Porcine epidemic diarrhea virus specific monoclonal antibody, applications and test strips

Abstract

The application discloses a porcine epidemic diarrhea virus specific monoclonal antibody, application and test strip, relates to the technical field of biotechnology, and aims to solve the problem of porcine epidemic diarrhea virus detection, the CDR1, CDR2 and CDR3 of the heavy chain variable region of the monoclonal antibody or antigen binding fragment have the amino acid sequences shown in SEQ ID NO:9-11 respectively, the CDR1, CDR2 and CDR3 of the light chain variable region of the monoclonal antibody or antigen binding fragment have the amino acid sequences shown in SEQ ID NO:12, DTS and SEQ ID NO:13 respectively.The test strip has the characteristics of high specificity and high sensitivity for detecting porcine epidemic diarrhea virus.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology. More particularly, the present application relates to a porcine epidemic diarrhea virus specific monoclonal antibody, application and test strip. BACKGROUND

[0002] Porcine epidemic diarrhea virus (PEDV) is the pathogen causing porcine epidemic diarrhea (PED). The clinical features of the disease are vomiting, acute diarrhea and dehydration. The mortality rate of newborn piglets infected within 7 days can reach 90-100%; the mortality rate of infected nursery pigs drops to 30-50%, but the development is retarded; the mortality rate of infected growing pigs and sows is less than 5%, but the foraging and lactation are reduced, and the growth is stagnant.

[0003] PEDV infection inhibits intestinal mucosal immunity, leading to secondary bacterial or viral infection and aggravating the disease. Because its clinical symptoms are very similar to other pig-derived enteroviruses, such as porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine delta coronavirus, etc., it is difficult to distinguish, which brings difficulties to the diagnosis of PEDV. The diagnosis techniques of PEDV include: traditional methods (such as virus isolation, ELISA), molecular detection (RT-PCR, recombinase polymerase amplification) and immunochromatography technology (colloidal gold test strip), etc. Among them, the traditional method and the molecular method are suitable for the laboratory, which requires professional instruments and equipment and professional personnel; the immunochromatography technology is more suitable for the field demand, which has the advantages of simple operation, fast detection speed and easy result interpretation.

[0004] However, there is still a lack of specific and sensitive detection antibodies and tools. SUMMARY

[0005] An object of the present application is to provide a porcine epidemic diarrhea virus specific monoclonal antibody, application and test strip, which has the characteristics of strong specificity and high sensitivity for detecting porcine epidemic diarrhea virus.

[0006] In order to achieve these objects and other advantages of the present application, the present application provides a porcine epidemic diarrhea virus specific monoclonal antibody or antigen binding fragment thereof, the CDR1, CDR2 and CDR3 of the heavy chain variable region of the monoclonal antibody or antigen binding fragment thereof have the amino acid sequences shown in SEQ ID NO: 9-11, respectively; the CDR1, CDR2 and CDR3 of the light chain variable region of the monoclonal antibody or antigen binding fragment thereof have the amino acid sequences shown in SEQ ID NO: 12, DTS and SEQ ID NO: 13, respectively.

[0007] Further, the monoclonal antibody or antigen-binding fragment thereof has an amino acid sequence shown in SEQ ID NO: 1 for the heavy chain variable region, and an amino acid sequence shown in SEQ ID NO: 2 for the light chain variable region.

[0008] The present application also provides a nucleic acid encoding the specific monoclonal antibody or antigen-binding fragment thereof for porcine epidemic diarrhea virus.

[0009] The present application also provides a recombinant vector comprising the nucleic acid.

[0010] The present application also provides a recombinant host cell comprising the recombinant vector.

[0011] The present application also provides use of the specific monoclonal antibody or antigen-binding fragment thereof for porcine epidemic diarrhea virus in the preparation of a reagent or kit for detecting porcine epidemic diarrhea virus.

[0012] The present application also provides a test strip for detecting porcine epidemic diarrhea virus, comprising a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorption pad, the nitrocellulose membrane being provided with a detection line and a quality control line, the conjugate pad being sprayed with a nano-enzyme-labeled capture antibody, and the detection line being coated with a detection antibody; wherein the capture antibody or the detection antibody is the specific monoclonal antibody or antigen-binding fragment thereof for porcine epidemic diarrhea virus.

[0013] Further, the capture antibody and the detection antibody are selected from the specific monoclonal antibody or antigen-binding fragment thereof for porcine epidemic diarrhea virus and antibody B, respectively; wherein the CDR1, CDR2 and CDR3 of the heavy chain variable region of antibody B have the amino acid sequences shown in SEQ ID NOs: 14-16, respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region of antibody B have the amino acid sequences shown in SEQ ID NOs: 17, KAS and SEQ ID NO: 18, respectively.

[0014] Further, the heavy chain variable region of antibody B has an amino acid sequence shown in SEQ ID NO: 5, and the light chain variable region of antibody B has an amino acid sequence shown in SEQ ID NO: 6.

[0015] Further, the quality control line is coated with goat anti-mouse IgG; the nanoscale enzyme is washed with deionized water, after centrifugation, the supernatant is discarded, the precipitate is resuspended with MES buffer solution, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide are added, after mixing, it is placed in a shaker for activation; after activation, it is washed with MES buffer solution, then resuspended with MES buffer solution, to obtain activated nanoscale enzyme; the capture antibody is incubated with the activated nanoscale enzyme solution overnight; Tris buffer is added to terminate the reaction, after magnetic separation, it is resuspended with Tris buffer, to obtain the nanoscale enzyme labeled capture antibody.

[0016] The present application at least includes the following beneficial effects:

[0017] The test strip provided by the present application has the following significant beneficial effects: 1. Strong specificity, can accurately identify PEDV, and has no cross reaction with other pathogens such as porcine transmissible gastroenteritis virus and porcine delta coronavirus similar to clinical symptoms; 2. Extremely high sensitivity, the minimum detection amount of virus is 49 TCID 50 / mL, far more than the traditional colloidal gold test strip; 3. Simple and fast operation, no professional equipment is needed, the result can be judged by naked eye within 10 minutes, which is very suitable for on-site instant detection, and provides an efficient and reliable technical means for early diagnosis and prevention and control of PEDV.

[0018] Other advantages, objects, and features of the present application will be apparent from the following description, and will be understood by those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is an SDS-PAGE identification result graph of the purified N protein of porcine epidemic diarrhea virus, M: protein Marker; 1: purified recombinant pET32a-N protein.

[0020] Figure 2 It is a structural schematic diagram of the porcine epidemic diarrhea virus test strip.

[0021] Figure 3 It is a schematic diagram of the detection result of the porcine epidemic diarrhea virus test strip.

[0022] Figure 4 It is a sensitivity detection result graph of the porcine epidemic diarrhea virus test strip.

[0023] Figure 5 It is a negative anal swab detection result graph.

[0024] Figure 6 It is a specificity detection result graph of the porcine epidemic diarrhea virus test strip. DETAILED DESCRIPTION

[0025] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0026] It should be understood that terms such as "having," "comprising," and "including" used in the embodiments of this application do not exclude the presence or addition of one or more other elements or combinations thereof. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are only used to explain the relative positional relationship and movement of components in a specific posture. If the specific posture changes, the directional indication will also change accordingly. When an element is referred to as "fixed to" or "set on" another element, it can be directly on the other element or may have an intervening element present. When an element is referred to as "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element through an intervening element. Descriptions involving "first," "second," etc., in the embodiments of this application are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features.

[0027] It should be noted that the technical solutions of the various embodiments of this application can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this application.

[0028] The embodiments of this application provide a porcine epidemic diarrhea virus-specific monoclonal antibody or its antigen-binding fragment thereof, comprising: a monoclonal antibody (hereinafter also referred to as antibody A, protein A) or its antigen-binding fragment heavy chain variable region CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NO: 9-11 respectively; and a monoclonal antibody or its antigen-binding fragment light chain variable region CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NO: 12, DTS and SEQ ID NO: 13 respectively.

[0029] The amino acid sequences of the CDR1, CDR2 and CDR3 of the heavy chain and light chain variable region of the monoclonal antibody or antigen-binding fragment thereof are shown in Table 1. The amino acid sequences shown in SEQ ID NOs: 9-11 are GYSFTAYF, FNPYNGET, GSGNYADGGFVY, respectively. The amino acid sequences shown in SEQ ID NOs: 12-13 are SSVSY, FQGSGYPFT, respectively. The antigen-binding fragment is a Fab fragment, a F(ab)2 fragment or a single-chain Fv fragment (scFv). The constant region of the heavy chain of the above monoclonal antibody is of IgG1 type, and the constant region of the light chain is of Kappa type.

[0030] Table 1

[0031]

[0032] In another embodiment, the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO: 2.

[0033] The amino acid sequence of SEQ ID NO: 1 is:

[0034] EVQLQQSGPESLKPGASVKISCKASGYSFTAYFMNWMKQSHGKSLEWIGRFNPYNGETFYNQKFKDKATLTVDKSSSTAHMELLSLTSEDSAVYYCGSGNYADGGFVYWGQGTLVTV

[0035] The amino acid sequence of SEQ ID NO: 2 is:

[0036] EIVLTQSPAIMSVSPGEKVTMTCSASSSVSYMHWYQQKSSTSPKLWIYDTSKLASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPFTFGSGTKLELK

[0037] The embodiments of the present application also provide a nucleic acid encoding the specific monoclonal antibody or antigen-binding fragment thereof of porcine epidemic diarrhea virus. The DNA encoding the heavy chain variable region of the monoclonal antibody or antigen-binding fragment is shown in SEQ ID NO: 3; and the DNA encoding the light chain variable region of the monoclonal antibody or antigen-binding fragment is shown in SEQ ID NO: 4.

[0038] Specifically, SEQ ID NO: 3 is:

[0039] GAGGTCCAGCTGCAACAGTCTGGACCTGAATCACTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTTACTGCCTACTTTATGAACTGGATGAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGACGTTTTAATCCTTACAATGGTGAGACTTTCTACAACCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCTAGCACAGCCCACATGGAGCTCCTGAGCCTGACATCTGAGGACTCTGCAGTCTATTATTGTGGAAGCGGGAATTACGCCGACGGGGGTTTTGTTTATTGGGGCCAAGGGACTCTGGTCACTGTCTC

[0040] SEQ ID NO: 4 is:

[0041] GAAATTGTGCTCACCCAGTCTCCAGCAATCATGTCTGTATCTCCAGGGGAAAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAAGCACCTCCCCCAAACTCTGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCAGGTCGCTTCAGTGGCAGTGGGTCTGGAAACTCTTACTCTCTCACGATCAGCAGCATGGAGGCTGAAGATGTTGCCACTTATTACTGTTTTCAGGGGAGTGGGTACCCATTCACGTTCGGCTCGGGGACCAAGCTGGAGCTGAAA

[0042] The embodiments of the present application also provide a recombinant vector comprising the nucleic acid. Illustratively, primers are designed according to the sequence of Genbank accession number AF353511.1, and the PCR amplification product is inserted into the prokaryotic expression vector pET32a to obtain a recombinant vector. The embodiments of the present application also provide a recombinant host bacterium comprising the recombinant vector. Illustratively, the recombinant vector is transformed into E. coli BL21 to obtain a recombinant host bacterium.

[0043] The embodiments of the present application also provide application of the specific monoclonal antibody or antigen-binding fragment thereof for PEDV in preparation of a reagent or kit for detecting PEDV. Exemplarily, as a detection reagent, the reagent comprises a capture antibody and a detection antibody, the capture antibody is selected from at least one of the specific monoclonal antibody or antigen-binding fragment thereof for PEDV N protein and antibody B or antigen-binding fragment thereof; or, the detection antibody is selected from at least one of the specific monoclonal antibody or antigen-binding fragment thereof for PEDV N protein and antibody B or antigen-binding fragment thereof; or, the capture antibody is the specific monoclonal antibody or antigen-binding fragment thereof for PEDV N protein, and the detection antibody is the specific monoclonal antibody B or antigen-binding fragment thereof for PEDV N protein; or, the capture antibody is antibody B or antigen-binding fragment thereof, and the detection antibody is the specific monoclonal antibody or antigen-binding fragment thereof for PEDV N protein.

[0044] The embodiments of the present application also provide a test strip for detecting PEDV, which comprises a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad, the nitrocellulose membrane is provided with a detection line and a quality control line, the binding pad is sprayed with nano-enzyme-labeled capture antibodies, and the detection line is coated with detection antibodies; wherein the capture antibodies or the detection antibodies are the specific monoclonal antibody or antigen-binding fragment thereof for PEDV.

[0045] Specifically, the test strip comprises a PVC bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad, the sample pad, the binding pad, the nitrocellulose membrane and the water absorption pad are sequentially overlapped on the PVC bottom plate; the nitrocellulose membrane is provided with a detection line T and a quality control line C, the detection line T is arranged at one end close to the binding pad, and the quality control line C is arranged at one end close to the water absorption pad; the binding pad is sprayed with nano-enzyme-labeled capture antibodies, the detection line T is coated with detection antibodies, and the quality control line C is coated with goat anti-mouse IgG.

[0046] The capture antibody and the detection antibody specifically include the following cases: the binding pad is sprayed with the above-mentioned specific PEDV N protein monoclonal antibody combined with nanoscale enzyme labels, the detection line T is coated with antibody B; or the binding pad is sprayed with antibody B labeled with nanoscale enzymes, and the detection line T is coated with the above-mentioned specific PEDV N protein monoclonal antibody; or the binding pad is sprayed with the above-mentioned specific PEDV N protein monoclonal antibody combined with nanoscale enzyme labels, and the detection line T is coated with other existing specific PEDV monoclonal antibody; or the binding pad is sprayed with antibody B labeled with nanoscale enzymes, and the detection line T is coated with other existing specific PEDV monoclonal antibody; or the binding pad is sprayed with other existing specific PEDV monoclonal antibody combined with nanoscale enzyme labels, and the detection line T is coated with the above-mentioned specific PEDV N protein monoclonal antibody; or the binding pad is sprayed with other existing specific PEDV monoclonal antibody combined with nanoscale enzyme labels, and the detection line T is coated with antibody B.

[0047] The CDR1, CDR2 and CDR3 of the heavy chain variable region of antibody B have the amino acid sequences shown in SEQ ID NO: 14-16, respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region of antibody B have the amino acid sequences shown in SEQ ID NO: 17, KAS, SEQ ID NO: 18, respectively. The amino acid sequences of the CDR1, CDR2 and CDR3 of the heavy chain and light chain variable region of antibody B are shown in Table 2, and the amino acid sequences shown in SEQ ID NO: 14-16 are GYTFTTYY, IYPGNINT, ARGISSALPY, respectively, and the amino acid sequences shown in SEQ ID NO: 17-18 are QNINIW, QQGQSYPLT, respectively.

[0048] Table 2

[0049]

[0050] Further, the heavy chain variable region of antibody B has the amino acid sequence shown in SEQ ID NO: 5; and the light chain variable region of antibody B has the amino acid sequence shown in SEQ ID NO: 6.

[0051] SEQ ID NO: 5 is:

[0052] QVQLKESGPELVKPGASVRISCKASGYTFTTYYIHWLRQRPGQGLEWIGWIYPGNINTKYHENFKDKATLTADKYSNTAHMQLSSLTSEDSAVYFCARGISSALPYWGQGTLVTV SEQ ID NO: 6 is:

[0053] DIQMTQSPSSLSASLGDTITITCHASQNINIWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTINSLQPEDIATYYCQQGQSYPLTFGGGTKLELK

[0054] The DNA encoding the heavy chain variable region of the monoclonal antibody or antigen binding fragment is set forth in SEQ ID NO: 7; the DNA encoding the light chain variable region of the monoclonal antibody or antigen binding fragment is set forth in SEQ ID NO: 8.

[0055] SEQ ID NO: 7 is:

[0056] CAGGTGCAGCTGAAGGAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGCAAGGCTTCTGGCTACACCTTCACAACCTACTATATACACTGGTTGAGACAGAGGCCTGGACAGGGACTTGAATGGATAGGATGGATTTATCCTGGAAATATTAATACTAAGTACCATGAGAACTTCAAGGACAAGGCCACACTAACTGCAGACAAATACTCCAACACAGCCCACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGGGGCATTAGTTCGGCCCTTCCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTC

[0057] SEQ ID NO: 8 is:

[0058] GACATCCAGATGACACAGTCTCCATCCAGTCTGTCTGCTTCCCTTGGAGACACAATTACCATCACTTGCCATGCCAGTCAGAACATTAATATTTGGTTAAGCTGGTACCAGCAGAAACCAGGAAATATTCCTAAACTGTTGATCTATAAGGCTTCCAACTTGCACACAGGCGTCCCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACATTAACCATCAACAGCCTGCAGCCTGAAGACATTGCCACTTACTACTGTCAACAGGGTCAAAGTTATCCTCTCACGTTCGGAGGGGGGACCAAGCTGGAGCTGAA

[0059] The quality control line is coated with goat anti-mouse IgG; the nanozyme is washed with deionized water, centrifuged, the supernatant is discarded, the precipitate is resuspended with a MES buffer solution, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide are added, after mixing, the mixture is placed in a shaker for activation; after activation, the mixture is washed with a MES buffer solution, then resuspended with a MES buffer solution to obtain activated nanozyme; the capture antibody is incubated with the activated nanozyme solution overnight; Tris buffer is added to terminate the reaction, after magnetic separation, the mixture is resuspended with Tris buffer to obtain the nanozyme-labeled capture antibody. The concentration of the nanozyme is 5 mg / mL; the concentration of the Tris buffer is 0.05 mol / L, and the pH is 7.2; the protection buffer is 5% BSA-Tirs; the composition of the PEDV N protein monoclonal antibody nanozyme labeling solution is: 60 µg of PEDV N protein monoclonal antibody is contained in 1 mL of the nanozyme labeling solution. The quality control line C is coated with 1 mg / mL of goat anti-mouse IgG.

[0060] The method for preparing the nanozyme immunochromatographic test strip comprises the following steps:

[0061] (1) preparing a conjugate pad coated with the nanozyme-labeled PEDV N protein monoclonal antibody;

[0062] (2) spraying the antibody B (1 mg / mL) and the goat anti-mouse IgG antibody (1 mg / mL) on the nitrocellulose membrane with a spacing of 5-8 mm, as the detection line T and the quality control line C, respectively;

[0063] (3) sequentially lapping the sample pad, the conjugate pad, the nitrocellulose membrane and the water absorption pad on the PVC bottom plate to obtain the nanozyme immunochromatographic test strip for detecting the porcine epidemic diarrhea virus.

[0064] The method for using the test strip is as follows:

[0065] (1) Take the sample to be tested.

[0066] (2) Take out the test strip and balance it to room temperature, add the sample to be tested to the sample addition hole, stand at room temperature, and determine the result.

[0067] (3) Result determination: after the reaction is completed, the positive and negative results are qualitatively determined by the naked eye.

[0068] The following is illustrated with specific examples.

[0069] Example 1: Preparation of PEDV N protein monoclonal antibody and amplification of antibody sequence

[0070] 1.1 Preparation of PEDV N protein

[0071] According to the sequence of Genbank accession number: AF353511.1, primers were designed, and the PCR product was inserted into the prokaryotic expression vector pET32a. The recombinant plasmid was named pET32a-N, which was transformed into E. coli BL21 (DE3). The engineered bacteria expressing PEDV N protein were inoculated into 100 mL LB containing ampicillin. When the OD 600 of the culture was about 0.8, IPTG was added to a final concentration of 1 mmol / L for induction expression. After 5 h, the precipitate was collected by centrifugation at 12000 rpm for 10 min. The precipitate was resuspended with an appropriate amount of PBS, and then ultrasonicated for 10 min (ultrasonic for 3 s, stop for 3 s). After ultrasonication, the supernatant was collected by centrifugation at 12000 rpm for 10 min at 4°C. The product was purified by nickel column, and the purified product was PEDV N protein. The identification result is shown in Figure 1 . After determining the protein concentration of the protein sample by BCA kit (Bi Yun Tian), the sample was aliquoted and stored at -80°C for standby.

[0072] 1.2 Preparation of PEDV N protein monoclonal antibody and amplification of antibody sequence

[0073] The purified PEDV N recombinant protein was used to immunize Babl / c mice at a dosage of 30 μg per mouse. For the first immunization, the PEDV N protein was mixed with an equal volume of Freund's complete adjuvant and emulsified, and then injected subcutaneously on the back in multiple points. The booster immunization was performed every 2 weeks for a total of four times. For the booster immunization, the PEDV N recombinant protein was mixed with an equal volume of Freund's incomplete adjuvant and emulsified, and then injected subcutaneously on the back in multiple points. The method of immunization was the same as that for the first immunization. One week after the fourth immunization, the mice were bled from the tail, and the antibody titer was determined by ELISA. The spleen cells of a mouse with the highest antibody titer were taken, and the hybridoma cell lines specifically secreting the PEDV N protein monoclonal antibody were screened by the conventional method. After expansion culture, the mouse was injected intraperitoneally, and the ascites obtained was subjected to affinity chromatography according to the PIERCE company NAb TM Protein G Spin Purification Kit.

[0074] On the other hand, the RNA of the positive hybridoma cells was extracted, and cDNA was synthesized by reverse transcription using Oligo-dt or random primers (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A). The antibody variable region gene was amplified by nested PCR. First, the antibody variable region gene was amplified using the first round of mouse antibody IgG and kappa light chain primers as the template, and then the antibody variable region gene was amplified using the second round of mouse antibody IgG and kappa light chain primers as the template. The PCR reaction system was as follows: PrimeSTAR Max Premix (2x) 25 μL, P1 and P2 each 1 μL, cDNA 1 μL, and ddH2O was added to 50 μL. The reaction program was as follows: 98 ℃ pre-denaturation for 2 min; 98 ℃ denaturation for 10 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 30 cycles; 72 ℃ extension for 10 min. The primers for amplifying the antibody variable region gene were referred to the literature (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, A.D., Wang, Q., Gazumyan, A., Nussenzweig, M.C., 2016. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nature protocols 11, 1908-1923.). After amplification, 1% agarose gel electrophoresis was performed, and the heavy chain and kappa light chain variable region genes were about 300 bp in size. The target fragments were recovered by cutting the gel and sequenced. The sequencing results were compared and analyzed with the antibody gene library (IMGT). The sequencing results confirmed that the amplified sequences were the DNA sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody. Specifically, the DNA sequences encoding the heavy chain variable region of the PEDV N protein A and B monoclonal antibodies were as shown in SEQ ID NO: 3 and SEQ ID NO: 7, respectively; the DNA sequences encoding the light chain variable region of the PEDV N protein A and B monoclonal antibodies were as shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively. The amino acid sequences of the heavy chain variable region of the PEDV N protein A and B monoclonal antibodies were as shown in SEQ ID NO: 1 and SEQ ID NO: 5, respectively; the amino acid sequences of the light chain variable region of the PEDV N protein A and B monoclonal antibodies were as shown in SEQ ID NO: 2 and SEQ ID NO: 6, respectively.

[0075] Example 2: Preparation of nano-enzyme immunochromatographic test strip for porcine epidemic diarrhea virus

[0076] 2.1 Preparation of nanosome-PEDV N protein monoclonal antibody probe

[0077] Take 500 μL of nanosome solution (preferably prepared according to the disclosure 202411276039.6 Nanosome and preparation method and application thereof), and wash the nanosome solution with 1 mL of deionized water for three times. After centrifugation, discard the supernatant. Resuspend the precipitate with 1 mL of MES buffer solution, and the pH of the 50 mM MES buffer solution is 6.0. Add 10 μL of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 20 μL of NHS (N-hydroxysuccinimide) at the same time. After mixing, activate for 30 min in a shaker. The concentration of EDC and NHS is 20 mg / mL. The rotation speed of the shaker is 70 rpm. After activation, wash with 1 mL of MES buffer solution once, and then resuspend with 500 μL of MES buffer solution to obtain the activated nanosome. Take 50 μg of PEDV N protein monoclonal antibody A and incubate with the activated nanosome solution overnight at 4°C. Incubate at room temperature for 30 min with 0.05 mol / L Tris buffer solution (pH 7.2), and terminate the reaction. After magnetic separation, resuspend with 0.5 mL of 5% BSA Tris buffer solution to obtain the nanosome-PEDV N monoclonal antibody probe.

[0078] 2.2 Preparation of nanosome binding pad

[0079] Spray the prepared nanosome-PEDV N protein monoclonal antibody A probe on the binding pad with a film sprayer, dry in a 37°C oven for 2-3 h, and seal for storage.

[0080] 2.2 Preparation of nitrocellulose membrane

[0081] Dilute PEDV N protein monoclonal antibody B and goat anti-mouse IgG antibody to 1 mg / mL, and spray them on the nitrocellulose membrane with a spacing of 5-8 mm, which are used as the test line (T) and the control line (C), respectively. After the lines are drawn, dry in a 37°C-38°C oven for 2-3 h.

[0082] 2.3 Assembly of test strip

[0083] Connect the sample pad, binding pad, nitrocellulose membrane, and water absorption pad in sequence on the PVC bottom plate to obtain the test strip. Cut the test strip according to the size of the test strip card shell, and then assemble the cut test strip into the test strip card shell. The structure diagram of the test strip is shown in Figure 2 .

[0084] Example 3: Application of nanosome immunochromatography test strip for porcine epidemic diarrhea virus

[0085] 3.1 Method for using the test strip

[0086] 3.1.1 Sample processing

[0087] (1) Collect the sample with a sample collection swab as a pig anal swab or pig fecal swab.

[0088] (2) Insert the swab after sampling into a sample tube (containing 1 mL of sample processing solution), take it out after fully stirring for 10-15 seconds, oscillate back and forth to mix, dissolve the sample and fully mix with the sample processing solution, then stand for 1 min to precipitate the undissolved sample. The sample processing solution is a 0.01 mol / L PBS solution with pH 7.4.

[0089] 3.1.2 Sample detection

[0090] Take out the sealed test strip, place it on a dry and stable table top, use a pipette to take an appropriate amount of supernatant in 3.1.1, slowly add 6-7 drops (about 100-150 μL) into the sample addition hole, start chromatography, time for 10 min, and observe the results. In order to amplify the detection signal, DAB, a substrate for peroxidase, can be added after 10 min, and the enzyme activity of the nanoenzyme will catalyze the chemical reaction of DAB to generate a large amount of brown precipitate.

[0091] 3.1.3 Judgment

[0092] (1) Two brown bands appear on the test strip (T: detection line, C: quality control line), which is judged as positive (see Figure 3 A).

[0093] (2) Only one brown band appears on the test strip (C: quality control line), which is judged as negative (see Figure 3 B).

[0094] (3) No brown band appears at the quality control line of the test strip, which is judged as invalid (see Figure 3 C).

[0095] 3.2 Sensitivity of the test strip

[0096] Dilute the PEDV TCID 50 10 5.50 / mL virus by 1:100, then dilute by 2 times, and then use the nanoenzyme immunochromatographic test strip for porcine epidemic diarrhea virus established by the application to test the sensitivity.

[0097] The results show that when the virus is diluted by 6400 times, the nanoenzyme immunochromatographic test strip for porcine epidemic diarrhea virus established by the application is positive, and when diluted by 12800 times, it is negative (see Figure 4) ; it is illustrated that the sensitivity of the test strip prepared in the embodiment can reach 6400 times, that is, the minimum virus detection amount of the method of the present application is 49 TCID 50 / mL.

[0098] 3.3 Specificity of the test strip

[0099] 5 negative anal swabs are detected by the test strip, numbered as P1-P5, and the detection result shows that the quality control line C appears a brownish band, and no band is found at the T line, which indicates that the detection result is negative Figure 5 ) ; in addition, different pig virus such as porcine deltacoronavirus PDCoV, porcine transmissible gastroenteritis virus TGEV, porcine sapelovirus PSV and porcine rotavirus PoRV are detected by the test strip, and it is found that only PEDV is positive, and the rest are not reacted, which indicates that the method established in the present application can specifically recognize PEDV, as shown in Figure 6 Thus, it can be seen that the test strip established in the present application has strong specificity.

[0100] Although the embodiments of the present application have been disclosed as above, it is not limited to the application listed in the specification and the embodiments, and it can be fully applied to various fields suitable for the present application, and other modifications can be easily realized by those skilled in the art, and therefore the present application is not limited to specific details and the embodiments shown and described herein, without departing from the general concept defined by the claims and the equivalent scope.

Claims

1. A porcine epidemic diarrhea virus specific monoclonal antibody or antigen binding fragment thereof, characterized in that, The CDR1, CDR2 and CDR3 of the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof are respectively the amino acid sequences shown in SEQ ID NO: 9-11; The CDR1, CDR2 and CDR3 of the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof are respectively the amino acid sequences shown in SEQ ID NO: 12, DTS and SEQ ID NO:

13.

2. The porcine epidemic diarrhea virus specific monoclonal antibody or antigen binding fragment thereof of claim 1, characterized in that, The heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO: 1; The light chain variable region of the monoclonal antibody or antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO:

2.

3. A nucleic acid encoding the porcine epidemic diarrhea virus specific monoclonal antibody or antigen-binding fragment thereof of claim 1.

4. A recombinant vector comprising the nucleic acid of claim 3.

5. A recombinant host cell comprising the recombinant vector of claim 4.

6. Use of the porcine epidemic diarrhea virus specific monoclonal antibody or antigen-binding fragment thereof of claim 1 in the preparation of a reagent or kit for detecting porcine epidemic diarrhea virus.

7. A test strip for detecting porcine epidemic diarrhea virus, characterized by, The test strip comprises a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorption pad, the nitrocellulose membrane is provided with a detection line and a quality control line, the conjugate pad is sprayed with a nano-enzyme labeled capture antibody, and the detection line is coated with a detection antibody; The capture antibody or the detection antibody is the porcine epidemic diarrhea virus specific monoclonal antibody or antigen-binding fragment thereof of claim 1.

8. The test strip for detecting porcine epidemic diarrhea virus according to claim 7, wherein, The capture antibody and the detection antibody are respectively selected from the porcine epidemic diarrhea virus specific monoclonal antibody or antigen-binding fragment thereof of claim 1 and antibody B; The CDR1, CDR2 and CDR3 of the heavy chain variable region of antibody B have respectively the amino acid sequences shown in SEQ ID NO: 14-16, and the CDR1, CDR2 and CDR3 of the light chain variable region of antibody B have respectively the amino acid sequences shown in SEQ ID NO: 17, KAS, SEQ ID NO:

18.

9. The test strip for detecting porcine epidemic diarrhea virus according to claim 8, characterized in that, The heavy chain variable region of antibody B has the amino acid sequence shown in SEQ ID NO: 5; The light chain variable region of antibody B has the amino acid sequence shown in SEQ ID NO:

6.

10. The test strip for detecting porcine epidemic diarrhea virus according to claim 7, wherein the virus is porcine epidemic diarrhea virus. The quality control line is coated with a goat anti-mouse IgG; The nano-enzyme is washed with deionized water, centrifuged, the supernatant is discarded, the precipitate is resuspended with a MES buffer solution, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide are added, mixed, and then activated in a shaker; after activation, the nano-enzyme is washed with a MES buffer solution, then resuspended with a MES buffer solution to obtain activated nano-enzyme; the capture antibody is incubated with the activated nano-enzyme solution overnight; Tris buffer is added to terminate the reaction, and after magnetic separation, the nano-enzyme labeled capture antibody is resuspended with Tris buffer.

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