CN117683124A

CN117683124A - Blocking ELISA kit for detecting N protein antibody of porcine epidemic diarrhea virus

Info

Publication number: CN117683124A
Application number: CN202311407892.2A
Authority: CN
Inventors: 王�琦; 曹丽艳; 郑海学; 孔祥雨; 万颖; 张宇; 袁聪; 段月月; 施磊
Original assignee: Lanzhou Veterinary Research Institute of CAAS; Institute of Urban Agriculture of Chinese Academy of Agricultural Sciences
Current assignee: Lanzhou Veterinary Research Institute of CAAS; Institute of Urban Agriculture of Chinese Academy of Agricultural Sciences
Priority date: 2023-10-27
Filing date: 2023-10-27
Publication date: 2024-03-12

Abstract

The invention belongs to the technical field of biology, and relates to a blocking ELISA kit for detecting an N protein antibody of Porcine Epidemic Diarrhea Virus (PEDV). The kit comprises: an ELISA plate coated with PEDV N protein, an HRP-labeled PEDV N protein monoclonal antibody, a positive serum control and a negative serum control. The PEDV N protein monoclonal antibody is 1F10, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1F10 is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4. The kit has no cross reaction with the common porcine diarrhea related virus positive serum, and the intra-batch and inter-batch repeated variation coefficient is less than 10%. The coincidence rate of the detection of the blocking ELISA kit and the foreign commercialized kit is 98.79%, the Kappa value is 0.95, and the detection of the blocking ELISA kit and the foreign commercialized kit are highly consistent.

Description

Blocking ELISA kit for detecting N protein antibody of porcine epidemic diarrhea virus

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a blocking ELISA kit for detecting an N protein antibody of porcine epidemic diarrhea virus.

Background

Porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) belongs to the order of the niporoviridae (Nidovirales), the family of Coronaviridae (Coronaviridae), the subfamily of Coronaviridae (Coronavirinae), the genus alpha coronavirus (Alpha coronavirus), and is the main pathogen causing porcine intestinal diseases. The disease is characterized by acute watery diarrhea, dehydration and vomiting. Pigs of all ages can be infected, but the suckling piglets are the most serious and have a mortality rate as high as 100%. Over a long period of time PEDV evolved a mutant strain with greater virulence, transmissibility and pathogenicity, rendering the existing vaccine insufficient to protect swine herds from PEDV infection.

PEDV is a single-stranded positive strand RNA virus with a genome size of about 28kb, a Cap structure (Cap) at the 5 'end, a Poly (a) tail at the 3' end, and at least 7 open reading frames (ORF 1a, ORF1b, and ORF 2-6). ORFs 1a and 1b encode nonstructural proteins (Nonstructural protein, NSPs), ORF3 encodes one nonstructural auxiliary protein (ORF 3), and the remaining ORFs encode four major structural proteins, including spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The N protein is a main component of PEDV nucleocapsid, is composed of 441 amino acids, has a molecular weight of 55-58 kDa, contains a plurality of potential phosphorylation sites, is rich in serine and has high isoelectric point, is a multifunctional alkaline phosphoprotein, has high expression and strong conservation in infected cells, and is a key protein for virus serodiagnosis and immunology detection.

The enzyme-linked immunosorbent assay (ELISA) has the advantages of simple operation, strong specificity, high sensitivity and the like, and is widely applied to detection of human and animal diseases.

Disclosure of Invention

The monoclonal antibody has high homogeneity and specificity, and the invention aims to screen a strain of PEDV N protein monoclonal antibody and establish a blocking ELISA kit for detecting PEDV N protein antibody with good sensitivity and specificity by using the monoclonal antibody. The coincidence rate of the blocking ELISA kit and the imported kit established by the invention is as high as 98.79%, and the Kappa value is 0.95, so that the blocking ELISA kit can be used for clinical detection of PEDV N protein antibodies, and has wide market prospect and good economic benefit.

The invention utilizes a prokaryotic expression system to obtain soluble PEDV N protein with immunogenicity and reactivity, uses the soluble PEDV N protein as an immune source to immunize mice, and utilizes cell fusion and subclone screening technology to obtain a specific monoclonal antibody which is named as 1F10. The invention obtains the antibody variable region gene sequence by using a nested PCR amplification technology, and can be obtained by using a genetic engineering or protein engineering method subsequently. The blocking ELISA kit for detecting the PEDV serum antibody, which is prepared based on the PEDV N protein specific monoclonal antibody, has the advantages of strong specificity and high sensitivity.

The blocking ELISA kit for detecting PEDV N protein antibody comprises: an ELISA plate coated with PEDV N protein, an HRP-labeled PEDV N protein monoclonal antibody, positive control serum and negative control serum.

The preparation method of the PEDV N protein comprises the following steps: according to PEDV CV777 strain N protein (Genbank accession number: AF 353511.1) as a reference, a specific primer is designed, an N gene is obtained through PCR amplification, the N gene is connected with a prokaryotic expression vector pET32a carrier, a recombinant plasmid pET32a-N is constructed, escherichia coli BL21 (DE 3) is transformed, recombinant N protein is obtained through IPTG induced expression, and then a nickel column is used for purification to obtain the purified recombinant N protein. The amino acid sequence of the PEDV N protein is shown as SEQ ID NO. 1, and the DNA sequence of the encoded PEDV N protein is shown as SEQ ID NO. 2.

Furthermore, the preparation method of the PEDV N protein monoclonal antibody comprises the following steps: balb/c mice were immunized with purified recombinant N protein as an immune source in an amount of 30. Mu.g/g. For the first immunization, the recombinant N protein and Freund's complete adjuvant are uniformly mixed in equal volume and then emulsified, and the multi-point subcutaneous injection is adopted for the back and the abdomen. Boosting was performed every 2 weeks for four total immunizations. The boosting is to uniformly mix the recombinant N protein and Freund's incomplete adjuvant in equal volume for emulsification, and the immunization method is the same as the first immunization. After the fourth immunization, taking the spleen cells of the mice and the SP2/0 cells of the myeloma cells to fuse and prepare hybridoma cells, performing indirect ELISA and indirect Immunofluorescence (IFA) verification on cell supernatants, screening to obtain positive clones, and injecting the hybridoma cells into the mice to prepare ascites after three subcloning, so as to obtain the PEDV N protein monoclonal antibody.

The amino acid sequence of the heavy chain variable region of the PEDV N protein monoclonal antibody is shown as SEQ ID NO. 3; the amino acid sequence of the light chain variable region of the PEDV N protein monoclonal antibody is shown as SEQ ID NO. 4. Wherein, the amino acid sequences of the heavy chain and light chain variable regions CDR1, CDR2 and CDR3 of the monoclonal antibody are as follows:

——	CDR1	CDR2	CDR3
				heavy chain VH	GYTFTTYY	IYPGNINT	ARISSALPY
Light chain VL	RSLLNTSSQKSY	FAS	QQHFSTPPT

The DNA sequence of the heavy chain variable region of the PEDV N protein monoclonal antibody is shown as SEQ ID NO. 5; the DNA sequence of the light chain variable region of the PEDV N protein monoclonal antibody is shown as SEQ ID NO. 6.

The positive control serum in the blocking ELISA kit is pig serum collected after PEDV artificial immunity; the negative control serum was porcine serum free of PEDV pathogen.

The blocking ELISA kit for detecting the PEDV N protein antibody further comprises a coating liquid, a blocking liquid, a sample diluent, an enzyme-labeled antibody diluent, a washing liquid, a color development liquid and a termination liquid.

The coating solution was 0.05M carbonate buffer (1.5 g Na ₂ CO ₃ ，2.92g NaHCO ₃ Added to deionized water, adjusted to pH 9.6, and then fixed to volume 1L).

The blocking solution was a PBST solution containing 5% skim milk, 5% BSA or 2% trehalose, wherein 5% skim milk, 5% BSA and 2% trehalose represent the addition of 5g skim milk, 5g BSA and 2g trehalose per 100mL PBST solution, respectively.

The sample dilution was a 0.01M PBS solution (0.2 g KH) pH7.4 with 1% BSA ₂ PO ₄ 、2.9g Na ₂ HPO ₄ ·12H ₂ O, 8g of NaCl, 0.2g of KCl were dissolved in 800mL of ultrapure water, the pH was adjusted to 7.4 with NaOH, and the volume was adjusted to 1L with water).

The enzyme-labeled antibody diluent is PBS solution of 4% sucrose, 0.05% Tween-20 and 0.01M pH7.4 of 8% glycerol; the wash was 20-fold concentrated PBST (5.4 g KH) ₂ PO ₄ 、28.4g Na ₂ HPO ₄ ·12H ₂ O, 160g NaCl, 4g KCl, 10mL Tween-20, and deionized water were added, and after the pH was adjusted to 7.4, the volume was fixed to 1L).

The color development liquid is TMB, the stop solution is 2M H ₂ SO ₄ 。

The blocking ELISA kit for detecting the PEDV N protein antibody comprises the following operation steps:

(1) Coating: diluting the purified PEDV N protein with a coating liquid, coating an ELISA plate at 4 ℃ overnight, removing the liquid in the plate, washing for 4 times, and drying by taking a piece of absorbent paper;

(2) Closing: adding a sealing liquid, sealing the ELISA plate, removing the liquid in the plate, washing for 4 times, and beating with water-absorbing paper;

(3) Sample adding: adding the serum to be detected after the dilution of the sample diluent, reacting, setting negative control, positive control and blank control holes, removing the liquid in the plate, washing for 4 times, and drying by taking a piece of absorbent paper;

(4) Adding enzyme-labeled secondary antibodies: adding HRP-marked PEDV N protein monoclonal antibody diluted by enzyme-labeled antibody diluent for reaction, removing liquid in the plate, washing for 4 times, and drying by taking a piece of absorbent paper;

(5) Color development: adding TMB substrate to develop color in dark;

(6) And (3) terminating: add 2M H ₂ SO ₄ Terminating the reaction;

(7) Reading: enzyme-labeled instrument for measuring OD ₄₅₀ ；

(8) Calculating the blocking rate: pi= (negative control OD value-serum OD value to be detected)/negative control OD value x 100%, and when the detected sample PI is greater than or equal to 38.65%, positive is determined, and PI < 38.65% is determined as serum antibody negative.

As a preferred embodiment, the PEDV N protein has a coating concentration of 0.5. Mu.g/mL.

As a preferable scheme, the dilution ratio of the serum sample to be detected is 1:4.

As a preferred embodiment, the dilution factor of the HRP-labeled PEDV N protein monoclonal antibody is 1:8000.

The beneficial effects of the invention are as follows:

the invention uses the purified PEDV N protein as antigen, the PEDV N protein monoclonal antibody as detection antibody, and establishes a blocking ELISA kit and a detection method for detecting the PEDV N protein antibody through a series of reaction conditions and reagent optimization. The blocking ELISA kit established by the invention has better specificity, sensitivity, intra-batch and inter-batch repeatability and stability. The coincidence rate with the imported kit is as high as 98.79%, and the Kappa value is 0.95, which shows that the blocking ELISA kit established by the invention can be used for clinical detection of PEDV N protein antibodies, and has wide market prospect and good economic benefit.

The invention patent application publication No. CN 110016078A discloses a detection method of blocking ELISA based on PEDV N protein specific nano-antibody and application thereof, wherein the coincidence rate is 94%, and the coincidence rate of the establishment method and an imported ELISA kit (abbexa, UK) is as high as 98.79%, which is obviously superior to the method.

Drawings

FIG. 1 shows the result of PCR amplification of PEDV N gene and enzyme digestion identification of pET32a-N recombinant plasmid;

A.M: DL2000 relative molecular mass standard; 1: water control; 2: n gene, the size is about 1300bp.

B.M: DL5000 relative molecular mass standard; 1: the pET32a-N recombinant plasmid was digested with BamHI and XhoI.

FIG. 2 shows the expression and purification results of pET32a-N recombinant protein;

A.M: protein markers; 1: pre-induction of pET32 a-N; 2: after pET32a-N induction; 3: pET32a-N cleavage supernatant; 4: precipitation after cleavage of pET32 a-N;

B.M: protein markers; 1: a supernatant; 2: flow through; 3-4: eluting with 25 μm imidazole; 5 to 6:50 μm imidazole elution; 7-8: eluting with 75 μm imidazole; 9-10: 100 μm imidazole elution; 11: eluting with 150 μm imidazole; 12: eluting with 200 μm imidazole; 13: eluting with 300 μm imidazole; 14: 500. Mu.M imidazole elution.

FIG. 3 is a graph showing the results of ELISA for detecting the titer of polyclonal antibodies against PEDV N protein.

FIG. 4 is a schematic illustration of an indirect immunofluorescence assay to verify the reactivity of monoclonal antibodies.

A.1f10 hybridoma cell supernatant; SP2/0 cell supernatant.

FIG. 5 is a diagram showing the reactivity of a Westernblot to identify monoclonal antibodies;

m: protein Marker, mock: veroE6 cell control; PEDV: PEDV infects VeroE6 cells.

FIG. 6 shows the results of monoclonal antibody subclass identification.

FIG. 7 shows SDS-PAGE identification after monoclonal antibody purification.

M: protein markers; 1F10: purified monoclonal antibodies.

FIG. 8 shows the results of PCR amplification of the variable region of the monoclonal antibody 1F 10;

A. heavy chain variable region PCR amplification results. M: DL2000 Marker; VH:1F10 heavy chain variable region PCR amplification product.

B. Results of PCR amplification of the light chain K variable region. M: DL2000 Marker; VL:1F10 light chain variable region PCR amplification product.

Fig. 9 is a graph plotting ROC curves.

FIG. 10 shows the specific detection results of PEDV N protein antibody detection blocking ELISA method.

Detailed Description

The following detailed description of the present invention is provided to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of the present invention.

EXAMPLE 1 construction, expression and purification of recombinant plasmid of PEDV N protein

1.1 construction of the pET32a-N recombinant plasmid

Specific primers were designed, upstream P1, with reference to the PEDV CV777 strain N protein (Genbank accession number: AF 353511.1): 5' -CGCGGATCCATGGCTTCTGTCAGCTTTCA-3′Downstream P2:5' -CCGCTCGAGTTAATTTCCTGTATCGAAGATC-3', bamHI and XhoI cleavage sites (underlined) were introduced at the 5' end of the upstream and downstream primers. After primer synthesis, PCR amplification was performed using cDNA of the PEDV CV777 strain as a template. The PCR reaction system is as follows: primeSTAR Max Premix (2X) 25. Mu.L, 1. Mu.L each of P1 and P2, 1. Mu.L cDNA, ddH ₂ O was added to 50. Mu.L. The reaction procedure is: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 55℃for 30s, extension at 72℃for 2min,30 cycles; extending at 72℃for 10min. The PCR products were detected by 1% agarose gel electrophoresis, and the results are shown in FIG. 1A.

Gel recovery was performed according to the instructions of Omega company EZNAGel Extraction Kit to obtain N gene PCR gel recovery products. The recovered N gene PCR gel product was digested with BamHI and XhoI, and ligated to pET32a vector to transform DH 5. Alpha. Competent cells. And (3) carrying out double enzyme digestion identification on the constructed recombinant plasmid, and carrying out sequencing identification on the plasmid with positive enzyme digestion identification, wherein the positive recombinant plasmid is named pET32a-N. The results of the double cleavage assay are shown in FIG. 1B.

1.2 Induction expression and purification of recombinant N proteins

The recombinant plasmid pET32a-N is transformed into escherichia coli BL21 (DE 3), and single colony is picked in 5mL of ampicillin resistant LB for OD ₆₀₀ About 0.8, IPTG was added at a final concentration of 1mmol/L to induce expression. After induction, the pellet was collected by centrifugation at 12000rpm for 2min, resuspended in an appropriate amount of PBS, sonicated for 5min (3 s, 3s stopped), centrifuged at 12000rpm for 10min at 4℃after sonication, and the supernatant and pellet were collected, respectively. The supernatant and the precipitate were added to a 5×loading buffer, and boiled in boiling water for 10min, and SDS-PAGE was performed. The results of the identification are shown in FIG. 2A.

Expanding the induced expression quantity of pET32a-N protein according to the method, taking the supernatant after ultrasonic treatment, and purifying by a nickel column. The specific operation steps are as follows:

(1) And (3) resin filling: taking an empty column, adding 2mL of nickel column NTA resin, washing the column once by using distilled water with 5 column volumes when the preservation solution descends to the surface of the resin, and balancing the column by using balancing solution (20 mM Tris-HCl,500mM NaCl,5mM imidazole, pH 7.4) with 5 column volumes;

(2) Loading: when the equilibrium liquid is lowered to the surface of the resin, 3mL of cleavage supernatant containing recombinant protein is added, the sample is repeatedly loaded for 2-3 times, each time of action is carried out for 2min, and sample flow through liquid is collected; adding 5 times of column volume of balance liquid to wash the column for 1 time;

(3) Protein elution: and (3) preparing imidazole with different concentrations by using a balancing solution to elute the protein sample.

(4) And (5) cleaning and preserving the column: the column was washed once with 5 volumes of 0.5M NaOH, then once with distilled water, followed by the addition of appropriate amount of 70% absolute ethanol, and stored in a refrigerator at 4 ℃.

Samples collected each time were identified by SDS-PAGE. The result of the authentication is shown in FIG. 2B.

EXAMPLE 2 preparation of PEDV N protein monoclonal antibody and amplification of antibody sequences

2.1 immunization of mice

Purified recombinant N protein was immunized against Babl/c mice in an amount of 30. Mu.g/g. For the first immunization, the recombinant N protein and Freund's complete adjuvant are uniformly mixed in equal volume and then emulsified, and subcutaneous injection is adopted at 1 point on the back and 2 points on the abdomen. Boosting is carried out every 2W for four times, wherein the boosting is to uniformly mix recombinant N protein and Freund's incomplete adjuvant in equal volume for emulsification, and the immunization method is the same as the first immunization.

2.2ELISA detection of antibody titers

After 1 week of the fourth immunization, mice were bled at their tails and antibody titers were determined. The inactivated PEDV virus solution and the coating solution are diluted 1:1 to coat ELISA plates, 50 mu L/well, coated at 37 ℃ for 1h, and then coated with PBST (K) ₂ HPO ₄ 0.26g、Na ₂ HPO ₄ ·12H ₂ 2.89g of O, 8.50g of NaCl and 0.5mL of Tween-20, adding water to fix the volume to 1L, and pH 7.4), and adding 2% of trehalose for sealing at 37 ℃ for 1h. Diluting the mouse positive serum of immune recombinant N protein and the mouse negative serum of non-immune recombinant N protein with PBST ratio, diluting with gradient from 1:100 to 1:204800, diluting with 12 gradients, standing at 37deg.C for 30min, washing with PBST for four times, adding HRP-labeled goat anti-mouse IgG (1:20000 dilution), standing at 37deg.C for 30min, washing with PBST for four times, developing TMB, and measuring OD on enzyme-labeling instrument ₄₅₀ 。

Results showShown, at 1:12800 dilution of serum, immunized mice OD numbers 1, 2, and 3 ₄₅₀ /NC>2.0, indicating that the antibody titer can reach above 1:12800 (FIG. 3).

2.3 preparation of monoclonal antibodies

Spleen cells of a recombinant N protein immunized mouse are mixed with SP20 cells (ratio of 5:1), and cell fusion is carried out under the action of fusion agent PEG. The fused cells were plated in 96-well cell plates at 37℃with CO ₂ Culturing in an incubator. To confluent the hybridoma cells to 1/10 of the bottom of the cell plate, the supernatant was aspirated for ELISA detection (ELISA coating method same as 2.2). Wells identified as positive by ELISA were then validated by indirect immunofluorescence assay (IFA). ELISA and IFA positive holes are selected, and the selected positive hybridoma cells are subcloned by a limiting dilution method. Wells were visualized under an inverted microscope, where only individual clones were grown, and supernatants were taken for antibody detection using the ELISA and IFA methods described above. Positive cells entered the next round of subcloning, performed three times in total.

IFA test: will be 1X 10 ⁴ Individual VeroE6 cells were plated in 96 well cell plates and after confluence of the cells with a monolayer, 0.01MOI PEDV was seeded. 24h after inoculation, cells were fixed with 4% paraformaldehyde at room temperature for 30min, and with 0.01mol/L PBS (K ₂ HPO ₄ 0.26g、Na ₂ HPO ₄ ·12H ₂ 2.89g of O, 8.50g of NaCl, water to 1L, pH 7.4) for 3 times; then 0.1% Triton X-100 is used for permeation, and the reaction is carried out for 10min at room temperature, and PBS is used for washing 3 times; hybridoma cell supernatants and goat anti-mouse FITC-IgG were then incubated and the results were observed under a fluorescence microscope after completion of the reaction.

2.4 monoclonal antibody specificity identification

After 3 subcloning, the cell supernatant secreted by the obtained single cell strain is subjected to IFA and western blot detection. IFA assay showed that specific green fluorescent signal was detected by 1F10 cell supernatant on PEDV-infected VeroE6 cells (fig. 4A), whereas no fluorescent signal was seen by SP2/0 cell supernatant on PEDV-infected cells (fig. 4B). Virus-infected and uninfected VeroE6 cells were collected, lysed, subjected to SDS-PAGE, and subsequently protein gels were transferred to NC membranes for western blot validation. 1F10 cell supernatant was used as primary antibody, anti-mouse HRP-IgG was used as secondary antibody at room temperature for 1h, and after each reaction was completed, PBST was used for washing 4 times, followed by exposure and development. Westernblot results showed that 1F10 was able to recognize N protein in virus infected cells (FIG. 5).

2.5 monoclonal antibody subclass identification

SBA cloning according to Southern Biotech ^TM System/HRP antibody subclass identification kit operation shows that the obtained monoclonal antibodies are subjected to antibody subclass identification. As a result, FIG. 6 shows that the 1F10 monoclonal antibody heavy chain constant region is of the IgG1 type and the light chain constant region is of the Kappa type.

2.6 preparation and purification of ascites

Balb/c mice of 10-12W (weeks) were taken, each of which was intraperitoneally injected with Freund's incomplete adjuvant 0.5mL, and after 1W, each of which was intraperitoneally injected with 5X 10 ⁵ After 7-10 days, the abdominal cavity of the mouse obviously swells by the hybridoma cells (0.2 mL), and ascites is collected and split charging is carried out. Purification of ascites according to PIERCE Corp. NAb ^TM Protein G Spin Purification Kit affinity chromatography, purification followed by SDS-PAGE electrophoresis to identify purity (FIG. 7).

2.7 PCR amplification and sequence determination of monoclonal antibody variable region Gene

RNA from 1F10 hybridoma cells was extracted and reverse transcribed into cDNA using Oligo-dt or random primers, respectively (PrimeScript II 1st Strand cDNA Synthesis Kit,TAKARA,6210A).

The antibody variable region gene was amplified by nested PCR. The cDNA is first used as template to amplify the variable region gene of antibody with the first round of mouse antibody IgG and kappa light chain primer, and the first round of product is then used as template to amplify the variable region gene of antibody with the second round of mouse antibody IgG and kappa light chain primer. The PCR reaction system is as follows: primeSTAR Max Premix (2X) 25. Mu.L, 1. Mu.L each of P1 and P2, 1. Mu.L cDNA, ddH ₂ O was added to 50. Mu.L. The reaction procedure is: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 55℃for 30s, elongation at 72℃for 30s,30 cycles; extending at 72℃for 10min. Primer references for gene amplification of antibody variable regions (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 the amplification, 1% agarose gel electrophoresis was performed, the gene size of the heavy chain and kappa light chain variable region was about 300bp (FIGS. 8A and 8B), and the desired fragment was recovered by gel cutting. The recovered target fragment was inserted into a pMD-18T vector, and sequence was determined.

The sequencing results were compared with the antibody gene library (IMGT) and confirmed that the amplified sequences were DNA of the heavy and light chain variable regions of the monoclonal antibody.

Specifically, the DNA sequence of the heavy chain variable region of the PEDV N protein monoclonal antibody 1F10 is shown as SEQ ID NO. 5; the DNA sequence of the light chain variable region of the PEDV N protein monoclonal antibody 1F10 is shown as SEQ ID NO. 6. The amino acid sequence of the heavy chain variable region of the PEDV N protein monoclonal antibody 1F10 is shown as SEQ ID NO. 3; the amino acid sequence of the light chain variable region of the PEDV N protein monoclonal antibody 1F10 is shown as SEQ ID NO. 4. The amino acid sequences of the heavy chain and light chain variable regions CDR1, CDR2, and CDR3 of monoclonal antibody 1F10 are shown in table 1 below.

TABLE 1 amino acid sequences of the heavy and light chain variable regions CDR1, CDR2 and CDR3 of monoclonal antibody 1F10

Example 3 establishment of blocking ELISA method for detecting PEDV N protein antibody

3.1 serum preparation

(1) Preparation of PEDV-positive porcine serum (standard positive serum): healthy piglets of about 35 days of age were selected for primary immunization with 2mL of PEDV (10 ⁴ TCID ₅₀ /mL) back sea point injection; at intervals of 2 weeks, inactivating the virus with 2 times of the first immunity dose, and performing back sea point injection; after 2 weeks, booster immunizations were performed, and the immunization dose was the same as the second immunization. Serum was isolated 1 week after boost and identified as PEDV positive serum by ELISA kit (abbexa, uk).

(2) Preparation of PEDV-negative porcine serum (standard negative serum): serum was prepared by ELISA kit identification screening negative pigs without PEDV infection.

3.2 selection of optimal reaction conditions for blocking ELISA

3.2.1 determination of optimal antigen coating concentration and optimal dilution of serum samples

(1) PEDV N protein was diluted in the ratio of 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 μg/mL with coating solution, and the ELISA plate was added laterally, 50 μl per well, and coated for 1h at 37 ℃. After spin-drying, the plate is washed with PBST for 4 times.

(2) 2% trehalose was added and the mixture was blocked at 37℃for 1 hour. And (3) washing for three times after spin-drying, wherein the method is the same as that of (1).

(3) The negative and positive serum were diluted 1:2, 1:4, 1:8, 1:16, and 1:32 times and then added to ELISA plate longitudinally, and allowed to act at 37℃for 30min. And (3) washing for three times after spin-drying, wherein the method is the same as that of (1).

(4) HRP-labeled mab 1F10 (HRP-1F 10) was diluted (1:8000-fold) with antibody dilution and added to ELISA plates, 50. Mu.L per well, and allowed to react at 37℃for 30min. And (3) washing for three times after spin-drying, wherein the method is the same as that of (1).

(5) 50. Mu.L of TMB substrate chromogenic solution was added to each well and the mixture was allowed to act at 37℃for 10min.

(6) 50 mu L of 2M H are added to each well ₂ SO ₄ The reaction was terminated. Reading OD ₄₅₀ nm values. PI values were calculated, pi= (negative control OD value-serum OD value to be tested)/negative control OD value x 100%.

As a result, the optimal coating concentration of PEDV N protein was 0.5. Mu.g/mL and the optimal dilution of serum was 1:4 when the PI value was maximized, as shown in Table 2.

TABLE 2 selection of optimal coating concentration of antigen and optimal dilution of serum

3.2.2 determination of optimal coating conditions

The ELISA plates were coated according to the optimal antigen coating concentration of PEDV N protein determined at 3.2.1, and were coated at 37℃for 1h, 2h, 3h and 4h, and overnight at 4 ℃. According to OD ₄₅₀ The nm value and PI value determine the optimal coating conditions. The results are shown in Table 3, and the PI value of the sample tested was the largest when coated overnight at 4℃indicating that the optimal coating condition was 4℃overnight.

TABLE 3 optimal coating conditions for antigens

3.2.3 determination of optimal blocking solution and action conditions

The ELISA plates were coated according to the optimal coating concentration of PEDV N protein as determined in 3.2.1 and 3.2.2 and the coating conditions. 5% skim milk, 5% BSA or 2% trehalose was selected as blocking solution and allowed to act at 37℃for 1h. According to OD ₄₅₀ The nm value and the PI value determine the optimal blocking solution. As shown in Table 4, the PI value of the test sample was higher than that of 5% skim milk and 5% BSA by 77.79% when the sample was blocked with 2% trehalose. Therefore, 2% trehalose has the best blocking effect. 5% skim milk, 5% BSA and 2% trehalose represent the addition of 5g skim milk, 5g BSA, respectively, per 100mL PBST solution2g of trehalose.

TABLE 4 selection of optimal blocking solution

Sealing liquid	5% skim milk	5％BSA	2% trehalose
				PI％	74.22	75.05	77.79

ELISA plates were blocked with optimal blocking solution, blocking at 37℃for 1h, 2h, 3h and 4h, and overnight blocking at 4 ℃. According to OD ₄₅₀ The nm value and PI value determine the optimal blocking conditions. As shown in Table 5, the PI value of the sample was maximum when the sample was blocked at 37℃for 3 hours, indicating that the optimal blocking condition was blocked at 37℃for 3 hours.

TABLE 5 determination of optimal closing time

3.2.4 determination of the optimal time of action of the serum to be tested

After coating and sealing ELISA plates using optimal conditions, serum to be tested with optimal dilution concentration is added, and the reaction is performed at 37 ℃ for 15min, 30min, 45min, 60min and 90min. As shown in Table 6, the PI value was maximum when the reaction time of the serum to be tested was 60min, and thus the optimal action time of the serum to be tested was 60min.

TABLE 6 determination of time of serum to be tested

Serum duration of action	15min	30min	45min	60min	90min
						PI％	58.65	73.16	77.14	78.53	76.35

Determination of 3.2.5 enzyme-labeled antibody working concentration and reaction time

After serum to be tested is acted under the optimal condition, ELISA plates are washed, HRP-1F10 monoclonal antibodies are diluted by antibody diluents of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000 and 1:64000, and are acted for 30min at 37 ℃. According to OD ₄₅₀ The nm value and the PI value determine the optimal working concentration of the enzyme-labeled antibody. As shown in Table 7, the optimal working concentration of the enzyme-labeled antibody was 1:8000 times, when the PI value was maximum at 1:8000 times dilution of HRP-1F 10.

TABLE 7 optimal working concentration of enzyme-labeled antibodies

After the working concentration of the optimal enzyme-labeled antibody is determined, the enzyme-labeled antibody is selected to act for 15min, 30min, 45min and 60min at 37 ℃. According to OD ₄₅₀ The nm value and the PI value determine the reaction time of the enzyme-labeled antibody. As shown in Table 8, the PI value was maximized when the enzyme-labeled antibody was allowed to act at 37℃for 30min, and therefore, the optimal conditions for the enzyme-labeled antibody were found to act at 37℃for 30min.

TABLE 8 optimal time of action of enzyme-labeled antibodies

HRP-1F10 action time	15min	30min	45min	60min
					PI％	80.04	82.63	74.37	74.37

3.2.6 determination of the color development time

After the enzyme-labeled antibody reacts under the optimal action condition, the ELISA plate is washed, and color development is performed at 37 ℃ for 5min, 10min, 15min and 20min. According to OD ₄₅₀ The nm value and the PI value determine the optimal substrate action time. As shown in table 9, PI value was maximum when the development time was 15 min.

TABLE 9 determination of color development time

Color development time	5min	10min	15min	20min
					PI％	77.13	78.34	78.87	78.42

3.3 determination of critical value

85 PEDV positive serum samples and 160 negative serum samples are detected according to an optimized blocking ELISA method, and OD (optical density) of the serum samples is measured after detection is finished ₄₅₀ nm, and the blocking rate (PI value) was calculated, and ROC curves were plotted using SPSS17.0 software (fig. 9). When the Youden index was maximum (0.981), the specificity was 100%, the sensitivity was 98.1%, and the corresponding PI was 38.65. Therefore, when the PI value is larger than or equal to 38.65%, the result is positive; when the PI value of the sample is less than 38.65%, the sample is judged as negative.

3.4 specificity assay

SADS-CoV, TGEV, PDCoV, porcine Sapelo Virus (PSV) and porcine rotavirus (PRoV) positive sera were detected by an optimized blocking ELISA method. 3 replicates were set for each sample, with PEDV negative and positive serum as control. As shown in FIG. 10, only PEDV serum samples were positive and none of the others had cross-reactions, as detected by the optimized blocking ELISA method, demonstrating the good specificity of the method established by the present invention.

3.5 repeatability test

5 serum samples (positive serum 3 parts P1, P2 and P3; negative serum 2 parts N1 and N2) were selected for testing to assess the in-batch and inter-batch reproducibility of the blocking ELISA method, according to OD ₄₅₀ The value at nm calculates the PI value. Then calculating the variation coefficient through PI valueThe results are shown in Table 10, and the variation coefficient of the batch and batch-to-batch reproducibility tests are below 10%, which indicates that the established blocking ELISA method has good reproducibility.

Table 10 blocking reproducibility of ELISA detection methods

3.6 blocking ELISA method of the invention and commercial kit comparison test

The PEDV N protein antibody blocking ELISA method established by the invention and a commercial kit (abbexa, UK) are used for jointly detecting 165 field pig serum samples, and then the coincidence rate of the detection results of the blocking ELISA method and the commercial kit is calculated. The detection result of the commercialized kit shows that 26 parts of positive serum and 139 parts of negative serum are used; positive serum 26 parts and negative 139 parts were tested using the blocking ELISA method of the invention. The total coincidence rate is 98.79%, and the coincidence rate is higher.

Further statistics were carried out on both methods, and Kappa values were calculated, and the result showed that Kappa value was 0.95 (Kappa. Gtoreq.0.75), indicating that both detection methods were almost completely consistent (Table 11). The PEDV N protein antibody blocking ELISA method established by the invention has the same detection effect as a commercial kit.

TABLE 11 blocking ELISA and commercial kit comparison results of the invention

The coincidence rate and Kappa value are calculated as follows:

the coincidence rate= [ (a+d)/n ]. 100;

P _A ＝(a+d)/n；P _e ＝[(a+b)(a+c)+(c+d)(b+d)]/n ² ；Kappa＝(P _A -P _e )/(1-P _e )。

the above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention, so that all equivalent changes or modifications of the structure, characteristics and principles described in the claims should be included in the scope of the present invention.

Claims

1. The porcine epidemic diarrhea virus N protein monoclonal antibody is characterized in that the heavy chain variable region of the monoclonal antibody comprises CDR1 with an amino acid sequence GYTFTTYY, CDR2 with an amino acid sequence IYPGNINT and CDR3 with an amino acid sequence ARISSALPY;

the light chain variable region of the monoclonal antibody comprises a CDR1 with an amino acid sequence of RSLLNTSSQKSY, a CDR2 with an amino acid sequence of FAS and a CDR3 with an amino acid sequence of QQHFSTPPT.

2. The monoclonal antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the monoclonal antibody are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

3. A gene encoding the porcine epidemic diarrhea virus N protein monoclonal antibody of claim 1.

4. The gene according to claim 3, wherein the heavy chain variable region and the light chain variable region of the monoclonal antibody have the gene sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

5. A recombinant expression vector comprising the gene of claim 3.

6. A host cell comprising the recombinant expression vector of claim 5.

7. The use of the monoclonal antibody according to claim 1 or 2, the gene according to claim 3 or 4, the recombinant expression vector according to claim 5, the host cell according to claim 6 in any one of the following aspects,

(1) The application in preparing products for treating diseases related to porcine epidemic diarrhea virus infection and infection thereof; the product is a drug or vaccine;

(2) The application in preparing a porcine epidemic diarrhea virus detection reagent or a kit.

8. A blocking ELISA kit for detecting porcine epidemic diarrhea virus N protein antibodies, comprising: an ELISA plate coated with the N protein of the porcine epidemic diarrhea virus, an HRP-labeled monoclonal antibody of the N protein of the porcine epidemic diarrhea virus according to claim 1 or 2, positive control serum and negative control serum.

9. The kit of claim 8, further comprising a coating solution, a blocking solution, a sample diluent, an enzyme-labeled antibody diluent, a washing solution, a color developing solution, and a stop solution.

10. The kit according to claim 9, wherein,

the coating liquid is carbonate buffer liquid of 0.05M;

the blocking solution is PBST solution containing 5% skimmed milk, 5% BSA or 2% trehalose, wherein 5% skimmed milk, 5% BSA or 2% trehalose respectively represent that 5g skimmed milk, 5g BSA and 2g trehalose are added into every 100mL PBST solution;

the sample dilution was a 0.01M PBS solution pH7.4 of 1% BSA;

the enzyme-labeled antibody diluent is a PBS solution of 4% sucrose, 0.05% Tween-20 and 8% glycerol, 0.01M pH 7.4; the wash solution was 20-fold concentrated PBST;

the color development liquid is TMB, the stop solution is 2M H ₂ SO ₄ 。