PEDV N protein monoclonal antibody as well as preparation method and application thereof

Abstract

The invention provides a PEDV (porcine epidemic diarrhea virus) N protein monoclonal antibody as well as a preparation method and application thereof, and the method comprises the following steps: performing prokaryotic expression on PEDV N protein, and purifying to obtain the PEDV N protein; emulsifying the purified PEDV N protein and an isopyknic Freund's adjuvant, injecting the emulsified PEDV N protein into 8-week-old female BALB / c mice at multiple points, and immunizing once every two weeks; the method comprises the following steps: uniformly mixing splenocytes of a mouse subjected to boosted immunization for 3 days with SP2 / 0 cells in a logarithmic phase according to a ratio of 5: 1, carrying out cell fusion under the action of PEG 1500, and culturing a fused cell mixed solution by using an HAT culture medium; screening out positive pores with strong antibody secretion capacity by adopting an indirect ELISA (Enzyme-Linked Immunosorbent Assay) method, and carrying out subcloning by adopting a limited dilution method to obtain six cell strains for secreting antibodies; the PEDV N protein mAb is prepared by adopting an in-vivo ascites inducing method. The prepared PEDV N protein monoclonal antibody can be specifically and efficiently combined, and can be used as an antibody tool for researching PEDV virus infection and anti-infection mechanisms.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibody preparation, and in particular to a PEDV N protein monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious disease caused by porcine epidemic diarrhea virus (PEDV) in pigs. The main clinical symptoms are acute diarrhea, dehydration, vomiting, acute enteritis, etc. The virus has the characteristics of sudden onset, rapid spread, and wide range of epidemics. It can infect pigs of all ages, and is most harmful to unweaned piglets under two weeks of age, which can cause a mortality rate of 90-100%. At present, the disease has been reported in all pig-raising countries in the world, among which the most serious ones are European and Asian countries, causing serious economic losses to the breeding industry in various places.

[0003] PEDV is a member of the order Nephrovirales, family Coronaviridae, genus Coronavirus. The genome is about 28kb long and consists of 2 non-structural proteins and 4 structural proteins. PEDV nucleocapsid protein (N protein) is one of the structural proteins of PEDV and is a core component of coronavirus. This protein plays an important role in inducing cellular immunity in infected organisms. In the early stages of viral infection, it can stimulate the body to produce humoral immunity and induce the body to produce a large number of anti-N protein antibodies. PEDV N protein is an important target protein for early detection of PEDV infection and an important antigen. Therefore, it is often used in serological testing. This requires excellent PEDV N monoclonal antibodies as the basis. Accordingly, it is necessary to establish a cell line that stably produces monoclonal antibodies. Whether a suitable cell line can be constructed depends on effective PEDV N recombinant proteins and monoclonal antibody preparation methods. Summary of the invention

[0004] The purpose of the present invention is to provide a PEDV N protein monoclonal antibody and a preparation method and application thereof, so as to at least solve one problem in the background technology.

[0005] In a first aspect, the present invention provides a method for preparing a monoclonal antibody against PEDV N protein, the preparation method comprising:

[0006] The PEDV N protein is expressed in prokaryotes and purified using a HIS-tag nickel column affinity chromatography column to obtain a purified PEDV N protein;

[0007] The purified PEDV N protein was emulsified with an equal volume of Freund's adjuvant and injected into 8-week-old female BALB / c mice at multiple sites, once every two weeks for a total of 3 immunizations;

[0008] The spleen cells of mice that had been boosted for 3 days were mixed with SP2 / 0 cells in the logarithmic growth phase at a ratio of 5:1, and the cells were fused under the action of PEG1500. The fused cell mixture was added to the feeder cells prepared one day in advance and cultured in HAT medium;

[0009] The indirect ELISA method was used to screen the positive wells with strong antibody secretion ability, and the limiting dilution method was used for subcloning to obtain 6 antibody-secreting cell lines;

[0010] The mAb against PEDV N protein was prepared by in vivo ascites induction method.

[0011] Furthermore, the step of prokaryotically expressing the PEDV N protein comprises:

[0012] The PEDV N gene was amplified by RT-PCR using PEDV N-specific primers and nucleic acid extracted from allantoic fluid of chicken embryos infected with PEDV strains as templates.

[0013] The PEDV N gene was integrated into the plasmid vector by homologous recombination to obtain a recombinant plasmid;

[0014] The recombinant plasmid was transformed into the host bacteria BL21 to obtain bacterial liquid;

[0015] Recombinant bacteria carrying the PEDV N gene were screened from the bacterial solution and expressed the PEDV N protein under IPTG induction.

[0016] Furthermore, the nucleotide sequence of the PEDV N protein is shown in SEQ ID NO.1.

[0017] Furthermore, the amino acid sequence of the PEDV N protein is shown in SEQ ID NO.2.

[0018] Furthermore, the nucleotide sequences of the amplified primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0019] Furthermore, the plasmid vector is pCold III, which carries a His tag and a cold shock inducible promoter, and utilizes low temperature induction to reduce the excessive folding of proteins to form inclusion bodies, thereby increasing the soluble expression of proteins.

[0020] Furthermore, the step of integrating the PEDV N gene into a plasmid vector by homologous recombination to obtain a recombinant plasmid comprises:

[0021] The PEDV N gene was cloned into the prokaryotic expression vector pCold III using the homologous recombinase of Quanshijin, transformed into the competent Escherichia coli cell DH5α, and transformed into the prokaryotic expression strain BL21 after plasmid sequencing verification to obtain the recombinant expression bacteria carrying the PEDV N gene.

[0022] Furthermore, the step of expressing the PEDV N protein in a prokaryotic manner and purifying the protein using a HIS-tagged nickel column affinity chromatography column to obtain the purified PEDV N protein comprises:

[0023] The supernatant protein was obtained by purification using a HIS-tag nickel column affinity chromatography;

[0024] The protein supernatant was added to the HIS-tagged nickel affinity chromatography column and incubated at 4°C overnight;

[0025] Use 10 mM imidazole wash solution to elute impurities and 100 mM to elute the target protein;

[0026] The eluted PEDV N protein was centrifuged through an ultrafiltration tube to remove imidazole.

[0027] In a second aspect, the present invention further provides a PEDV N protein monoclonal antibody, wherein the PEDV N protein monoclonal antibody is prepared according to the above-mentioned method for preparing the PEDV N protein monoclonal antibody.

[0028] In a third aspect, the present invention also proposes a use of a PEDV N protein monoclonal antibody in the prevention of PEDV virus infection.

[0029] In summary, according to the above-mentioned method for preparing PEDV N protein monoclonal antibodies, the prepared PEDV N protein monoclonal antibodies can bind specifically and efficiently, and can be used as antibody tools for studying PEDV virus infection and anti-infection mechanisms. In addition, the prepared monoclonal antibodies meet the requirements for raw materials for the development of immunochromatographic test strips. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0031] Figure 1 This is a schematic diagram of SDS-PAGE electrophoresis detection of PEDV N protein induced expression in one embodiment of the present invention;

[0032] Figure 2 This is a schematic diagram of Western blot detection of PEDV N protein expression in one embodiment of the present invention;

[0033] Figure 3 This is a schematic diagram of SDS-PAGE electrophoresis detection of PEDV N protein after purification in one embodiment of the present invention;

[0034] Figure 4 This is a schematic diagram of Western blot identification of PEDV N monoclonal antibody in one embodiment of the present invention;

[0035] Figure 5 This is a schematic diagram of IFA identification of PEDV N monoclonal antibodies in one embodiment of the present invention. DETAILED DESCRIPTION

[0036] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0037] The following detailed description is an exemplary description, which is intended to provide further detailed description of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The terms used in the present invention are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present invention.

[0038] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional channels such as ordinary biochemical reagent stores.

[0039] Example 1

[0040] Construction of prokaryotic expression plasmid for PEDV N protein

[0041] The PEDV N gene was amplified by RT-PCR using PEDV N-specific primers and nucleic acid extracted from the allantoic fluid of chicken embryos infected with PEDV strains as a template. Primers with homology arms of the pCold III vector were designed to obtain the PEDV N gene sequence with homology arms by PCR amplification. The PEDV N gene was cloned into the prokaryotic expression vector pCold III using the homologous recombinase 2×Basic Assembly Mix of Full-Form Gold, transformed into Escherichia coli competent cells DH5α, and after verification by plasmid sequencing, it was transformed into the prokaryotic expression strain BL21 to obtain the recombinant expression bacteria with the PEDV N gene.

[0042] The nucleotide sequence of the PEDV N gene is shown in SEQ ID NO: 1; the amino acid sequence encoded by the PEDV N gene is shown in SEQ I NO: 2, specifically:

[0043] SEQ ID NO: 1 Nucleotide sequence of PEDV N gene

[0044] ATGGCTTCTGTCAGCTTTCAGGATCGTGGCCGCAAACGGGTGCCATTATCCCTCTATGCCCCTCTTAGGGTTACTAATGACAAACCCCTTTCTAAGGTACTTGCAAATAATGCTGTACCCACTAATAAAGGAAGTAAGGACCAGCAAATTGGATACTGGAATGAGCAAATTCGCTGGCGCATGCGCCGTGGTGAGCGAATTGAGCAACCTTCCAATTGGCATTTCTACTACCTCGGAACAGGACCTCACGCCGACCTTCGCTATAGGACTCGTACTGAGGGTGTTTTCTGGGTTGCTAAAGAAGGCGCAAAGACTGAACCCACTAACCTGGGTGTCAGAAAGCCGTCTGAAAAGCCAATCATTCCAAATTTCTCTCAACAGCTTCCCAGCGTAGTTGAGATTGTTGAACCTAACACACCTCCTACTTCACGTGTAAATTCACGTAGCAGGAGTCGTGGTAATGGCAACAATAGGTCCAGATCTCCAAGTAACAACAGAGGCAATAACCAGTCCCGCGGTAATTCACAGAATCGTGGAAATAACCAGGGTCGTGGAGCTTCTCAGAACAGAGGAGGCAATAATAATAACAATAACAATTCTCGTAACCAGTCCAAGAACAGAAACCAGTCAAATGACCGTGGTGGAATGACATCACGCGATGATCTGGTGGCTGCTGTCAAGGATGCCCTTAAATCTTTGGGTATTGGAGAAAATCCTGATAGGCTTAGGCAACAACAGAAGCCTAAGCAGGAAAAGTCTGACAACGGCGGCAAAAATACACCTAAGAAGAACAAATCCAGGGCCACTTCGAAGGAACGTGACCTCAAGGACATCCCAGAGTGGAGGAGAATTCCCAAGGGCGAAAATAGCGTAGCAGCTTGCTTCGGACCCAGGGGGGGCTTCAAAAATTTTGGAGATGCGGAGTTTGTCGAAAAAGGTGTTGATGCCTCAGGCTATGCTCAGATCGCCAGTTTGGCACCAAATGTTGCAGCATTGCTCTTTGGTGGTAATGTGGCTGTTCGCGAGCTAGCGGACTCTTACGAGATTACATACAATTATAAAATGACTGTGCCAAAGTCTGATCCAAATGTTGAGCTTCTTGTTTCACAGGTTGATGCATTTAAAACTGGTAATGCAAAACCCCAGAGAAAGAAGGAAAAGAAGAACAAGCGTGAAACCACGCAGCAGCTTAATGAAGATGCCATCTACGATGACGTGGGTGTGTCATCTGATGTGACTCATGCCAATTTGGAATGGGACACAGCTGTTGATGGTGGTGACACGGCCGTTGAAATTATCAACGAGATCTTCGATACAGGAAATTAA。

[0045] SEQ ID NO: 2 Amino acid sequence of the PEDV N gene

[0046] MASVSFQDRGRKRVPLSLYAPLRVTNDKPLSKVLANNAVPTNKGSKDQQIGYWNEQIRWRMRRGERIEQPSNWHFYYLGTGPHADLRYRTRTEGVFWVAKEGAKTEPTNL GVRKPSEKPIIPNFSQQLPSVVEIVEPNTPPTSRVNSRSRSRGNGNNRSRSPSNNRGNNQSRGNSQNRGNNQGRGASQNRGGNNNNNNNSRNQSKNRNQSNDRGGMTSRDD LVAAVKDALKSLGIGENPDRLRQQQKPKQEKSDNGGKNTPKKNKSRATSKERDLKDIPEWRRIPKGENSVAACFGPRGGFKNFGDAEFVEKGVDASGYAQIASLAPNVAA LLFGGNVAVRELADSYEITYNYKMTVPKSDPNVELLVSQVDAFKTGNAKPQRKKEKKNKRETTQQLNEDAIYDDVGVSSDVTHANLEWDTAVDGGDTAVEIINEIFDTGN*

[0047] The primer pairs for amplifying the PEDV N gene are as follows:

[0048] SEQ ID NO.3:AATCACAAAGTGCATATGATGGCTTCTGTCAGCTTTC

[0049] SEQ ID NO.4:ATTCGGATCCCTCGAGCtcagtggtggtggtggtggtgc

[0050] Example 2

[0051] Expression and purification of PEDV N protein

[0052] (1) After activation, the recombinant expression bacteria constructed in Example 1 were transferred to LB liquid medium containing ampicillin and cultured on a shaker (37°C, 220 rpm) overnight. The bacterial liquid was transferred to a new LB medium containing ampicillin at a volume of 1:100 and cultured on a shaker until the OD 600value ≈ 0.8, cool at room temperature for 30 minutes, add IPTG to final concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1 mM, respectively, and induce expression at 16°C and 220 rpm for 24 hours; collect the cells by centrifugation at 4°C and 8000 rpm for 10 minutes, wash the cells twice with PBS, and finally resuspend the cells with 1 / 10 volume of the original bacterial solution; ultrasonically clarify the bacterial solution with an ultrasonic disruptor at 200W, working for 3s and resting for 6s; centrifuge at 4°C and 12000 rpm for 20 minutes to separate the supernatant and precipitate; aspirate 100 μL of the supernatant for SDS-PAGE electrophoresis, and verify the protein expression by Western blot. Figure 1 and Figure 2 As shown, for Figure 1 : M is the standard molecular weight of protein, 1 is the expression of pCold III-PEDV N protein without IPTG; 2-7 are the supernatants of pCold III-PEDV N expression induced by 0.1, 0.2, 0.4, 0.6, 0.8, and 1 mM IPTG, and 8-13 are the precipitates of pCold III-PEDV N expression induced by 0.1, 0.2, 0.4, 0.6, 0.8, and 1 mM IPTG; Figure 2 : M is the standard molecular weight of protein, 1 is the supernatant of pCold III-PEDV N induced expression; 2 is the precipitate of pCold III-PEDV N induced expression. The results showed that PEDV N recombinant protein (about 51 KDa) can be expressed in E. coli BL21.

[0053] (2) Purify the supernatant protein obtained in (1) using a HIS-tagged nickel column affinity chromatography column. Add the protein supernatant to a HIS-tagged nickel column affinity chromatography column and incubate at 4°C overnight; elute the impurities with 10mM imidazole washing solution and elute the target protein with 100mM; the eluted high-purity PEDV N protein is centrifuged through an ultrafiltration tube to remove the imidazole. The protein purification status is detected by SDS-PAGE, as shown in FIG. Figure 3 As shown, for Figure 3 : M is the standard molecular weight of protein, 1 is the flow-through of PEDV N protein after hanging on the column, 2-6 are all washed with 10mM imidazole; 7-11 are all eluted with 100mM imidazole. The results show that the PEDV N recombinant protein can be effectively purified.

[0054] Example 3

[0055] Preparation of monoclonal antibody against PEDV N protein

[0056] (1) Antibody titer detection method

[0057] Use the coating solution to dilute the PEDV N protein to 0.5μg / mL, and add 100μL / well to a 96-well ELISA plate, coat overnight at 4℃, wash the plate 3 times with PBST and pat dry. Add 200μL / well of freshly prepared 5% skim milk powder, block at 37℃ for 2h, pat dry and store at 4℃ for later use. Add the processed samples to a 96-well ELISA plate, set up positive and negative serum controls, incubate at 37℃ for 1h, wash the plate 3 times with PBST and pat dry. Dilute the goat anti-mouse IgG-HRP 1:5000 with PBS, add 100μL / well to a 96-well ELISA plate, incubate at 37℃ for 1h, wash 5 times with PBST and pat dry.

[0058] Add enzyme substrate TMB reaction solution, 100 μL / well, react at room temperature for 5-8 minutes. Add 2M H2SO4 stop solution, 100 μL / well, and quickly detect the OD of each well with an ELISA reader after adding the stop solution. 450 If the OD of the hole to be measured is 450 The value is 2.1 times greater than or equal to that of the negative control, that is, the P / N value is >2.1, which is a positive result.

[0059] (2) Hybridoma cell preparation

[0060] Take the purified PEDV N protein (~50 μg / mouse) and add an equal volume of Freund's adjuvant (CFA for the first immunization and IFA for the second immunization), emulsify it and inject it subcutaneously into two 8-week-old female BALB / c mice at multiple points. Mice injected with PBS and an equal volume of adjuvant emulsified were set as controls. Immunization was performed once every two weeks for a total of 3 times. In addition, to ensure the effect of immunization, mice can be intraperitoneally boosted with PEDV N protein (100 μg / mouse) three days before fusion.

[0061] One day before fusion, an unimmunized and healthy Kunming mouse was killed by cervical scission, and the feeder cells were added to a 96-well plate. The boosted immunized mice were killed by removing their eyes and bleeding, and the serum was collected as a positive control. The mouse spleen was removed and placed on a sterile large dish, and then an appropriate amount of DMEM culture medium was drawn with a syringe, and the spleen was repeatedly blown thin to prepare a single cell suspension, and the spleen cell suspension was fused with SP2 / 0 cells at a ratio of 5:1. The cells were gently resuspended in HAT selection medium, added to the feeder cells prepared one day in advance at 100μL / well, and placed flat in a 37℃ 5% CO2 incubator. The state of the cells was observed after about 4 days.

[0062] (3) Subclone screening

[0063] When the hybridoma cell state reached 30%, the indirect ELISA method was used to screen out the positive wells with strong antibody secretion ability, and the limiting dilution method was used for subcloning. After three subclones, six cell lines with stable antibody secretion were obtained, namely 1A1, 1G1, 2A3, 2C5, 3G11 and 3H1.

[0064] (4) Large-scale preparation of monoclonal antibodies

[0065] The mAb of PEDV N protein was prepared in large quantities by inducing ascites in vivo. 1 mL of ascites adjuvant was intraperitoneally inoculated into BALB / c multiparous female mice 2 weeks in advance. The cells in logarithmic growth state were resuspended and counted, and 1x10 6 cells. About 10 days later, ascites was collected and purified. The titer of purified ascites was 1:160,000 to 1:320,000 by indirect ELISA (Table 1). The monoclonal antibody subclass was identified as IgG1 using a monoclonal antibody detection kit (biodragon) (Table 2).

[0066] Table 1 Anti-PEDV N monoclonal antibody titer determination

[0067] Dilution 1A1 1G1 2A3 2C5 3G11 3H1 Positive Negative 1:5000 3.950 4.000 4.000 3.920 3.800 3.900 3.880 0.323 1:10000 3.900 3.950 3.890 3.800 3.580 3.610 3.800 0.317 1:20000 3.900 3.750 3.800 3.540 3.270 2.940 3.960 0.343 1:40000 3.480 3.130 3.540 2.630 2.530 2.240 3.000 0.323 1:80000 2.530 2.050 2.670 1.430 1.430 1.360 3.999 0.303 1:160000 1.630 1.300 1.860 0.827 0.800 0.713 3.998 0.283 1:320000 0.853 0.718 1.050 0.431 0.408 0.369 3.997 0.263 1:640000 0.514 0.387 0.607 0.259 0.240 0.215 3.996 0.243

[0068] Table 2 Identification of subtypes of anti-PEDV N monoclonal antibodies

[0069]

[0070] Example 4 Identification and application of monoclonal antibodies

[0071] (1) WB identification of monoclonal antibodies

[0072] When Vero cells grow to 80% to 90%, they are inoculated with PEDV virus for infection. After 24 hours, RIPA lysis buffer is added to the cells to obtain the supernatant, and SDS-PAGE electrophoresis is performed. The 1A1 strain high titer purified antibody is selected as the primary antibody, diluted 1:2000, and incubated at 4°C overnight. The color development results are as follows Figure 4 ,for Figure 4 : Mock is the cell lysate not infected with PEDV virus, and PEDV is the cell lysate infected with PEDV virus.

[0073] (2) IFA identification of monoclonal antibodies

[0074] When Vero cells grow to 80% to 90%, they are inoculated with PEDV virus. After 24 hours, the culture medium is discarded, and the cells are fixed with formaldehyde acetone and blocked with 1% BSA in PBST at room temperature. The 1A1 strain high titer purified antibody is selected as the primary antibody, diluted 1:4000, and incubated at 4°C overnight. The staining and photography results are shown in the figure below. Figure 5 ,for Figure 5 : Mock is the cell lysate not infected with PEDV virus, and PEDV is the cell lysate infected with PEDV virus.

[0075] according to Figure 4 and Figure 5 The results showed that the prepared anti-PEDV N monoclonal antibodies can bind specifically and efficiently, and can be used as antibody tools for studying PEDV virus infection and anti-infection mechanisms. In addition, the prepared monoclonal antibodies meet the requirements for raw materials for the development of immunochromatographic test strips.

[0076] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a monoclonal antibody against PEDV N protein, characterized in that: The preparation method comprises: The PEDV N protein is expressed in prokaryotes and purified using a HIS-tag nickel column affinity chromatography column to obtain a purified PEDV N protein; The purified PEDV N protein was emulsified with an equal volume of Freund's adjuvant and injected into 8-week-old female BALB / c mice at multiple sites, once every two weeks for a total of 3 immunizations; The spleen cells of mice that had been boosted for 3 days were mixed with SP2 / 0 cells in the logarithmic growth phase at a ratio of 5:1, and the cells were fused under the action of PEG1500. The fused cell mixture was added to the feeder cells prepared one day in advance and cultured in HAT medium; The indirect ELISA method was used to screen the positive wells with strong antibody secretion ability, and the limiting dilution method was used for subcloning to obtain 6 antibody-secreting cell lines; The mAb against PEDV N protein was prepared by in vivo ascites induction method.

2. The method for preparing a monoclonal antibody against PEDV N protein according to claim 1, characterized in that: The step of prokaryotically expressing the PEDVN protein comprises: The PEDV N gene was amplified by RT-PCR using PEDV N-specific primers and nucleic acid extracted from allantoic fluid of chicken embryos infected with PEDV strains as templates. The PEDV N gene was integrated into the plasmid vector by homologous recombination to obtain a recombinant plasmid; The recombinant plasmid was transformed into the host bacteria BL21 to obtain bacterial liquid; Recombinant bacteria carrying the PEDV N gene were screened from the bacterial solution and expressed the PEDV N protein under IPTG induction.

3. The method for preparing a monoclonal antibody against PEDV N protein according to claim 2, characterized in that: The nucleotide sequence of PEDV N protein is shown in SEQ ID NO.

1.

4. The method for preparing a monoclonal antibody against PEDV N protein according to claim 2, characterized in that: The amino acid sequence of PEDV N protein is shown in SEQ ID NO.

2.

5. The method for preparing a monoclonal antibody against PEDV N protein according to claim 2, characterized in that: The nucleotide sequences of the primer pair for amplification are shown in SEQ ID NO.3 and SEQ ID NO.

4.

6. The method for preparing a monoclonal antibody against PEDV N protein according to claim 2, characterized in that: The plasmid vector is pColdIII, and the plasmid vector carries a His tag and a cold shock inducible promoter.

7. The method for preparing a monoclonal antibody against PEDV N protein according to claim 2, characterized in that: The step of integrating the PEDVN gene into the plasmid vector by homologous recombination to obtain the recombinant plasmid comprises: The PEDV N gene was cloned into the prokaryotic expression vector pCold III using the homologous recombinase of Quanshijin, transformed into the competent Escherichia coli cell DH5α, and transformed into the prokaryotic expression strain BL21 after plasmid sequencing verification to obtain the recombinant expression bacteria carrying the PEDV N gene.

8. The method for preparing a monoclonal antibody against PEDV N protein according to claim 2, characterized in that: The steps of expressing the PEDV N protein in a prokaryotic manner and purifying the protein using a HIS-tagged nickel column affinity chromatography column to obtain the purified PEDV N protein include: The supernatant protein was obtained by purification using a HIS-tag nickel column affinity chromatography; The protein supernatant was added to the HIS-tagged nickel affinity chromatography column and incubated at 4°C overnight; Use 10 mM imidazole wash solution to elute impurities and 100 mM to elute the target protein; The eluted PEDV N protein was centrifuged through an ultrafiltration tube to remove imidazole.

9. A PEDV N protein monoclonal antibody, characterized in that: The PEDV N protein monoclonal antibody is prepared according to the method for preparing a PEDV N protein monoclonal antibody according to any one of claims 1 to 8.

10. Use of the PEDV N protein monoclonal antibody according to claim 9 in anti-infection of PEDV virus.

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