Porcine delta coronavirus n protein monoclonal antibody and latex immunochromatography test strip containing the same

Abstract

This invention belongs to the field of virus detection technology, specifically relating to a monoclonal antibody against porcine delta coronavirus N protein and a latex immunochromatographic test strip containing this antibody. This invention uses prokaryotically expressed PDCoV N protein as an immunogen to immunize mice, and screens for specific monoclonal antibodies 2G3 and 9C4 through cell fusion and subcloning techniques. The test strip includes a PVC base plate, a sample pad, a conjugation pad, a nitrocellulose membrane (NC membrane), and an absorbent pad; a control line C and a detection line T are respectively set on the NC membrane. This test strip can specifically recognize PDCoV, with a detection limit as low as 10. 3.0 TCID 50 The virus has a concentration of 1000 μL / mL and does not cross-react with other common clinical diarrhea viruses. The results can be interpreted in just 15 minutes, making it fast, sensitive, and easy to operate.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, specifically relating to a monoclonal antibody against the N protein of porcine delta coronavirus (PDCoV) and a latex immunochromatographic test strip containing the antibody. Background Technology

[0002] Porcine deltacoronavirus (PDCoV) is a newly discovered porcine enteric coronavirus belonging to the genus δ-coronavirus in the family Coronaviridae. PDCoV primarily causes intestinal infection in newborn piglets, with clinical symptoms including diarrhea, vomiting, and dehydration, which can lead to death in severe cases. This virus has the potential to spread across species, infecting not only pigs but also mice, chickens, cattle, and even humans, thus posing significant public health risks.

[0003] Establishing rapid and accurate diagnostic methods is crucial for the prevention and control of PDCoV. Currently, commonly used PDCoV pathogen detection methods mainly include virus isolation and identification, RT-PCR, and quantitative RT-PCR (qRT-PCR). However, these methods are often time-consuming, require specialized instruments and experimental facilities, and have high technical requirements for operators. Therefore, they are mostly limited to laboratory testing and are difficult to implement for rapid diagnosis in farms.

[0004] Lateral immunochromatography is a technique suitable for rapid on-site diagnosis. Its principle involves conjugating signal markers such as colloidal gold or latex microspheres with antigens or antibodies, amplifying the signal to obtain visually visible detection results. This technique is simple to operate, quick to interpret (typically within 15 minutes), and requires no additional instruments. It is currently widely used in animal disease inspection and quarantine, environmental monitoring, and healthcare. Regarding the selection of signal markers, latex microspheres generally offer higher detection sensitivity, better physicochemical stability, and superior preservation properties compared to colloidal gold, making them more suitable for qualitative analysis of antigens / pathogens and clinical screening. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody against porcine delta coronavirus N protein and a latex immunochromatographic test strip containing this antibody. This invention uses prokaryotically expressed PDCoV N protein as an immunogen to immunize mice. Specific monoclonal antibodies 2G3 and 9C4 are screened through cell fusion and subcloning techniques. These two antibodies are used as capture and labeling antibodies, respectively. Based on the double-antibody sandwich principle, a latex immunochromatographic test strip for detecting PDCoV N antigen is established. Using this test strip to detect PDCoV is simple, time-efficient, and highly sensitive.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] This invention provides an antibody or antigen-binding fragment against the porcine delta coronavirus N protein, wherein the antibody or antigen-binding fragment is any one of the following:

[0008] A: The variable region of the heavy chain of the antibody or antigen binding fragment includes CDR1 with the amino acid sequence GYTFTDYS (SEQ ID NO:33), CDR2 with the amino acid sequence INTETGEP (SEQ ID NO:34), and CDR3 with the amino acid sequence ARDGSWYVIYFDY (SEQ ID NO:35); the variable region of the light chain of the antibody or antigen binding fragment includes CDR1 with the amino acid sequence ENIYSN (SEQ ID NO:36), CDR2 with the amino acid sequence SAT, and CDR3 with the amino acid sequence QHFWVTPYT (SEQ ID NO:37).

[0009] B: The heavy chain variable region of the antibody or antigen binding fragment includes CDR1 with the amino acid sequence VYTFTTYY (SEQ ID NO:40), CDR2 with the amino acid sequence INPSNGDS (SEQ ID NO:41), and CDR3 with the amino acid sequence TRSYYYGLFDY (SEQ ID NO:42); the light chain variable region of the antibody or antigen binding fragment includes CDR1 with the amino acid sequence QDINSY (SEQ ID NO:43), CDR2 with the amino acid sequence RAN, and CDR3 with the amino acid sequence LQYDEFPPT (SEQ ID NO:44).

[0010] In a further embodiment, the antibody or antigen-binding fragment is any one of the following:

[0011] A: The amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment is QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWINTETGEPTYADDFEGRFAFSLETSASTAYLQINKLKNEDTATYFCARDGSWYVIYFDYWGQGTTLTVSS (SEQ ID NO:31), and the amino acid sequence of the light chain variable region of the antibody or antigen binding fragment is DIQMTQSPASLSVSVGETVTIACRASENIYSNLAWYQQKQGKSPQLLVYSATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWVTPYTFGGGTKLEIK (SEQ ID NO:32).

[0012] B: The amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment is QVQLQQPGAELVKPGASVKLSCKASVYTFTTYYMYWVKQRPGQGLEWIGGINPSNGDSNLNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTRSYYYGLFDYWGQGTTLTVSS (SEQ ID NO:38), and the amino acid sequence of the light chain variable region of the antibody or antigen binding fragment is DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGEDYSLTISSLEYEDMGIYYCLQYDEFPPTFGGGTKLEIK (SEQ ID NO:39).

[0013] In a further embodiment, the antibody is a monoclonal antibody. Specifically, this invention has screened monoclonal antibody 2G3 containing the complementarity-determining region described in A and monoclonal antibody 9C4 containing the complementarity-determining region described in B.

[0014] The present invention also provides a gene encoding the antibody or antigen-binding fragment.

[0015] In a further embodiment, the gene encoding the antibody or antigen-binding fragment is any one of the following:

[0016] A: The gene sequence encoding the variable region of the heavy chain of the antibody or antigen-binding fragment is CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAAATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTCAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCAACATATGCAGATGACTTCGAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATAAACAAACTCAAAAATGAGGACACGGCTACATATTTCTGTGCTCGCGACGGTAGTTGGTACGTAATCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID) NO:27), the gene sequence encoding the variable region of the light chain of the antibody or antigen binding fragment is GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCACCATCGCATGTCGAGCAAGTGAGAATATTTACAGTAATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATTCTGCAACAAACTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTATTCCCTCAAGATCAACAGCCTGCAGTCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGGTTACTCCGTACACGTTCGGAGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO:28).

[0017] B: The gene sequence encoding the variable region of the heavy chain of the antibody or antigen-binding fragment is CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGTCTACACCTTCACCACCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGGGGGATTAATCCTAGCAATGGTGACTCTAACCTCAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACGGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGATCCTATTACTACGGCCTCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID) NO:29), the gene sequence encoding the variable region of the light chain of the antibody or antigen binding fragment is GACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGTGCAAACAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGGAAGATTATTCTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGAGTTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO:30).

[0018] The present invention provides biological materials containing the gene, wherein the biological material is an expression cassette, transposon, plasmid vector, viral vector or host cell.

[0019] The antibody or antigen-binding fragment of the present invention can be used to detect porcine delta coronavirus. Therefore, the antibody or antigen-binding fragment, the gene, or the biological material can be used to prepare porcine delta coronavirus detection reagents or kits.

[0020] In addition, the present invention also provides a detection reagent or kit for porcine delta coronavirus containing the antibody or antigen-binding fragment, the gene or the biological material.

[0021] Furthermore, the test kit is a latex immunochromatographic test strip for detecting porcine delta coronavirus, wherein the antibody or antigen-binding fragment in the test strip is used as a capture antibody and / or a labeling antibody.

[0022] The test strip consists of a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), and an absorbent pad. The sample pad, conjugate pad, NC membrane, and absorbent pad are arranged sequentially and overlapped on the PVC base plate. A control line C and a test line T are set on the NC membrane. The conjugate pad is coated with monoclonal antibody 9C4 labeled with red latex microspheres. The test line T is coated with monoclonal antibody 2G3. The control line C is coated with goat anti-mouse IgG.

[0023] Furthermore, the sample pad is treated by soaking in a sample pad sealing solution, which consists of 25 mM Tris, 150 mM NaCl, 1% Tween-20, 1% BSA, 3% trehalose, and 0.1% ProClin 300 dissolved in ultrapure water.

[0024] Furthermore, the method for preparing the conjugate pad is as follows:

[0025] (1) The conjoint pad is soaked in conjoint pad sealing solution and dried for later use;

[0026] (2) The antibody solution labeled with latex microspheres is uniformly sprayed onto the blocked conjugate pad. The preparation method of the antibody solution labeled with latex microspheres includes: taking the latex microsphere suspension and adding it to the coupling buffer, sonicating and centrifuging, discarding the supernatant, resuspending it with coupling buffer and sonicating it evenly, adding EDC solution and NHS solution respectively for activation, vortexing and standing for 30 min, centrifuging after activation, resuspending it with coupling buffer again and sonicating it evenly; adding an appropriate amount of monoclonal antibody, incubating at room temperature, adding latex microsphere blocking solution for blocking, incubating at room temperature, centrifuging; resuspending the microspheres with latex microsphere preservation solution, sonicating it evenly, and obtaining the monoclonal antibody latex microsphere labeled solution.

[0027] Preferably, the specific preparation method of the above-mentioned solutions is as follows:

[0028] (1) The coupling buffer: 10 mM 2-morpholinoethanesulfonic acid (MES), 0.05% Proclin 300, dissolved in ultrapure water;

[0029] (2) The EDC solution: 10 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), dissolved in coupling buffer;

[0030] (3) The NHS solution: 10 mg / mL N-hydroxysuccinimide (NHS), dissolved in coupling buffer;

[0031] (4) The latex microsphere blocking solution: 5mM H3BO3, 10mM Na2B4O7·10H2O, 0.05% Tween-20, 1% BSA, 0.25% C2H7NO, dissolved in ultrapure water;

[0032] (5) The latex microsphere preservation solution: 25mM Tris, 150mM NaCl, 0.05% Tween-20, 1% BSA, 3% trehalose, 0.1% ProClin 300, dissolved in ultrapure water;

[0033] (6) The monoclonal antibody latex microsphere labeling solution: each 1 mL of monoclonal antibody latex microsphere labeling solution contains 10 μg of antibody.

[0034] (7) The binding pad sealing solution is the same as the latex microsphere preservation solution.

[0035] Furthermore, the NC membrane is prepared by spraying PDCoV N protein monoclonal antibody and goat anti-mouse IgG antibody onto the NC membrane at a spacing of 4-6 mm, which serve as the detection line T and the quality control line C, respectively. After the membrane is coated, it is dried for later use.

[0036] Furthermore, the detection line T is coated with 0.5 mg / mL of PDCoV N protein monoclonal antibody;

[0037] Furthermore, the quality control line C is coated with 0.5 mg / mL of goat anti-mouse IgG.

[0038] This invention provides a method for preparing a latex immunochromatographic test strip for detecting porcine delta coronavirus:

[0039] (1) The sample pad was soaked in the sample pad blocking solution and a binding pad coated with latex microsphere labeled antibody was prepared.

[0040] (2) Spray PDCoV N protein monoclonal antibody (0.5 mg / mL) and goat anti-mouse IgG antibody (0.5 mg / mL) at intervals of 4-6 mm onto the NC membrane;

[0041] (3) Arrange the sample pad, conjugate pad, NC membrane and absorbent pad in sequence and overlap them on the PVC base plate to obtain a complete latex immunochromatographic test strip for detecting porcine δ coronavirus.

[0042] The detection method of the test strip of the present invention comprises the following steps:

[0043] (1) Sample collection and processing;

[0044] (2) Add the sample to be tested into the sample well and let it stand for 15 minutes;

[0045] (3) Result determination: If no red band appears at the control line C, the result is invalid; if a red band appears at the control line C and no red band appears at the test line T, the result is negative; if red bands appear at both the control line C and the test line T, the result is positive.

[0046] The present invention has the following beneficial effects:

[0047] The latex immunochromatographic test strip of the present invention can specifically recognize PDCoV, with a detection limit as low as 10. 3.0 TCID 50 The concentration of the virus is 100 μL / mL and it does not cross-react with other common viruses that cause diarrhea in clinical practice, such as PEDV, TGEV, and PoRV, demonstrating good sensitivity and specificity.

[0048] The latex immunochromatographic test strip of the present invention has a short detection time, and the results can be observed with the naked eye in just 15 minutes. The detection itself does not depend on the environment or equipment, and the operation is simple. Attached Figure Description

[0049] Figure 1 SDS-PAGE electrophoresis results for identifying the expression form of PDCoV N protein. M: Protein molecular weight standard (Protein Marker); 1: Induction by pET-15b empty vector bacteria; 2: pET15b-N recombinant bacteria before induction; 3: pET15b-N recombinant bacteria after induction; 4: precipitate after lysis of pET15b-N recombinant bacteria; 5: supernatant after lysis of pET15b-N recombinant bacteria.

[0050] Figure 2 The image shows the SDS-PAGE electrophoresis result of PDCoV N protein purified by Ni column affinity chromatography; M: protein molecular weight standard (Protein Marker); 1: purified PDCoV N protein.

[0051] Figure 3 To verify the reactivity of monoclonal antibodies with PDCoV-infected LLC-PK1 cells using an indirect immunofluorescence assay; A: 2G3 hybridoma cell supernatant; B: 9C4 hybridoma cell supernatant; C: negative hybridoma cell supernatant (Mock).

[0052] Figure 4 The image shows the SDS-PAGE electrophoresis results of the purified monoclonal antibodies 2G3 and 9C4.

[0053] Figure 5To determine the optimal amount of antibody labeled on latex microspheres; A: Test strips with different antibody labeling amounts were used to detect strongly positive samples; B: Test strips with different antibody labeling amounts were used to detect weakly positive samples; C: Test strips with different antibody labeling amounts were used to detect negative samples.

[0054] Figure 6 To determine the optimal T-line antibody coating concentration (scratch concentration); A: Test strips with different T-line antibody coating concentrations for detecting strongly positive samples; B: Test strips with different T-line antibody coating concentrations for detecting weakly positive samples; C: Test strips with different T-line antibody coating concentrations for detecting negative samples.

[0055] Figure 7 The results show the sensitivity of the PDCoV latex immunochromatographic test strip.

[0056] Figure 8 The detection principle and structure of PDCoV latex immunochromatographic test strips.

[0057] Figure 9 This is a specific detection result for PDCoV latex immunochromatographic test strips. Detailed Implementation

[0058] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0059] The antigens, hybridoma cells, monoclonal antibodies, etc. in this invention will be described in detail below with reference to specific embodiments.

[0060] In the examples, female BALB / c mice were purchased from Three Gorges University, pMD18-T vector was purchased from Nanjing Novizan Biotechnology Co., Ltd., and BL21(DE3) competent cells were purchased from Beijing TransGen Biotech Co., Ltd.

[0061] The PDCoV strongly positive, weakly positive, and negative samples were clinical pig anal swab samples collected from different breeding enterprises, and were confirmed to be strongly positive, weakly positive, or negative for PDCoV by RT-qPCR testing.

[0062] Example 1: Expression and purification of PDCoV N recombinant protein

[0063] 1. Determination of PDCoV N protein expression form

[0064] The N gene of PDCoV (SEQ ID NO:1) was cloned into the multiple cloning site of the prokaryotic expression vector pET-15b using homologous recombination technology, constructing a recombinant plasmid pET15b-N expressing the PDCoV N protein. This plasmid, along with the empty vector (pET-15b), was transformed into BL21(DE3) competent cells. The cells were plated on LB agar plates containing ampicillin and incubated upside down at 37°C for 12-18 hours. Single colonies were then picked and inoculated into LB liquid medium containing ampicillin. When the bacterial culture reached OD... 630nm When the value reached 0.4-0.6, isopropyl thio-β-D-galactopyranoside (IPTG) was added to a final concentration of 1 mM for induction expression. Induction was performed at 20℃ for 12 h. The induced empty vector-transformed bacterial culture, as well as the recombinant vector-transformed bacterial culture before and after induction, were collected. The culture was centrifuged at 12000 r / min for 10 min, resuspended in an appropriate amount of PBS, and the bacterial cells were disrupted using an ultrasonic homogenizer. After centrifugation at 12000 r / min for 10 min, the supernatant and bacterial pellet were collected. 5×SDS-PAGE Loading Buffer was added, and the culture was incubated in a boiling water bath for 10 min, followed by an ice bath for 5 min. The treated samples were then subjected to SDS-PAGE analysis to determine the protein expression pattern. The results showed that the target protein was expressed at approximately 40 kDa, and the protein was expressed in both the supernatant and the pellet. Figure 1 ).

[0065] 2. Large-scale expression and purification of PDCoV N protein

[0066] After determining the expression form of PDCoV N protein, the recombinant bacterial culture was expanded, and PDCoV N protein was induced to express in large quantities according to the above induction method. After induction, the bacterial cells were collected by centrifugation, resuspended in PBS, and then disrupted with an autoclave until clear. The supernatant was collected by centrifugation, purified by Ni column affinity chromatography, and then detected by SDS-PAGE electrophoresis. The results showed that the target band was single and the size was consistent with the expectation. Figure 2 This indicates that highly pure PDCoV N protein was obtained.

[0067] Example 2: Preparation of PDCoV N protein monoclonal antibody

[0068] 1. Immunization and antibody titer determination in mice

[0069] The PDCoV N protein purified in Example 1 was emulsified with an equal volume of QuickAntibody-Mouse5W (Biodragon) and administered to 4-6 week old female BALB / c mice via intramuscular injection in the leg, 50 μg / mouse. A second immunization was performed 14 days after the first immunization, following the same procedure. Blood was collected from the mice 14 days after the second immunization, and serum was separated. PDCoV N protein was coated onto an ELISA plate, and the antibody titer against the N protein in the mouse serum was measured by indirect ELISA. When the serum antibody titer was greater than 1:12800, a booster immunization (100 μg / mouse) was administered via intraperitoneal injection. If the antibody titer did not reach 1:12800, a third intramuscular injection in the leg was administered.

[0070] 2. Screening of positive hybridoma cell lines

[0071] Three days after booster immunization, mouse spleen cells were aseptically isolated and fused with mouse myeloma cells (SP2 / 0). After fusion, the cells were cultured in complete medium containing HAT for 4–5 days, then in complete medium containing HT. When the cell colonies reached 1 / 4 of the well bottom area, the cell supernatant was detected by indirect ELISA. Positive cells were transferred to 48-well plates for further culture. When the cell colonies reached approximately 1 / 4 of the well bottom area, the cell supernatant was collected as the primary antibody for indirect immunofluorescence (IFA) verification against PDCoV-infected LLC-PK1 cells. IFA-positive hybridoma cells were subcloned using a limiting dilution method. After colony expansion, the culture supernatant from the single-clone cell wells was collected and IFA verification was performed again. Positive cells were used for the next round of subcloning. This process was repeated three times to obtain stable antibody-secreting hybridoma cell lines 2G3 and 9C4. The indirect immunofluorescence results are shown below. Figure 3 As shown.

[0072] 3. Preparation and purification of mouse ascites fluid

[0073] Female BALB / c mice aged 8-10 weeks were selected and pre-stimulated with Freund's incomplete adjuvant intraperitoneally (500 μL / mouse). Seven days later, hybridoma cells 2G3 and 9C4 were injected intraperitoneally, respectively, at doses of 5 × 10⁶. 5Cells / mouse. Ascites fluid was collected 7–10 days later based on the degree of abdominal distension in mice. The fluid was centrifuged at 12000 r / min for 10 min, and the clear yellow layer was collected. The ascites fluid monoclonal antibodies 2G3 and 9C4 were purified according to the instructions of a Protein A+G column (purchased from Beyotime Biotechnology Co., Ltd.). The purified monoclonal antibodies were then detected by SDS-PAGE. The results showed that both monoclonal antibodies exhibited distinct bands at approximately 50 kDa and 25 kDa, respectively, consistent with the size of the antibody heavy and light chains. Figure 4 This indicates that two monoclonal antibodies, 2G3 and 9C4, with high purity were obtained.

[0074] Example 3: Amplification and Sequencing of the Variable Region Gene of a Monoclonal Antibody

[0075] 1. Monoclonal antibody subtype identification

[0076] The prokaryotically expressed PDCoV N protein was coated onto an ELISA plate. Two purified monoclonal antibodies, 2G3 and 9C4, were used as primary antibodies. The antibody subtypes of monoclonal antibodies 2G3 and 9C4 were identified using the Bio-Long mouse monoclonal antibody subtype identification kit. The results showed that the heavy chain subtype of both monoclonal antibodies was IgG1 and the light chain subtype was κ.

[0077] 2. Amplification and Sequencing of the Variable Region Gene of Monoclonal Antibodies

[0078] After identifying the subtypes of the two monoclonal antibodies, 12 upstream degenerate primers and 1 downstream degenerate primer were designed to amplify the heavy chain variable region gene of monoclonal antibodies 2G3 and 9C4, and 11 upstream degenerate primers and 1 downstream degenerate primer were designed to amplify the light chain variable region gene of monoclonal antibodies 2G3 and 9C4. First, total RNA was extracted from hybridoma cell lines 2G3 and 9C4, and the RNA was reverse transcribed into cDNA using the HiScript II 1st Strand cDNA Synthesis Kit. The cDNA was then used as a template for PCR amplification. The heavy chain variable region degenerate primer sequences are shown in Table 1, and the light chain variable region degenerate primer sequences are shown in Table 2.

[0079] Table 1. Primers for amplifying the variable region of the heavy chain of monoclonal antibodies

[0080]

[0081] Table 2. Primers for amplifying the variable region of the light chain of monoclonal antibodies

[0082]

[0083] The results showed that the primer pairs capable of amplifying the heavy chain variable region and light chain variable region of monoclonal antibody 2G3 were MHV6 / MHCG1 and MKV3 / MKC, respectively, and the primer pairs capable of amplifying the heavy chain variable region and light chain variable region of monoclonal antibody 9C4 were MHV2 / MHCG1 and MKV6 / MKC, respectively. The amplified target fragments were recovered, purified, ligated into the pMD18-T vector, and sequenced. The sequencing results were compared with the antibody gene library (IMGT) to obtain the gene sequences of the heavy chain variable region and light chain variable region of both monoclonal antibodies. The heavy chain variable region and light chain variable region of monoclonal antibody 2G3 are 360 ​​bp and 321 bp in size, respectively. The gene sequence of the heavy chain variable region of monoclonal antibody 2G3 is shown in SEQ ID NO:27, and the gene sequence of the light chain variable region is shown in SEQ ID NO:28. The heavy chain variable region and light chain variable region of monoclonal antibody 9C4 are 354 bp and 321 bp in size, respectively. The gene sequence of the heavy chain variable region of monoclonal antibody 9C4 is shown in SEQ ID NO:29, and the gene sequence of the light chain variable region is shown in SEQ ID NO:30.

[0084] The amino acid sequences of the heavy chain variable region of monoclonal antibody 2G3 are shown in SEQ ID NO:31, and the amino acid sequences of the light chain variable region are shown in SEQ ID NO:32. The heavy chain variable region of monoclonal antibody 2G3 includes CDR1 (as shown in SEQ ID NO:33), CDR2 (as shown in SEQ ID NO:34), and CDR3 (as shown in SEQ ID NO:35); the light chain variable region of monoclonal antibody 2G3 includes CDR1 (as shown in SEQ ID NO:36), CDR2 (with the amino acid sequence SAT), and CDR3 (as shown in SEQ ID NO:37).

[0085] The amino acid sequence of the heavy chain variable region of monoclonal antibody 9C4 is shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:39. The heavy chain variable region of monoclonal antibody 9C4 includes CDR1 (amino acid sequence shown in SEQ ID NO:40), CDR2 (amino acid sequence shown in SEQ ID NO:41), and CDR3 (amino acid sequence shown in SEQ ID NO:42); the light chain variable region of monoclonal antibody 9C4 includes CDR1 (amino acid sequence shown in SEQ ID NO:43), CDR2 (amino acid sequence RAN), and CDR3 (amino acid sequence shown in SEQ ID NO:44).

[0086] Example 4: Preparation of latex immunochromatographic test strip for detecting porcine delta coronavirus

[0087] 1. Preparation of required reagents

[0088] (1) Coupling buffer: 10 mM 2-morpholinoethanesulfonic acid (MES), 0.05% Proclin 300 (dissolved in ultrapure water);

[0089] (2) EDC solution: 10 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide (dissolved in coupling buffer, freshly prepared and used);

[0090] (3) NHS solution: 10 mg / mL N-hydroxysuccinimide (dissolved in coupling buffer, freshly prepared and used);

[0091] (4) Blocking solution for latex microspheres: 5mM H3BO3, 10mM Na2B4O7·10H2O, 0.05% Tween-20, 1% BSA, 0.25% C2H7NO (dissolved in ultrapure water);

[0092] (5) Preservative solution for latex microspheres: 25mM Tris, 150mM NaCl, 0.05% Tween-20, 1% BSA, 3% trehalose, 0.1% ProClin 300 (dissolved in ultrapure water);

[0093] (6) Binding pad sealing solution: same as latex microsphere preservation solution;

[0094] (7) Sample pad sealing solution: 25mM Tris, 150mM NaCl, 1% Tween-20, 1% BSA, 3% trehalose, 0.1% ProClin 300 (dissolved in ultrapure water).

[0095] (8) Sample dilution solution: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4, 1% Tween-20, 0.5% TX-100, 0.1% ProClin 300 (dissolved in ultrapure water).

[0096] 2. Activation of latex microspheres

[0097] (1) Take 10 μL of microsphere (Suzhou Weidu Biotechnology, 4% solid content microsphere) suspension and add it to a 2 mL centrifuge tube containing 1 mL of coupling buffer. After sonication, centrifuge at 15℃ and 12000 r / min for 10 min and discard the supernatant.

[0098] (2) Add 1 mL of coupling buffer, sonicate to mix, centrifuge at 15℃ and 12000 r / min for 10 min, and discard the supernatant;

[0099] (3) Add 1 mL of coupling buffer and sonicate to mix well;

[0100] (4) Add 1µL of EDC solution, vortex to mix, then add 2µL of NHS solution and vortex to mix.

[0101] (5) Let stand at room temperature for 30 minutes;

[0102] (6) Centrifuge at 15℃ and 12000 r / min for 10 min, and discard the supernatant;

[0103] (7) Add 1.5 mL of coupling buffer, sonicate to mix, centrifuge at 15 °C and 12000 r / min for 10 min, and discard the supernatant;

[0104] (8) Add 1.5 mL of coupling buffer and repeat the washing step in (7).

[0105] 3. Conjugation of latex microspheres with monoclonal antibodies

[0106] (1) Add 750 μL of coupling buffer to the activated latex microspheres and sonicate to mix.

[0107] (2) Add 10µg of monoclonal antibody 9C4 to 250μL of coupling buffer to prepare the coupling solution;

[0108] (3) Add the antibody-containing conjugate solution to the mixed microspheres and vortex to mix.

[0109] (4) Incubate at room temperature for 2 hours.

[0110] 4. Encapsulation and preservation of latex microspheres

[0111] (1) Add 500 μL of latex microsphere blocking solution to the coupled microspheres and vortex mix.

[0112] (2) Sealed at room temperature for 1 hour;

[0113] (3) Centrifuge at 15℃ and 12000r / min for 10min, and discard the supernatant;

[0114] (4) Add 1 mL of latex microsphere preservation solution, ultrasonically disperse evenly, and store at 2-8℃ for later use.

[0115] 5. Determination of the optimal amount of antibody labeled with latex microspheres

[0116] The PDCoV N protein monoclonal antibody 9C4 was conjugated to activated latex microspheres. Antibody labeling levels of 5 μg, 10 μg, 15 μg, and 20 μg were set while maintaining a constant microsphere dosage (10 μL, 4% solid content). Comparison of test strips with different monoclonal antibody labeling levels for different clinical samples (strongly positive, weakly positive, and negative samples) revealed that at a labeling level of 5 μg, the T-line color of positive samples was generally lighter. At labeling levels of 15 μg and 20 μg, the T-line color of positive samples showed no significant change compared to the 10 μg labeling level. However, the negative samples corresponding to 15 μg and 20 μg antibody labeling levels showed slight nonspecific bands, while the negative samples corresponding to 10 μg antibody labeling level showed no nonspecific bands. Therefore, the optimal antibody labeling level for red latex microspheres was determined to be 10 μg. The test results are as follows: Figure 5 As shown.

[0117] 6. Determination of the optimal T-line antibody coating concentration

[0118] To determine the optimal coating concentration of the T-line antibody, PDCoV N protein monoclonal antibody 2G3 was diluted to 0.25, 0.5, 0.75, and 1 mg / mL and sprayed onto the T-line of different NC membranes. By comparing the detection results of different clinical samples (strongly positive, weakly positive, and negative samples) at different antibody coating concentrations, it was found that when the antibody coating concentration was 0.25 mg / mL, the detection bands for positive samples were weak. At antibody coating concentrations of 0.75 and 1 mg / mL, the colorimetric effect was not significantly improved compared to 0.5 mg / mL. At an antibody coating concentration of 1 mg / mL, weak nonspecific bands appeared for negative samples. Considering cost, the optimal antibody coating concentration was determined to be 0.5 mg / mL. The detection results are as follows: Figure 6 As shown.

[0119] 7. Preparation of the binding pad

[0120] (1) The conjoint pad is soaked in conjoint pad sealing solution and dried overnight at 37°C for later use;

[0121] (2) Spray the antibody solution labeled with latex microspheres evenly onto the conjugate pad after the blocking treatment.

[0122] 8. Preparation of sample pads

[0123] The sample pad was treated by immersing it in a sample pad sealing solution.

[0124] 9. Preparation of nitrocellulose membranes (NC membranes)

[0125] Spray 0.5 mg / mL of PDCoV N protein monoclonal antibody 2G3 and 0.5 mg / mL of goat anti-mouse IgG antibody onto a nitrocellulose membrane at 4-6 mm intervals, serving as the detection line T and control line C, respectively. After the membrane is coated, it is dried overnight at 37°C for later use.

[0126] 10. Assembly of test strips

[0127] The sample pad, conjugate pad, NC membrane, and absorbent pad are sequentially attached to the PVC base plate. The strips are then cut into 2.9mm wide strips using a strip cutter. The cut strips are then installed in the plastic housing, and finally the plastic cover is fastened to complete the assembly of the test strip. The detection principle and structure of the PDCoV latex immunochromatographic test strip are as follows: Figure 8 As shown.

[0128] 11. How to use the test strips and interpret the results

[0129] Dilute the sample to be tested (pig anal swab, intestinal contents) with 1 mL of sample diluent, mix well, and add 90 μL of the mixture to the sample well of the test strip. Let it stand at room temperature for 15 min and observe the results.

[0130] Interpretation criteria: If no red band appears at control line C, the result is invalid; if a red band appears at control line C but no red band appears at test line T, the result is negative; if red bands appear at both control line C and test line T, the result is positive.

[0131] 12. Sensitivity test

[0132] With a viral titer of 10 6.0 TCID 50 PDCoV virus solution was serially diluted 10-fold to 10 / mL. 3.0 TCID 50 / mL, the established test strip was used to detect samples with different viral titers, with three replicates for each titer. The results showed that the test strip could stably detect 10 / mL. 3.0 TCID 50 The virus concentration was / mL, and the results were consistent across three tests. Subsequently, 10... 3.0 TCID 50 The virus solution was serially diluted 2-fold to 10⁶ / mL. 1.8 TCID 50 / mL, and then tested with the established test strip. The results showed that weak bands could still be observed in some samples, but the repeatability was poor. Therefore, the limit of detection of this test strip was determined to be 10. 3.0 TCID 50 / mL of PDCoV virus, such as Figure 7 As shown.

[0133] (1) Specificity test

[0134] The established test strips were used to detect four different viruses causing swine diarrhea: PDCoV (samples included early Chinese lineage, Chinese 1.1 lineage, and Chinese 1.2 lineage), PEDV (samples included G1a, G1b, G2a, G2b, and G2c), TGEV, and PoRV (samples included G4, G5, and G9). The results showed that the detection method was only positive for PDCoV and showed no cross-reactivity with other viruses, indicating that the test strip has good specificity. Figure 9 As shown.

[0135] (2) Compliance rate test

[0136] The developed test strip was used to test 142 clinical porcine anal swab samples. The results were compared with RT-PCR, and the concordance rates of the two methods were compared. The results are shown in Table 3. The concordance rate between the latex immunochromatographic test strip and RT-PCR for positive samples was 94.9%, and the concordance rate for negative samples was 100%. These results demonstrate the accuracy and reliability of the latex immunochromatographic test strip in the detection of PDCoV in clinical samples.

[0137] Table 3. Comparison of latex immunochromatographic test strips and RT-PCR in clinical sample testing.

[0138]

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or antigen-binding fragment against porcine delta coronavirus N protein, characterized in that, The antibody or antigen-binding fragment is any one of the following A or B: A: The variable region of the heavy chain of the antibody or antigen-binding fragment includes CDR1 with an amino acid sequence as shown in SEQ ID NO:33, CDR2 with an amino acid sequence as shown in SEQ ID NO:34, and CDR3 with an amino acid sequence as shown in SEQ ID NO:35; the variable region of the light chain of the antibody or antigen-binding fragment includes CDR1 with an amino acid sequence as shown in SEQ ID NO:36, CDR2 with an amino acid sequence of SAT, and CDR3 with an amino acid sequence as shown in SEQ ID NO:

37. B: The variable region of the heavy chain of the antibody or antigen-binding fragment includes CDR1 with an amino acid sequence as shown in SEQ ID NO:40, CDR2 with an amino acid sequence as shown in SEQ ID NO:41, and CDR3 with an amino acid sequence as shown in SEQ ID NO:42; the variable region of the light chain of the antibody or antigen-binding fragment includes CDR1 with an amino acid sequence as shown in SEQ ID NO:43, CDR2 with an amino acid sequence of RAN, and CDR3 with an amino acid sequence as shown in SEQ ID NO:

44.

2. The anti-porcine delta coronavirus N protein antibody or antigen-binding fragment according to claim 1, characterized in that, The antibody or antigen-binding fragment is any one of the following A or B: A: The amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment is shown in SEQ ID NO:31, and the amino acid sequence of the light chain variable region of the antibody or antigen binding fragment is shown in SEQ ID NO:

32. B: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment is shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment is shown in SEQ ID NO:

39.

3. The anti-porcine delta coronavirus N protein antibody or antigen-binding fragment according to claim 1, characterized in that, The antibody is a monoclonal antibody.

4. A gene encoding the antibody or antigen-binding fragment of claim 1.

5. The gene according to claim 4, characterized in that, The gene encoding the antibody or antigen-binding fragment is either A or B from the following: A: The gene sequence encoding the variable region of the heavy chain of the antibody or antigen binding fragment is shown in SEQ ID NO:27, and the gene sequence encoding the variable region of the light chain of the antibody or antigen binding fragment is shown in SEQ ID NO:

28. B: The gene sequence encoding the variable region of the heavy chain of the antibody or antigen-binding fragment is shown in SEQ ID NO:29, and the gene sequence encoding the variable region of the light chain of the antibody or antigen-binding fragment is shown in SEQ ID NO:

30.

6. A biomaterial containing the gene of claim 4, characterized in that, The biological material is a transposon, plasmid vector, viral vector, or host cell.

7. The use of the antibody or antigen-binding fragment according to any one of claims 1-3, the gene according to claim 4 or 5, or the biological material according to claim 6 in the preparation of a porcine delta coronavirus detection reagent or kit.

8. A detection reagent or kit for porcine delta coronavirus containing the antibody or antigen-binding fragment of any one of claims 1-3, the gene of claim 4 or 5, or the biological material of claim 6.

9. The porcine delta coronavirus detection reagent or kit according to claim 8, characterized in that, The kit is a latex immunochromatographic test strip for detecting porcine delta coronavirus.

10. The porcine delta coronavirus detection reagent or kit according to claim 9, characterized in that, In the test strip, the antibody or antigen-binding fragment is used as a capture antibody and / or a labeling antibody.

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