PEDV. Attached Figure Description

[0052] Figure 1 SDS-PAGE analysis was performed to determine the expression of PEDV N protein in BL21(DE3).

[0053] Figure 2 The results of the fifth and sixth purifications of PEDV N protein were analyzed by SDS-PAGE.

[0054] Figure 3 This is the fifth immune serum antibody titer test.

[0055] Figure 4 This is a flow cytometry sorting diagram of peripheral blood in alpacas.

[0056] Figure 5 This is a PCR amplification diagram of the heavy chain variable region.

[0057] Figure 6 The expression status of antibody at BL21(DE3).

[0058] Figure 7 The antibody purification results were analyzed using SDS-PAGE.

[0059] Figure 8 For antibody sensitivity and specificity detection. Detailed Implementation

[0060] This application discloses a nanobody against PEDV N protein. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve its effect. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this application. The methods and applications of this application have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this application to realize and apply the technology of this application.

[0061] The operations such as "coating", "sealing", "washing", "drying", "patting dry" and "incubation" mentioned in this application are all routine experimental operations in the field. Those skilled in the art should understand the specific meaning, implementation process and method of the operations.

[0062] Unless otherwise stated, all experimental methods involved in this application are conventional methods.

[0063] Unless otherwise specified, all reagents used in the experiments are of analytical grade, and the water used in the experiments shall meet the requirements of Grade I water in GB / T 6682.

[0064] To make the objectives, technical solutions, and advantages of this application clearer, the implementation schemes of this application will be further described in detail below with reference to the embodiments.

[0065] Example 1: Expression and purification of PEDV N protein:

[0066] (1-1) Expression and identification of PEDV N protein:

[0067] The plasmid containing the PEDV N gene was transformed into BL21(DE3) competent cells, then evenly spread onto LB agar plates (containing 50 μg / mL kanamycin sulfate), and incubated overnight at 37°C. Single colonies were selected from the transformed plates and inoculated into 4 mL of LB medium (containing 50 μg / mL kanamycin sulfate), and cultured until OD500 reached. 600 The concentration was 0.5-0.8. Isopropyl-β-d-galactoside (IPTG, CAS No. 367-93-1) was added to the culture medium in test tubes to a final concentration of 0.2 mM, and then the tubes were incubated at 15℃ and 37℃ to induce expression.

[0068] Centrifuge the induced culture medium at 12000 rpm for 5 min, discard the supernatant, resuspend the precipitate in PBS buffer, and finally add SDS-PAGE protein loading buffer. Heat the sample at 100℃ for 10 min, then centrifuge and collect the supernatant for electrophoresis. Whole bacteria were lysed using 20 mM Tris (pH 8.0), 300 mM NaCl, 20 mM Imidazole containing 1% Triton X-100, 1 mM DTT, and 1 mM MPMSF by sonication. The supernatant and precipitate were then analyzed by SDS-PAGE. The results showed that PEDV N protein was expressed in both the supernatant and inclusion bodies after induction. Figure 1 ).

[0069] (1-2) Purification of PEDV N protein:

[0070] PEDV N protein in the supernatant was purified by affinity chromatography. Whole bacteria were lysed by sonication in a buffer of 50 mM Tris (pH 8.0), 300 mM NaCl, 20 mM Imidazole containing 1% Triton X-114, 1% Triton X-100, 1 mM DTT, and 1 mM PMSF. Simultaneously, the Ni-IDA affinity chromatography column was equilibrated with a buffer of 50 mM Tris (pH 8.0), 300 mM NaCl, and 20 mM Imidazole. The target protein was then eluted with equilibration buffers containing different concentrations of imidazole, and each eluted fraction was collected for SDS-PAGE analysis.

[0071] After five rounds of Ni-IDA affinity chromatography purification analysis, the target protein purity reached the expected level. The elution fraction from the sixth round was collected and dialyzed into 1×PBS, pH 7.4. After dialysis, the sample was filtered through a 0.22 μm filter, and the purified protein concentration was determined to be 0.5 mg / mL with a purity >90%. Figure 2 ).

[0072] Example 2: Rapid development of singleB monoclonal antibodies:

[0073] (2-1) Animal immunization:

[0074] One healthy, age-appropriate alpaca with no prior immunization was selected as the immunization subject. The antigen solution and Freund's adjuvant were mixed at a volume ratio of 1:1, and the injection dose was 0.5 mg per alpaca per dose. The alpaca was immobilized, and the drug was injected subcutaneously on both sides near the cervical lymph nodes at a 30-degree angle. The drug was injected slowly, and a total of 5 immunizations were administered, with each immunization two weeks apart. One week after the last immunization, serum antibody titers were measured. The results are as follows: Figure 3 As shown, the titer reaches 128K, indicating a good immune effect.

[0075] (2-2) B cell sorting:

[0076] Peripheral blood was collected from alpacas to prepare peripheral blood mononuclear cells (PBMCs). Transfer alpaca peripheral blood to 50mL centrifuge tubes, add an equal volume of SOL001 solution for dilution, and mix gently. Take an appropriate number of new sterile centrifuge tubes, first add Ficoll-Paque PLUS cell separation medium, then gently spread the diluted alpaca peripheral blood onto the Ficoll-Paque PLUS cell separation medium in the centrifuge tubes, with a volume ratio of separation medium to cell suspension of 1:2. Place the spread sample in a centrifuge at 2000 rpm for 20 minutes at room temperature. After centrifugation, remove the centrifuge tubes, aspirate the single nuclear layer and place it in a new 50mL sterile centrifuge tube, add SOL001 solution and bring the volume to 50mL, centrifuge at 1500 rpm for 10 minutes at room temperature. Discard the supernatant. Wash the cells with SOL024 solution once more. Resuspend the cells in 1mL SOL024 solution and store at 4℃ for sorting. PEDV N protein was fluorescently labeled. Using a flow cytometer, the fluorescence signal of the PEDV N protein antigen and the fluorescence signal after B cell staining were detected to sort antigen-affinity individual B cells into 96-well plates. Figure 4 As shown, 336 B cells that bind to the antigen were eventually isolated.

[0077] The SOL001 solution was 0.01 M PBS (pH=7.4): Weigh 8.0 g NaCl, 0.2 g KCl, 3.63 g Na2HPO4•12H2O, and 0.24 g KH2PO4, add pure water to 1000 mL, filter through a 0.22 μm filter, and adjust the pH to 7.4.

[0078] SOL023 solution is 0.5 M EDTA: Weigh 1.86 g of EDTANa2•2H2O and dissolve it in 10 mL of DEPC H2O. Adjust the pH to 8.0 until the sodium EDTA salt is completely dissolved. Filter through a 0.22 µm filter membrane and dispense for later use.

[0079] SOL024 solution is a FACS buffer (FB) solution: 10 mL of fetal bovine serum and 2 mL of SOL023 solution are added to 500 mL of SOL001 solution to prepare FB solution.

[0080] (2-3) B cell cloning and expression plasmid construction:

[0081] B cells were lysed to release RNA, which was then converted to cDNA via reverse transcription. Using the cDNA as a template, the variable region fragment was amplified by specific primers for alpaca heavy chain PCR. This variable region gene was ligated into the pcDNA3.4 vector containing the constant region of the alpaca heavy chain antibody. Colony PCR identification yielded 52 positive clones. Figure 5 ).

[0082] (2-4) High-throughput expression and cell supernatant detection:

[0083] The day before transfection, HEK293 cells were seeded to ensure that the cells grew to (3.0-5.0) × 10⁻⁶ on the day of transfection. 6 Cells / mL, viability 98%, cell suspension diluted with culture medium, 400 μl per well in 96-well plates. Transfection buffer was added to each well of a 96-well plate containing HEK293 cells, 100 μl per well; plasmid was added and mixed by pipetting; transfection reagent was added and mixed by pipetting; after incubation at 37°C for 10 min, the mixture was transferred to 96-well plates for further culture. 20 h after transfection, 25 μl of 293F Hi-exp feed (purchased from OPMA, catalog number AC601502) was added to each well of the 96-well plate. After expression for 4-5 days, the cells were centrifuged at 4000 rpm for 10 min, and the supernatant was collected for ELISA detection. The results showed 16 positive clones, one of which was selected for sequencing. The nucleotide sequence was obtained as shown in SEQ ID NO. 16.

[0084] Table 1. Nucleotide sequences:

[0085]

[0086] The nucleotide sequence SEQ ID NO.9 is expected to express the amino acid sequence SEQ ID NO.1;

[0087] Nucleotide sequence SEQ ID NO.10; Expected amino acid sequence SEQ ID NO.2;

[0088] Nucleotide sequence SEQ ID NO.11; Expected expressed amino acid sequence SEQ ID NO.3;

[0089] Nucleotide sequence SEQ ID NO.12; Expected amino acid sequence SEQ ID NO.4;

[0090] Nucleotide sequence SEQ ID NO.13; Expected amino acid sequence SEQ ID NO.5;

[0091] Nucleotide sequence SEQ ID NO.14; Expected amino acid sequence SEQ ID NO.6;

[0092] Nucleotide sequence SEQ ID NO.15; Expected amino acid sequence SEQ ID NO.7;

[0093] Nucleotide sequence SEQ ID NO.16 Expected amino acid sequence SEQ ID NO.8.

[0094] Table 2. Amino acid sequence:

[0095]

[0096] Example 3, Antibody Expression and Purification:

[0097] (3-1) Antibody expression:

[0098] Plasmids containing the antibody gene were transformed into BL21(DE3) competent cells and then evenly spread onto LB agar plates (containing 50 μg / mL kanamycin sulfate). The plates were then incubated overnight at 37°C. Single clones were selected from the transformed plates and inoculated into 4 mL of LB medium (containing 50 μg / mL kanamycin sulfate). After incubation until the OD600 reached 0.5-0.8, 0.2 mM IPTG was added to the culture medium in the tubes, and expression was induced at 15°C and 37°C, respectively. The induced culture medium was centrifuged at 12000 rpm for 5 min, the supernatant was removed, the precipitate was resuspended in PBS, and finally, SDS-PAGE loading buffer was added. The sample was heated at 100°C for 10 min, then centrifuged again, and the supernatant was collected for electrophoresis. Whole bacteria were lysed by sonication using 20 mM Tris (pH 8.0), 300 mM NaCl, 20 mM Imidazole containing 1% Triton X-100, 1 mM DTT, and 1 mM PMSF. The supernatant and precipitate were then analyzed by SDS-PAGE. Analysis of whole-bacterial lysis by sonication showed no significant expression after induction at 15℃, and no significant expression in the supernatant after induction at 37℃, but inclusion body expression was observed. Figure 6 ).

[0099] (3-2) Antibody purification:

[0100] Inclusion bodies were washed with 50 mM Tris (pH 8.0), 300 mM NaCl containing 1% Triton X-100, 2 mM EDTA, and 5 mM DTT. The inclusion bodies were then dissolved in a buffer of 50 mM Tris (pH 8.0), 300 mM NaCl, 8 M Urea, and 20 mM Imidazole while simultaneously equilibrating the Ni-IDA column. Finally, the target protein was eluted with equilibration buffers containing different concentrations of imidazole, and each eluted fraction was collected for SDS-PAGE analysis. Figure 7After purification analysis by Ni-IDA affinity chromatography, Lane 8-9 with relatively high purity was collected and added to the treated dialysis bag. The protein was dialyzed into buffer [1×PBS (pH 7.2), 4 mM GSH, 0.4 mM GSSG, 0.4 M L-Arginine, 1 M Urea] at 4°C for renaturation. After renaturation, the DT10099-1A-1M1E1 protein was finally dialyzed into a storage solution of 20 mm PBS, 150 mm NaCl, pH 7.2 for 6-8 h. After dialysis and renaturation, the supernatant was filtered through a 0.22 μm filter and aliquoted and frozen at -80°C.

[0101] Example 4: Detection of specificity and sensitivity of purified antibodies:

[0102] (4-1) Antigen coating: Dilute PEDV N protein and laboratory-preserved PRRSV GP5 protein to 2 μg / ml with 10 mM PBS buffer, add 100 μl to each well of a 96-well microplate, and incubate overnight at 4°C. Wash the coated microplate twice with a plate washer and dry it. Add 200 μl of blocking buffer to each well and incubate at 37°C for 2 h. Then wash the blocked microplate twice with a plate washer and dry it.

[0103] (4-2) Incubation of primary antibody: PBS is used as negative control. The antibody is diluted to 2 μg / mL and serially diluted 2 times. 50 μL is added to each well and incubated at 37℃ for 1.5 h. After incubation, the plate is washed 3 times in a plate washer and then dried.

[0104] (4-3) Incubation of secondary antibody: Add 100 μl of HRP-labeled VHH secondary antibody to each well and incubate at 37°C for 45 min. Wash the plate 5 times in a plate washer and then dry it.

[0105] (4-4) Color development: Add 100 μl of color development solution to each well of the 96-well plate and incubate at 37°C for 15 min.

[0106] (4-5) Termination and reading: Add 50 μl of stop solution to each well of the 96-well plate, and then place it in an ELISA reader for reading. Set the detection wavelength to 450 nm and read the test results. An OD value greater than 2.1 times that of the negative control is considered positive.

[0107] The results are as follows Figure 8 As shown, this nanobody can specifically recognize PEDV N protein, and its antibody titer is >512K, indicating high sensitivity.

[0108] The above provides a detailed description of the anti-PEDV N protein nanobody provided in this application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are merely for the purpose of helping to understand the method and core ideas of this application. It should be noted that those skilled in the art can make several improvements and modifications to this application without departing from the principles of this application, and these improvements and modifications also fall within the protection scope of the claims of this application.

Claims

1. A Nanobody against PEDV N protein, characterized in that, The nanobody comprises three complementarity-determining regions, CDR1, CDR2, and CDR3, wherein: The amino acid sequence of CDR1 is shown in SEQ ID NO.1; The amino acid sequence of CDR2 is shown in SEQ ID NO.2; The amino acid sequence of CDR3 is shown in SEQ ID NO.

3.

2. The Nanobody according to claim 1, characterized in that, The nanobody comprises four framework regions: FR1, FR2, FR3, and FR4, wherein: The FR1 includes an amino acid sequence as shown in SEQ ID NO.4, or an amino acid sequence containing one or more amino acid substitutions, deletions or insertions, or any combination thereof relative to SEQ ID NO.4; And / or, the FR2 comprises an amino acid sequence as shown in SEQ ID NO.5, or an amino acid sequence containing one or more amino acid substitutions, deletions or insertions or any combination thereof relative to SEQ ID NO.5; And / or, the FR3 comprises an amino acid sequence as shown in SEQ ID NO. 6, or an amino acid sequence containing one or more amino acid substitutions, deletions or insertions or any combination thereof relative to SEQ ID NO. 6; And / or, the FR4 comprises an amino acid sequence as shown in SEQ ID NO.7, or an amino acid sequence containing one or more amino acid substitutions, deletions or insertions, or any combination thereof relative to SEQ ID NO.

7.

3. The nanobody according to claim 2, characterized in that, The FR1 consists of an amino acid sequence as shown in SEQ ID NO.4; And / or, the FR2 consists of an amino acid sequence as shown in SEQ ID NO.5; And / or, the FR3 consists of an amino acid sequence as shown in SEQ ID NO. 6; And / or, the FR4 consists of an amino acid sequence as shown in SEQ ID NO.

7.

4. The Nanobody according to claim 1, characterized in that, The nanobody comprises an amino acid sequence as shown in SEQ ID NO.8, or an amino acid sequence containing one or more amino acid substitutions, deletions, or insertions, or any combination thereof, relative to SEQ ID NO.8 and other than SEQ ID NO.1, 2, and 3.

5. The Nanobody according to claim 4, characterized in that, The nanobody consists of an amino acid sequence as shown in SEQ ID NO.

8.

6. A nucleic acid molecule encoding the nanobody according to any one of claims 1 to 5.

7. The nucleic acid molecule according to claim 6, characterized in that, The nucleic acid molecule includes nucleotide sequence I encoding CDR1, nucleotide sequence II encoding CDR2, and nucleotide sequence III encoding CDR3, wherein: The nucleotide sequence of nucleotide sequence I is shown in SEQ ID NO.9; The nucleotide sequence of nucleotide sequence II is shown in SEQ ID NO.10; The nucleotide sequence of nucleotide sequence III is shown in SEQ ID NO.

11.

8. The nucleic acid molecule of claim 7, wherein, The nucleic acid molecule further includes nucleotide sequence IV encoding FR1, nucleotide sequence V encoding FR2, nucleotide sequence VI encoding FR3, and nucleotide sequence VII encoding FR4, wherein: The nucleotide sequence IV includes the nucleotide sequence shown in SEQ ID NO.12, or a nucleotide sequence containing one or more nucleotide substitutions, deletions or insertions relative to SEQ ID NO.12, or any combination thereof; And / or, the nucleotide sequence V comprises a nucleotide sequence as shown in SEQ ID NO.13, or a nucleotide sequence containing one or more nucleotide substitutions, deletions or insertions relative to SEQ ID NO.13, or any combination thereof; And / or, the nucleotide sequence VI includes the nucleotide sequence shown in SEQ ID NO.14, or a nucleotide sequence containing one or more nucleotide substitutions, deletions or insertions relative to SEQ ID NO.14, or any combination thereof; And / or, the nucleotide sequence VII comprises a nucleotide sequence as shown in SEQ ID NO.15, or a nucleotide sequence containing one or more substitutions, deletions or insertions of nucleotides relative to SEQ ID NO.15, or any combination thereof.

9. The nucleic acid molecule of claim 8, wherein, The nucleotide sequence IV consists of a nucleotide sequence as shown in SEQ ID NO. 12; And / or, the nucleotide sequence V consists of the nucleotide sequence shown in SEQ ID NO.13; And / or, the nucleotide sequence VI is composed of the nucleotide sequence shown in SEQ ID NO.14; And / or, the nucleotide sequence VII consists of a nucleotide sequence as shown in SEQ ID NO.

15.

10. The nucleic acid molecule according to claim 7, characterized in that, The nucleic acid molecule includes a nucleotide sequence as shown in SEQ ID NO. 16, or a nucleotide sequence fragment containing one or more nucleotide substitutions, deletions or insertions, or any combination thereof, relative to SEQ ID NO. 16 and other than SEQ ID NO. 9, 10, 11.

11. The nucleic acid molecule of claim 10, wherein The nucleic acid molecule consists of a nucleotide sequence as shown in SEQ ID NO.

16.

12. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in any one of claims 6 to 11.

13. A host cell characterized in that, The host cell comprises the nucleic acid molecule according to any one of claims 6 to 11 and / or the recombinant expression vector according to claim 12.

14. A host cell characterized in that, The host cell is transformed or transfected by the nucleic acid molecule according to any one of claims 6 to 11 and / or the recombinant expression vector according to claim 12.

15. The host cell of claim 13 or 14, characterized in that, The host cell is selected from prokaryotic cells or eukaryotic cells.

16. A detection reagent or a detection kit for detecting an antibody against PEDV, characterized in that, The detection reagent or detection kit includes the nanobody as described in any one of claims 1 to 5.

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