Latex immunochromatography test strip for rapidly detecting porcine epidemic diarrhea virus and preparation method of latex immunochromatography test strip

Abstract

The invention belongs to the technical field of biology, and relates to a latex immunochromatography test strip for rapidly detecting porcine epidemic diarrhea virus (PEDV) and a preparation method of the latex immunochromatography test strip. The test strip comprises a PVC (polyvinyl chloride) bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, a quality control line C and a detection line T are arranged on the nitrocellulose membrane. A latex microsphere labeled PEDV N protein monoclonal capture antibody is sprayed on the combination pad, a detection line T is coated with a PEDV N protein monoclonal detection antibody 1E1, and a quality control line C is coated with goat anti-mouse IgG. The test strip can specifically recognize PEDV, and does not react with other porcine intestinal coronaviruses with similar clinical symptoms. The test strip is good in specificity, relatively high in sensitivity and simple to operate, a result is interpreted within 10 minutes, and compared with a traditional colloidal gold detection technology, the stability and the color development effect are obviously improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a latex immunochromatographic test strip for rapid detection of porcine epidemic diarrhea virus and a preparation method thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious enteric disease caused by porcine epidemic diarrhea virus (PEDV), with clinical manifestations of vomiting, diarrhea, dehydration and enteritis. Although pigs of all ages are susceptible, piglets are most susceptible, with a mortality rate of up to 100%. Clinically, the prevention and control of PED is a systematic strategy that requires not only effective vaccine immunization, but also timely and accurate antibody and antigen detection. However, in recent years, with the changes in the homology of PEDV epidemic strains and vaccine strains, some vaccines have become ineffective and are difficult to provide adequate protection. Therefore, early and rapid diagnosis remains the focus of prevention and control of the disease.

[0003] Commonly used pathogen detection methods include virus isolation and culture, electron microscopy, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) and real-time quantitative PCR. These methods have their own unique advantages, but are time-consuming and require professional technicians. Otherwise, it is easy to cause differences in experimental results. Therefore, it is difficult to meet the requirements of rapid, cheap and efficient monitoring of field or clinical samples.

[0004] Lateral immunochromatography technology is a technology that couples different signal markers on the surface of antigens or antibodies to achieve macroscopic visualization or microscopic antigen-antibody reaction by signal amplification. This technology is easy to operate and can be completed without the need for professional personnel. At the same time, it does not require the use of specific instruments, and the test results can be obtained by the naked eye. It is also time-saving and can be completed within 15 minutes. It greatly shortens the detection time. Its commonly used signal markers are mainly colloidal gold particles and latex microspheres. Although there are a variety of markers on the market, colloidal gold particles and latex microspheres are still the ones that can be used for large-scale production.

[0005] Compared with colloidal gold, latex microspheres have a high tolerance to physical and chemical factors and are not easily affected by them, which is beneficial to the stability of the finished test strips. At the same time, latex microspheres are filled with oil-soluble color dyes, which have high contrast, deep color, and high visualization, and are also better than colloidal gold in color rendering effect. Summary of the invention

[0006] The present invention uses a prokaryotic expression system to obtain a soluble PEDV N protein with immunogenicity and reactivity, and uses it as an immunogen to immunize mice, and uses cell fusion and subclone screening technology to obtain specific monoclonal antibodies, which are named 1F10 and 1E1 respectively. The antibody variable region gene sequence is obtained by nested PCR amplification technology, and can be subsequently obtained by genetic engineering or protein engineering methods. Based on the PEDV N protein-specific monoclonal antibody, a convenient, rapid and highly sensitive latex immunochromatographic test strip and preparation method for detecting porcine epidemic diarrhea virus are developed.

[0007] The present invention specifically includes the following contents:

[0008] In a first aspect, the present invention provides a PEDV N protein monoclonal antibody, wherein the monoclonal antibodies are 1F10 and 1E1.

[0009] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO: 2. Among them, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain and light chain variable regions of monoclonal antibody 1F10 are as follows:

[0010] 1F10 CDR1 CDR2 CDR3 Heavy chain VH GYTFTTYY IYPGNINT ARISSALPY Light chain VL RSLLNTSSQKSY FAS QQHFSTPPT

[0011] The DNA sequence encoding the heavy chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO:3; the DNA sequence encoding the light chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO:4.

[0012] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1E1 is shown in SEQ ID NO: 5; the amino acid sequence of the light chain variable region of monoclonal antibody 1E1 is shown in SEQ ID NO: 6. Among them, the amino acid sequences of the heavy chain and light chain variable regions CDR1, CDR2 and CDR3 of monoclonal antibody 1E1 are as follows:

[0013] 1E1 CDR1 CDR2 CDR3 Heavy chain VH GYTFTAYV INPYNDRT ARDDDDYEEGFAY Light chain VL QKCQYIWLSL SCI SAHSGAYT

[0014] The DNA sequence encoding the heavy chain variable region of monoclonal antibody 1E1 is shown in SEQ ID NO:7; the DNA sequence encoding the light chain variable region of monoclonal antibody 1E1 is shown in SEQ ID NO:8.

[0015] The heavy chain constant regions of the above-mentioned PEDV N protein monoclonal antibodies are all of IgG1 type.

[0016] The light chain constant regions of the above-mentioned PEDV N protein monoclonal antibodies are all of Kappa type.

[0017] In the second aspect, the present invention provides a latex immunochromatographic test strip for rapid detection of porcine epidemic diarrhea virus, the test strip comprising a PVC bottom plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad, wherein the sample pad, the conjugation pad, the nitrocellulose membrane and the water-absorbing 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 conjugation pad, and the quality control line C is arranged at one end close to the water-absorbing pad; the conjugation pad is sprayed with a monoclonal capture antibody of PEDV N protein labeled with latex microspheres; the detection line T is coated with a monoclonal antibody 1E1 of PEDV N protein as a detection antibody; the quality control line C is coated with goat anti-mouse IgG. The capture antibody can adopt the PEDV N protein monoclonal antibody 1F10 of the present application.

[0018] Preferably, the preparation method of the conjugate pad is:

[0019] The conjugate pad is blocked with a conjugate pad blocking solution;

[0020] The latex microspheres were activated with N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), centrifuged, resuspended with MES buffer, and ultrasonicated to fully disperse the fluorescent microspheres. Then, PEDV N protein monoclonal capture antibody was added, incubated at room temperature, and then blocked with blocking solution, incubated at room temperature, and centrifuged; the precipitate was resuspended with a latex microsphere marker protective agent to obtain a PEDV N protein monoclonal capture antibody latex microsphere labeling solution;

[0021] The prepared PEDV N protein monoclonal capture antibody latex microsphere labeling solution is evenly sprayed on the blocked conjugate pad using an instrument;

[0022] The conjugate pad blocking solution is an ultrapure aqueous solution of 5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20 and 1.5% m / v PVP-40.

[0023] The latex microsphere marker blocking solution is an ultrapure aqueous solution containing 10% m / v BSA;

[0024] The latex microsphere marker protective agent is a Tris-HCl solution containing 3% m / v sucrose, 1% m / v PVP-40, and 1% v / v Tween-20.

[0025] The PEDV N protein monoclonal capture antibody latex microsphere labeling solution is composed of: 20 μg of PEDV N protein monoclonal capture antibody per 1 mL of latex microsphere labeling solution.

[0026] Preferably, the detection line T is coated with 1 mg / mL of PEDV N protein monoclonal detection antibody 1E1.

[0027] Preferably, the quality control line C is coated with 1 mg / mL of goat anti-mouse IgG.

[0028] Preferably, the sample pad is immersed in a sample pad blocking solution; the sample pad blocking solution is a Tris-HCl solution containing 2% BSA, 0.5% S17, 2% PEG20000 and 0.5% sodium caseinate.

[0029] In a third aspect, the present invention provides a method for preparing the above-mentioned latex microsphere immunochromatographic test strip for detecting porcine epidemic diarrhea virus, the method comprising the following steps:

[0030] (1) preparing a binding pad coated with latex microspheres labeled with a monoclonal capture antibody of PEDV N protein;

[0031] (2) PEDV N protein monoclonal detection antibody 1E1 (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) were sprayed on the nitrocellulose membrane at intervals of 5 to 8 mm, serving as the detection line T and the quality control line C, respectively;

[0032] (3) The sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent pad are overlapped on the PVC bottom plate in sequence to obtain a latex microsphere immunochromatographic test strip for detecting porcine epidemic diarrhea virus.

[0033] In a fourth aspect, the detection method of the test strip of the present invention comprises the following steps:

[0034] (1) Collect samples for inspection.

[0035] (2) Take out the test strip and equilibrate it to room temperature. Add the sample to be tested to the sample adding area, let it stand at room temperature, and determine the result.

[0036] (3) Result determination: After the reaction is completed, the positive and negative properties are qualitatively determined by the naked eye.

[0037] The beneficial effects of the present invention are:

[0038] The latex microsphere immunochromatographic test strip for detecting PEDV provided by the present invention can be used for the specific detection of PEDV pathogens, and the test strip can detect the virus in the culture at a minimum limit of 10 3.2 TCID 50 / 0.1mL, and did not react with other porcine enteric coronaviruses with similar clinical symptoms, such as SADS-CoV, TGEV and PDCoV.

[0039] The test strip prepared by the present invention has the advantages of good specificity, sensitivity, simple operation, short time, etc., and provides a rapid detection method for on-site or laboratory detection of porcine infectious diarrhea coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The results of PCR amplification of PEDV N gene and restriction enzyme digestion of pET32a-N recombinant plasmid were shown;

[0041] AM: DL2000 relative molecular mass standard; 1: water control; 2: N gene, with a size of about 1300 bp.

[0042] BM: DL5000 relative molecular mass standard; 1: pET32a-N recombinant plasmid was double-digested with BamHI and XhoI.

[0043] Figure 2 This is the result of pET32a-N recombinant protein expression and purification;

[0044] AM: protein marker; 1: before pET32a-N induction; 2: after pET32a-N induction; 3: pET32a-N lysis supernatant; 4: precipitate after pET32a-N lysis;

[0045] BM: protein marker; 1: supernatant; 2: flow-through; 3-4: 25 μM imidazole elution; 5-6: 50 μM imidazole elution; 7-8: 75 μM imidazole elution; 9-10: 100 μM imidazole elution; 11: 150 μM imidazole elution;

[0046] 12: 200 μM imidazole elution; 13: 300 μM imidazole elution; 14: 500 μM imidazole elution.

[0047] Figure 3 This is a graph showing the test results of ELISA testing the titer of anti-mouse PEDV N protein polyclonal antibodies.

[0048] Figure 4 The reactivity of the monoclonal antibodies was verified by indirect immunofluorescence assay.

[0049] A. 1F10 hybridoma cell supernatant; B. 1E1 hybridoma cell supernatant; C. SP2 / 0 cell supernatant.

[0050] Figure 5 To identify the reactivity of monoclonal antibodies for western blot;

[0051] M: protein marker, Mock: VeroE6 cell control; PEDV: PEDV infected VeroE6 cells.

[0052] Figure 6 This is the result of monoclonal antibody subclass identification.

[0053] Figure 7 The monoclonal antibody was purified and identified by SDS-PAGE.

[0054] M: protein marker; 1: 1F10 monoclonal antibody purification; 2: 1E1 monoclonal antibody purification.

[0055] Figure 8 This is the result of PCR amplification of the variable region of the monoclonal antibody;

[0056] A. PCR amplification results of heavy chain variable region. M: DL2000 Marker; 1F10: PCR amplification of heavy chain variable region;

[0057] 1E1: PCR amplification of the heavy chain variable region.

[0058] B. PCR amplification results of light chain K variable region. M: DL2000 Marker; 1F10: PCR amplification of light chain variable region;

[0059] 1E1: PCR amplification of light chain variable region.

[0060] Fig. 9 Determination of the optimal amount of antibody labeling on latex microspheres.

[0061] Fig.10 Determination of the optimal antibody labeling concentration at the T line.

[0062] Fig.11 Schematic diagram of test strip assembly.

[0063] Fig.12 A graph showing the sensitivity test results of the test strips.

[0064] Fig.13 Graph showing specificity test results of the test strip. DETAILED DESCRIPTION

[0065] The present invention is described in more detail below through specific implementation modes to facilitate understanding of the technical solution of the present invention, but is not intended to limit the protection scope of the present invention.

[0066] Example 1 Construction, expression and purification of PEDV N protein recombinant plasmid

[0067] 1.1 Construction of pET32a-N recombinant plasmid

[0068] Using the N protein of PEDV CV777 strain (Genbank accession number: AF353511.1) as a reference, specific primers were designed, upstream P1: 5′-CGC GGATCCATGGCTTCTGTCAGCTTTCA-3′, downstream P2: 5′-CCG CTCGAG TTAATTTCCTGTATCGAAGATC-3′, BamHI and XhoI restriction sites (underlined parts) were introduced at the 5′ end of the upstream and downstream primers. After primer synthesis, PCR amplification was performed using the cDNA of the PEDV CV777 strain as a template. The PCR reaction system was: PrimeSTAR Max Premix (2×) 25μL, 1μL each of P1 and P2, 1μL of cDNA, and ddH2O was added to 50μL. The reaction procedure was: pre-denaturation at 98℃ for 2min; denaturation at 98℃ for 10s, annealing at 55℃ for 30s, extension at 72℃ for 2min, 30 cycles; extension at 72℃ for 10min. The PCR product was detected by 1% agarose gel electrophoresis, and the results are as follows. Figure 1 As shown in A.

[0069] According to the instruction manual of Omega's EZNAGel Extraction Kit, gel recovery was performed to obtain the N gene PCR gel recovery product. The N gene PCR gel recovery product and the pET32a vector were double-digested with BamHⅠ and XhoI, and then connected and transformed into DH5α competent cells. The constructed recombinant plasmid was double-digested for identification, and the plasmids that were positive for enzyme digestion were sequenced and identified. The positive recombinant plasmid was named pET32a-N. The double-digestion identification results are shown in Figure 1. Figure 1 As shown in B.

[0070] 1.2 Induced expression and purification of PEDV N recombinant protein

[0071] The recombinant plasmid pET32a-N was transformed into Escherichia coli BL21 (DE3), and a single colony was picked and placed in 5 mL of ampicillin-resistant LB. 600 When the expression of 1mmol / L IPTG was about 0.8, the expression was induced by adding IPTG at a final concentration of 1mmol / L. 5h after induction, the precipitate was collected by centrifugation at 12000rpm for 2min, and the precipitate was resuspended with an appropriate amount of PBS and ultrasonicated for 5min (ultrasonication for 3s, stop for 3s). After ultrasonication, the supernatant and precipitate were centrifuged at 4℃ for 10min, and the supernatant and precipitate were collected respectively. The supernatant and precipitate were added to 5× loading buffer, boiled in boiling water for 10min, and identified by SDS-PAGE. The identification results are as follows: Figure 2 As shown in A.

[0072] According to the above method, the induced expression of pET32a-N protein was expanded, and the supernatant after ultrasonic lysis was taken for nickel column purification. The specific operation steps are as follows:

[0073] (1) Loading the resin: Take an empty column and add 2 mL of nickel column NTA resin. When the storage solution drops to the surface of the resin, wash the column once with 5 column volumes of distilled water, and then equilibrate the column with 5 column volumes of equilibration solution (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.4);

[0074] (2) Sample loading: When the equilibrium solution drops to the resin surface, add 3 mL of lysate containing the recombinant protein. Repeat the sample loading 2 to 3 times, each time for 2 minutes, and collect the sample flow-through; add 5 column volumes of equilibrium solution to rinse the column once;

[0075] (3) Protein elution: Use equilibrium solution to prepare different concentrations of imidazole to elute the protein sample.

[0076] (4) Column cleaning and storage: Rinse the column once with 5 volumes of 0.5 M NaOH, then rinse once with distilled water, then add an appropriate amount of 70% anhydrous ethanol and store in a refrigerator at 4°C.

[0077] Each collected sample was subjected to SDS-PAGE identification. The identification results are as follows Figure 2 As shown in B.

[0078] Example 2 Preparation of PEDV N protein monoclonal antibody and amplification of antibody sequence

[0079] 2.1 Immunization of mice

[0080] Purified PEDV N recombinant protein was used to immunize Babl / c mice at a dose of 30 μg / mouse. For the first immunization, PEDV N protein was mixed with Freund's complete adjuvant in equal volumes and then emulsified, and multiple back subcutaneous injections were performed. Booster immunization was performed every 2 weeks, for a total of four immunizations. Booster immunization was performed by mixing PEDV N recombinant protein and Freund's incomplete adjuvant in equal volumes and emulsifying them. The immunization method was the same as the first immunization.

[0081] 2.2ELISA detection of antibody titer

[0082] One week after the fourth immunization, the mice were tail-cut and blood was collected to determine the antibody titer. The inactivated PEDV virus solution was diluted with the coating solution 1:1 and coated on the ELISA plate, 50 μL / well, coated at 37°C for 1 hour, washed four times with PBST (K2HPO4 0.26g, Na2HPO4·12H2O2.89g, NaCl 8.50g, Tween-20 0.5mL, dilute to 1L with water, pH7.4), and then blocked with 2% trehalose at 37°C for 1 hour. The positive serum of mice immunized with N protein and the negative serum of mice not immunized with N protein were diluted with PBST in multiple ratios, with a dilution gradient from 1:100 to 1:204800, and 12 dilution gradients were added. The cells were incubated at 37°C for 30 min, washed four times with PBST, and then HRP-labeled goat anti-mouse IgG (1:20000 dilution) was added. The cells were incubated at 37°C for 30 min, washed four times with PBST, and developed with TMB. The OD was measured on a microplate reader. 450 .

[0083] The results showed that when the serum was diluted 1:12800, the OD of mice immunized with No. 1, 2 and 3 was 450 / NC>2.0, indicating that the antibody titer can reach 1:12800 or above ( Figure 3 ).

[0084] 2.3 Preparation of monoclonal antibodies

[0085] Take the spleen cells of a mouse immunized with PEDV N protein and mix them with SP20 cells (5:1 ratio), and then perform cell fusion under the action of the fusion agent PEG. The fused cells are plated in a 96-well cell plate and cultured in a 37°C, CO2 incubator. When the hybridoma cells cover 1 / 10 of the bottom of the cell plate, aspirate the supernatant for ELISA detection (ELISA coating method is the same as 2.2). The wells identified as positive by ELISA are then verified by IFA. Select the ELISA and IFA positive wells, and use the limiting dilution method to subclone the screened positive hybridoma cells. Observe under an inverted microscope, mark the wells where only a single clone grows, take the supernatant, and use the above-mentioned ELISA and IFA methods for antibody detection. Positive cells enter the next round of subcloning, which is performed three times in total.

[0086] IFA test: 1×10 4VeroE6 cells were plated in a 96-well cell plate, and after the cells were fully plated, 0.01 MOI PEDV was inoculated. 24 hours after inoculation, the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, and washed three times with 0.01 mol / L PBS (K2HPO4 0.26g, Na2HPO4·12H2O 2.89g, NaCl 8.50g, water to 1L, pH 7.4); then permeabilized with 0.1% TritonX-100, at room temperature for 10 minutes, and washed three times with PBS; then incubated with hybridoma cell supernatant and anti-mouse FITC-labeled goat anti-mouse IgG, and the results were observed under a fluorescence microscope after the reaction was completed.

[0087] 2.4 Monoclonal Antibody Specificity Identification

[0088] After three subclonings, the cell supernatants secreted by the obtained single cell lines were subjected to IFA and western blot detection. The IFA identification results showed that the obtained 1F10 and 1E1 cell supernatants could detect specific green fluorescence signals in PEDV-infected VeroE6 cells ( Figure 4 A and 4B), while no fluorescence signal was observed when the supernatant of SP2 / 0 cells was used to treat PEDV-infected cells ( Figure 4 C). Virus-infected and uninfected VeroE6 cells were collected, and SDS-PAGE was performed after cell lysis. The protein gel was then transferred to the NC membrane for western blot verification. 1F10 and 1E1 cell supernatants were used as primary antibodies and reacted at room temperature for 1 hour. Anti-mouse HRP-IgG was used as secondary antibody and reacted at room temperature for 1 hour. After each reaction, the cells were washed 4 times with PBST and then exposed for color development. Western blot results showed that 1F10 and 1E1 could recognize PEDV N protein in virus-infected cells ( Figure 5 ).

[0089] 2.5 Monoclonal Antibody Subclass Identification

[0090] According to Southern Biotech's SBA Clonotyping TM System / HRP Antibody Subclass Identification Kit Operating Instructions: The obtained monoclonal antibody was identified for antibody subclass. Figure 6 As shown, the heavy chain constant regions of 1F10 and 1E1 monoclonal antibodies are both of IgG1 type, and the light chain constant regions are both of Kappa type.

[0091] 2.6 Preparation and purification of ascites

[0092] Balb / c mice aged 10 to 12 weeks were selected and each mouse was intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. After 1 week, each mouse was intraperitoneally injected with 5×10 5hybridoma cells (0.2 mL). After 7 to 10 days, the abdominal cavity of the mouse was obviously bulging, and the ascites was collected and packaged. The ascites was purified according to the PIERCE NAb TM Protein G Spin Purification Kit was used for affinity chromatography, and the purity was determined by SDS-PAGE electrophoresis after purification. Figure 7 ).

[0093] 2.7 PCR amplification and sequence determination of monoclonal antibody variable region genes

[0094] RNA was extracted from 1F10 and 1E1 hybridoma cells and reverse transcribed into cDNA using Oligo-dt or random primers (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A).

[0095] The antibody variable region gene was amplified by nested PCR. First, the above cDNA was used as a template to amplify the antibody variable region gene using the first round of mouse antibody IgG and κ light chain primers. Then, the first round product was used as a template to amplify the antibody variable region gene using the second round of mouse antibody IgG and κ light chain primers. The PCR reaction system was: PrimeSTAR Max Premix (2×) 25μL, P1 and P2 1μL each, cDNA 1μL, ddH2O to 50μL. The reaction procedure was: 98℃ pre-denaturation for 2min; 98℃ denaturation for 10s, 55℃ annealing for 30s, 72℃ extension for 30s, 30 cycles; 72℃ extension for 10min. Primers for antibody variable region gene amplification refer to the literature (vonBoehmer, L., Liu, C., Ackerman, S., Gitlin, AD, Wang, Q., Gazumyan, A., Nussenzweig, MC, 2016. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nature protocols 11, 1908-1923.)

[0096] After amplification, 1% agarose gel electrophoresis was performed and the heavy chain and κ light chain variable region gene size was approximately 300 bp ( Figure 8 A and 8B), gel excision to recover the target fragment, and the recovered target fragment was inserted into the pMD-18T vector for sequence determination.

[0097] The sequencing results were compared and analyzed with the antibody gene library (IMGT), and the sequencing results confirmed that the amplified sequences were DNA sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody. Specifically, the DNA sequences encoding the heavy chain variable region of the PEDV N protein monoclonal antibodies 1F10 and 1E1 are 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 monoclonal antibodies 1F10 and 1E1 are 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 monoclonal antibodies 1F10 and 1E1 are 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 monoclonal antibodies 1F10 and 1E1 are shown in SEQ ID NO:2 and SEQ ID NO:6, respectively.

[0098] 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.

[0099] Table 1 Amino acid sequences of heavy chain and light chain variable regions CDR1, CDR2 and CDR3 of monoclonal antibody 1F10

[0100] 1F10 CDR1 CDR2 CDR3 Heavy chain VH GYTFTTYY IYPGNINT ARISSALPY Light chain VL RSLLNTSSQKSY FAS QQHFSTPPT

[0101] The amino acid sequences of the heavy chain and light chain variable regions CDR1, CDR2 and CDR3 of monoclonal antibody 1E1 are shown in Table 2 below.

[0102] Table 2 Amino acid sequences of heavy chain and light chain variable regions CDR1, CDR2 and CDR3 of monoclonal antibody 1E1

[0103] 1E1 CDR1 CDR2 CDR3 Heavy chain VH GYTFTAYV INPYNDRT ARDDDDYEEGFAY Light chain VL QKCQYIWLSL SCI SAHSGAYT

[0104] Example 3 Preparation of latex microsphere immunochromatographic test strips for detecting porcine epidemic diarrhea virus

[0105] 3.1 Preparation of conjugate pad

[0106] 3.1.1 Determination of the optimal amount of monoclonal capture antibody for PEDV N protein labeled with latex microspheres

[0107] The prepared PEDV N protein monoclonal antibody 1F10 was coupled to latex microspheres, so that the final concentrations of monoclonal antibody 1F10 were 20 μg / mL, 40 μg / mL, 60 μg / mL, and 80 μg / mL, respectively. The other four successfully labeled conjugates were used to detect the positive control and negative control, respectively, such as Fig. 9As shown, when the final antibody concentration is 20μg / mL, the positive and negative reactions are weaker than those of other groups, which will affect the later sensitivity of the test strip; at 80μg / mL, although the positive reaction is the strongest, obvious nonspecific bands appear in the negative reaction; at concentrations of 40μg / mL and 60μg / mL, the difference between positive and negative is obvious and there is no nonspecificity. Based on cost considerations, 40μg / mL was finally selected as the optimal latex microsphere-labeled antibody amount for this test strip.

[0108] 3.1.2 Preparation of PEDV N protein monoclonal capture antibody latex microsphere labeling solution

[0109] Take 975 μL of 0.1M pH5.6 MES buffer and add it to a 2mL centrifuge tube, then add 25 μL of latex microspheres with a solid content of 4% (Suzhou Weidu Biotechnology Co., Ltd.) and mix gently; then add 10 μL of 20 mg / mL EDC (purchased from sigma, product number: 03449) and 5 μL of 20 mg / mL NHS (purchased from sigma, product number: 8045180025), mix thoroughly and activate at room temperature for 20 minutes, and centrifuge at 17000r for 20 minutes; remove the supernatant solution, resuspend it with 1mL MES buffer and centrifuge it again at 17000r for 20 minutes; add 40 μg of PEDV N protein monoclonal antibody 1F10 and incubate at room temperature for 2 hours; add 100 μL of 10w / v% BSA solution for blocking, incubate at room temperature for 1 hour, and centrifuge at 17000r for 20 minutes. After centrifugation for 10 minutes, discard the supernatant; finally, resuspend with 1 mL of latex microsphere marker protective agent; if there is a precipitate that is obviously difficult to mix, sonicate at 100W for 3 seconds, stop for 3 seconds, and sonicate for a total of 1 minute, and store at 4°C for later use.

[0110] 3.1.3 Preparation of latex microsphere binding pad

[0111] At 37°C to 38°C, the conjugate pad was completely immersed in the conjugate pad blocking solution (5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20, 1.5% m / v PVP-40 and 0.05% Krovin 300 ultrapure aqueous solution) for 2 hours, and then taken out and dried in an oven for 2 to 4 hours. The prepared monoclonal antibody 1F10 latex microsphere labeling solution of the PEDV N protein was evenly sprayed on the conjugate pad using a film sprayer, placed in a 37°C oven to dry for 2 to 3 hours, and sealed for storage.

[0112] 3.2 Preparation of sample pad

[0113] At 37°C to 38°C, soak the sample pad in sample pad blocking solution (Tris-HCl solution of 2% BSA, 0.5% S17, 2% PEG200000, 0.5% sodium caseinate and 0.05% Krovin 300) for 2 hours, take it out and place it in an oven to dry for 2 to 4 hours, and then place it in an aluminum foil bag to dry for later use.

[0114] 3.3 Preparation of nitrocellulose membrane

[0115] To determine the optimal antibody coating concentration at the T line, the monoclonal antibody 1E1 of PEDV N protein was diluted to 0.5, 1.0, 1.5, and 2.0 mg / mL, sprayed on the T line, and PEDV cytotoxicity (positive control) and uninfected healthy cell supernatant (negative control) were added. The results were observed after 12 minutes of reaction. The test results are as follows Fig.10 As shown, when the concentration is 0.5 mg / mL, the overall reactivity is weak, which will affect the sensitivity of the test strip in the later stage; when the concentration is 2.0 mg / mL, although the overall reactivity is stronger than the other three, the negative control shows non-specificity; when the concentrations are 1.0 and 1.5 mg / mL, the reaction effects are almost the same. Considering the saving of raw materials, 1 mg / mL was finally selected as the optimal coating concentration at the T line.

[0116] 1 mg / mL of monoclonal antibody 1E1 against PEDV N protein and 0.5 mg / mL of goat anti-mouse IgG antibody were sprayed on the nitrocellulose membrane at intervals of 5 to 8 mm as the test line (T) and quality control line (C), respectively. After the lines were drawn, they were placed at 37°C to 38°C to dry for 2 to 3 hours.

[0117] 3.4 Assembly of test strips

[0118] The sample pad, the binding pad, the nitrocellulose membrane, and the absorbent pad are sequentially connected on the PVC bottom plate to obtain a test strip, and the test strip is cut into strips according to the size of the test strip card shell, and then the cut test strips are loaded into the test strip card shell. The structural schematic diagram of the test strip is shown in FIG. Fig.11 shown.

[0119] Example 4 Application of latex immunochromatographic test strips for detecting porcine epidemic diarrhea virus

[0120] 4.1 How to use the test strips

[0121] (1) Detection: Take out the test strip and equilibrate it to room temperature. Add the sample to be tested to the sample adding area, let it stand at room temperature, and determine the result.

[0122] (2) Result determination: Dilute the sample to be tested with sample diluent (1% BSA) at a ratio of 1:5. At room temperature, drop 100 μL of the sample on the sample pad and observe the result within 15 minutes. If both the test line (T line) and the quality control line (C line) appear red, it is positive. If the test line (T line) does not show color but the quality control line (C line) does, it is negative. If the quality control line (C line) does not show color, it can be determined that the test strip is invalid.

[0123] 4.2 Sensitivity of test strips

[0124] PEDV TCID 50 For 10 6.2 / 0.1mL of virus was diluted 1:5, and then 2-fold dilution was performed to test the sensitivity of the established test strip. 10 The test value is still positive after dilution, 1:2 11 The test value after dilution was negative. Therefore, the sensitivity of the test strip prepared in this embodiment can reach 1:2 10 , that is, the minimum virus detection amount of the method of the present invention is 10 3.2 TCID 50 / 0.1mL, such as Fig.12 shown.

[0125] 4.3 Specificity of test strips

[0126] The test strips were used to detect PEDV, TGEV, SADS-CoV, and PDCoV, and it was found that only PEDV was positive, while the others did not react, indicating that the method established by the present invention can specifically identify PEDV. Fig.13 shown.

[0127] 4.4 Stability test

[0128] Stability is an important factor in field applications. The test strips were stored at room temperature (18-25°C) and 4°C, and stability tests were performed on the test strips at 0, 1, 2, 3, 6, 9, 12, and 15 months. As shown in Table 3, the test strips can be stably stored for 9 months at room temperature and for 12 months at 4°C.

[0129] Table 3 Test strip stability test results

[0130]

[0131]

[0132] Note: -: negative; +, ++, +++: the degree of positivity increases in sequence.

[0133] The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structures, features and principles described in the patent scope of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A monoclonal antibody against the N protein of porcine epidemic diarrhea virus, characterized in that: The heavy chain variable region of the monoclonal antibody includes a CDR1 with an amino acid sequence of GYTFTAYV, a CDR2 with an amino acid sequence of INPYNDRT, and a CDR3 with an amino acid sequence of ARDDDDYEEGFAY; The light chain variable region of the monoclonal antibody includes a CDR1 with an amino acid sequence of QKCQYIWLSL, a CDR2 with an amino acid sequence of SCI, and a CDR3 with an amino acid sequence of SAHSGAYT.

2. The anti-porcine epidemic diarrhea virus N protein monoclonal antibody according to claim 1, characterized in that: 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: 5 and SEQ ID NO: 6, respectively.

3. A gene encoding the anti-porcine epidemic diarrhea virus N protein monoclonal antibody according to claim 1.

4. The gene according to claim 3, characterized in that The gene sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

5. A recombinant expression vector comprising the gene according to claim 3. A host cell comprising the recombinant expression vector according to claim 5.

7. 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, or the host cell according to claim 6 in any of the following aspects, characterized in that: (1) Use in the preparation of products for treating porcine epidemic diarrhea virus infection and related diseases caused by infection; the products are drugs or vaccines; (2) Application in the preparation of porcine epidemic diarrhea virus detection reagents or kits.

8. A latex immunochromatographic test strip for rapid detection of porcine epidemic diarrhea virus, characterized in that: The test strip comprises a PVC base plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad, wherein the sample pad, the conjugation pad, the nitrocellulose membrane and the water-absorbing pad are overlapped on the PVC base plate in sequence; a detection line T and a quality control line C are arranged on the nitrocellulose membrane, wherein the detection line T is arranged at one end close to the conjugation pad, and the quality control line C is arranged at one end close to the water-absorbing pad; the conjugation pad is sprayed with a monoclonal capture antibody of the N protein of porcine epidemic diarrhea virus labeled with latex microspheres; the detection line T is coated with the monoclonal antibody of the N protein of porcine epidemic diarrhea virus according to claim 1 as a detection antibody; and the quality control line C is coated with goat anti-mouse IgG.

9. The latex immunochromatographic test strip for rapid detection of porcine epidemic diarrhea virus according to claim 8, characterized in that: The heavy chain variable region of the monoclonal capture antibody of the N protein of porcine epidemic diarrhea virus includes CDR1 with the amino acid sequence of GYTFTTYY, CDR2 with the amino acid sequence of IYPGNINT, and CDR3 with the amino acid sequence of ARISSALPY; the light chain variable region includes CDR1 with the amino acid sequence of RSLLNTSSQKSY, CDR2 with the amino acid sequence of FAS, and CDR3 with the amino acid sequence of QQHFSTPPT.

10. A method for preparing a latex microsphere immunochromatographic test strip for rapid detection of porcine epidemic diarrhea virus according to claim 8 or 9, the method comprising the following steps: (1) preparing a binding pad coated with latex microspheres labeled with a monoclonal capture antibody for detecting porcine epidemic diarrhea virus N protein; (2) Spray the porcine epidemic diarrhea virus N protein monoclonal antibody and goat anti-mouse IgG antibody described in claim 1 onto a nitrocellulose membrane at intervals of 5 to 8 mm, as the test line T and the quality control line C, respectively; (3) The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are overlapped on the PVC base plate in sequence to obtain a latex microsphere immunochromatographic test strip for detecting porcine epidemic diarrhea virus.

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