Latex immunochromatographic test strip for rapid detection of porcine epidemic diarrhea virus and its preparation method

Abstract

The present invention belongs to the field of biotechnology and relates to a latex immunochromatographic test strip for rapid detection of porcine epidemic diarrhea virus (PEDV) and a preparation method thereof. The test strip includes a PVC bottom plate, a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad; a quality control line C and a test line T are provided on the nitrocellulose membrane. The conjugate pad is sprayed with a monoclonal capture antibody of PEDV N protein labeled with latex microspheres, the test line T is coated with a monoclonal detection antibody 1E1 of PEDV N protein, and the quality control line C is coated with goat anti-mouse IgG. This test strip can specifically recognize PEDV and does not react with other porcine intestinal coronaviruses with similar clinical symptoms. The test strip has good specificity, relatively high sensitivity, simple operation, and the result can be judged within 10 minutes. Compared with the traditional colloidal gold detection technology, the stability and color development effect have been significantly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly 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 intestinal disease caused by porcine epidemic diarrhea virus (PEDV). Clinically, it is often manifested as vomiting, diarrhea, dehydration and enteritis. Although pigs of all ages are susceptible, it is most harmful to piglets, with a fatality rate as high as 100%. The prevention and control of PED clinically is a systematic strategy, which not only requires effective vaccine immunization, but also needs to be combined with timely and accurate antibody and antigen detection. However, in recent years, due to the change in the homology between PEDV epidemic strains and vaccine strains, some vaccines have become ineffective and are difficult to provide sufficient protection. Therefore, early rapid diagnosis remains the key to preventing and treating this disease.

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

[0004] Lateral flow immunoassay technology is a technology that conjugates different signal markers on the surface of antigens or antibodies and realizes macroscopic visualization or microscopic antigen-antibody reactions through signal amplification. This technology is easy to operate, can be completed without professional personnel, and does not require specific instruments. The detection results can be obtained with the naked eye, and the time consumption is short, which can be completed within 15 minutes. The detection time is greatly shortened. Its commonly used signal markers are mainly colloidal gold particles and latex microspheres, etc. Although there are a variety of markers on the market currently, those that can be used for large-scale production are still colloidal gold particles and latex microspheres.

[0005] Compared with colloidal gold, latex microspheres have high tolerance to physics and chemistry and are not easily affected by them, which is beneficial to the stability of the finished test strip. At the same time, the inside of the latex microspheres is filled with oil-soluble color dyes, with high contrast, deep color and high visualization degree, and is also superior to colloidal gold in terms of color development effect. Summary of the Invention

[0006] The present invention utilizes a prokaryotic expression system to obtain a soluble PEDV N protein with immunogenicity and reactivity, immunizes mice with this as an immunogen, and obtains specific monoclonal antibodies by cell fusion and subcloning screening techniques, which are named 1F10 and 1E1 respectively. The gene sequences of the antibody variable regions are obtained by nested PCR amplification technology and can be obtained by subsequent genetic engineering or protein engineering methods. A latex immunochromatographic test strip and preparation method for detecting porcine epidemic diarrhea virus that are convenient, rapid, and highly sensitive are developed based on the PEDV N protein-specific monoclonal antibodies.

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

[0008] In the first aspect of the present invention, a PEDV N protein monoclonal antibody is provided, and 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 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 CDR1, CDR2, and CDR3 of the heavy and light chain variable regions 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 the IgG1 type.

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

[0017] Second aspect, the present invention provides a latex immunochromatographic test strip for rapid detection of porcine epidemic diarrhea virus. The test strip includes a PVC bottom plate, a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad. The sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent pad are sequentially lapped on the PVC bottom plate. The nitrocellulose membrane is provided with a test line T and a control line C. The test line T is arranged at one end close to the conjugate pad, and the control line C is arranged at one end close to the absorbent pad. The conjugate pad is sprayed with a monoclonal capture antibody of PEDV N protein labeled with latex microspheres. The test line T is coated with a monoclonal antibody 1E1 of PEDV N protein as a detection antibody. The 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 as follows:

[0019] The conjugate pad is treated by sealing with a conjugate pad sealing solution.

[0020] After the latex microspheres are activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), centrifuged, resuspended with MES buffer, and ultrasonically dispersed to make the fluorescent microspheres fully dispersed, then the monoclonal capture antibody of PEDV N protein is added, incubated at room temperature, and then sealed with a sealing solution, incubated at room temperature, and centrifuged. The precipitate is resuspended with a latex microsphere label protector to obtain a monoclonal capture antibody latex microsphere label solution of PEDV N protein.

[0021] The prepared monoclonal capture antibody latex microsphere label solution of PEDV N protein is evenly sprayed on the sealed conjugate pad by an instrument.

[0022] The conjugate pad sealing 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 label sealing solution is an ultrapure aqueous solution containing 10% m / v BSA.

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

[0025] The composition of the monoclonal capture antibody latex microsphere label solution of PEDV N protein is: each 1 mL of the latex microsphere label solution contains 20 μg of the monoclonal capture antibody of PEDV N protein.

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

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

[0028] Preferably, the sample pad is soaked and treated with 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, and the method includes the following steps:

[0030] (1) Prepare a conjugate pad coated with a latex microsphere-labeled monoclonal capture antibody of PEDV N protein.

[0031] (2) Spray the monoclonal detection antibody 1E1 (1 mg / mL) of PEDV N protein and the goat anti-mouse IgG antibody (1 mg / mL) on the nitrocellulose membrane at an interval of 5-8 mm, respectively, as the test line T and the control line C.

[0032] (3) Overlap the sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent pad 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 is as follows:

[0034] (1) Take the test sample.

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

[0036] (3) Result determination: After the reaction is completed, qualitatively judge the positive and negative by naked eyes.

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

[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. At the same time, the lowest limit of the virus that the test strip can detect in the culture is 10 3.2 TCID 50 / 0.1 mL, and it does 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 the on-site or laboratory detection of porcine infectious diarrhea coronavirus. Description of the Drawings

[0040] Figure 1 It is the result of PCR amplification of PEDV N gene and restriction enzyme digestion identification of pET32a-N recombinant plasmid;

[0041] A. M: DL2000 relative molecular mass standard; 1: water control; 2: N gene, about 1300bp in size.

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

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

[0044] A. M: protein Marker; 1: before induction of pET32a-N; 2: after induction of pET32a-N; 3: lysate supernatant of pET32a-N; 4: precipitate after lysis of pET32a-N;

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

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

[0047] Figure 3 It is the detection result diagram of ELISA for detecting the titer of polyclonal antibody against mouse PEDV N protein.

[0048] Figure 4 It is to verify the reactivity of monoclonal antibody by indirect immunofluorescence assay.

[0049] A. Supernatant of 1F10 hybridoma cells; B. Supernatant of 1E1 hybridoma cells; C. Supernatant of SP2 / 0 cells.

[0050] Figure 5 It is to identify the reactivity of monoclonal antibody by western blot;

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

[0052] Figure 6 Results of monoclonal antibody subclass identification.

[0053] Figure 7 SDS-PAGE identification of monoclonal antibody after purification.

[0054] M: Protein Marker; 1: Purified monoclonal antibody 1F10; 2: Purified monoclonal antibody 1E1.

[0055] Figure 8 Results of PCR amplification of monoclonal antibody variable region;

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

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

[0058] B. Results of PCR amplification 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] Figure 9 Determination of the optimal antibody labeling amount of latex microspheres.

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

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

[0063] Figure 12 Graph of the sensitivity test results of the test strip.

[0064] Figure 13 Graph of the specificity test results of the test strip. Specific implementation mode

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

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

[0067] 1.1 Construction of pET32a-N recombinant plasmid

[0068] Taking the N protein of PEDV CV777 strain (Genbank accession number: AF353511.1) as a reference, specific primers were designed. Upstream primer P1: 5′-CGC GGATCCATGGCTTCTGTCAGCTTTCA-3′, downstream primer P2: 5′-CCG CTCGAG TTAATTTCCTGTATCGAAGATC-3′. BamHI and XhoI restriction sites (underlined) were introduced at the 5′ ends 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 as follows: PrimeSTAR Max Premix (2×) 25 μL, 1 μL each of P1 and P2, 1 μL of cDNA, and ddH2O was added to make up to 50 μL. The reaction program was: pre-denaturation at 98 °C for 2 min; denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min, for 30 cycles; extension at 72 °C for 10 min. The PCR products were detected by 1% agarose gel electrophoresis, and the results were as Figure 1 shown in A.

[0069] Gel extraction was performed according to the instruction manual of the EZNAGel Extraction Kit from Omega Company to obtain the N gene PCR gel extraction product. After double digestion of the N gene PCR gel extraction product and the pET32a vector with BamHⅠ and XhoI, ligation and transformation of DH5α competent cells were carried out. The constructed recombinant plasmid was identified by double digestion, and the plasmid with a positive double digestion identification was further identified by sequencing. The positive recombinant plasmid was named pET32a-N. The double digestion identification results were as Figure 1 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). A single colony was picked and cultured in 5 mL of LB with ampicillin resistance until the OD 600 was approximately 0.8, and then IPTG with a final concentration of 1 mmol / L was added for induced expression. After 5 h of induction, the cells were centrifuged at 12000 rpm for 2 min to collect the precipitate. The precipitate was resuspended with an appropriate amount of PBS and then sonicated for 5 min (sonication for 3 s, pause for 3 s). After sonication, the mixture was centrifuged at 4 °C and 12000 rpm for 10 min to collect the supernatant and the precipitate respectively. The supernatant and the precipitate were added with 5×loading buffer and boiled in boiling water for 10 min for SDS-PAGE identification. The identification results were as Figure 2 shown in A.

[0072] According to the above method, the induced expression level of the pET32a-N protein was increased, and the lysate supernatant after sonication was taken for nickel column purification. The specific operation steps were as follows:

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

[0074] (2) Sample loading: When the equilibration buffer drops to the resin surface, add 3 mL of the lysate supernatant containing the recombinant protein, and repeat the sample loading 2 - 3 times, with each action lasting 2 min. Collect the sample flow-through; add 5 column volumes of equilibration buffer to wash the column once.

[0075] (3) Protein elution: Configure imidazole with different concentrations using the equilibration buffer to elute the protein sample.

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

[0077] The samples collected each time are identified by SDS-PAGE. The identification results are as Figure 2 shown in B.

[0078] Example 2 Preparation of Monoclonal Antibody Against PEDV N Protein and Amplification of Antibody Sequence

[0079] 2.1 Mouse immunization

[0080] Immunize Babl / c mice with the purified PEDV N recombinant protein at a dose of 30 μg per mouse. For the first immunization, mix the PEDV N protein and Freund's complete adjuvant in equal volumes and emulsify them, then perform multi-point subcutaneous injection on the back. Boost immunization is carried out every 2 weeks (weeks), for a total of four immunizations. The boost immunization is to mix the PEDV N recombinant protein and Freund's incomplete adjuvant in equal volumes and emulsify them, and the immunization method is the same as the first immunization.

[0081] 2.2 ELISA detection of antibody titer

[0082] One week after the fourth immunization, the mice were bled by tail amputation to measure the antibody titer. The inactivated PEDV virus solution was diluted 1:1 with the coating solution to coat the ELISA plate, 50 μL / well, and coated at 37 °C for 1 h. After washing four times with PBST (0.26 g of K2HPO4, 2.89 g of Na2HPO4·12H2O, 8.50 g of NaCl, 0.5 mL of Tween-20, made up to 1 L with water, pH 7.4), 2% trehalose was added and blocked at 37 °C for 1 h. The mouse positive serum immunized with N protein and the mouse negative serum not immunized with N protein were serially diluted with PBST, with dilution gradients from 1:100 to 1:204800, diluted in 12 gradients, incubated at 37 °C for 30 min, washed four times with PBST, then HRP-labeled goat anti-mouse IgG (diluted 1:20000) was added, incubated at 37 °C for 30 min, washed four times with PBST, developed with TMB, and the OD was measured on an enzyme-linked immunosorbent assay (ELISA) reader. 450 。

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

[0084] 2.3 Preparation of monoclonal antibodies

[0085] Spleen cells from a mouse immunized with PEDV N protein were mixed with SP20 cells (at a ratio of 5:1) and then cell fusion was carried out under the action of the fusogen PEG. The fused cells were plated in a 96-well cell culture plate and cultured in a 37 °C, CO2 incubator. When the hybridoma cells covered 1 / 10 of the bottom of the cell culture plate, the supernatant was aspirated for ELISA detection (the ELISA coating method was the same as in 2.2). The wells identified as positive by ELISA were further verified by IFA. The positive wells of ELISA and IFA were selected, and the selected positive hybridoma cells were subcloned by the limiting dilution method. Observed under an inverted microscope, the wells with only single clone growth were marked, the supernatant was taken, and the antibody was detected by the above ELISA and IFA methods. The positive cells entered the next round of subcloning, and this was carried out three times in total.

[0086] IFA test: 1×10 4Vero E6 cells were seeded in 96-well cell culture plates. After the cells grew to confluence, 0.01 MOI of PEDV was inoculated. At 24 h post-inoculation, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min and washed three times with 0.01 mol / L PBS (0.26 g of K2HPO4, 2.89 g of Na2HPO4·12H2O, 8.50 g of NaCl, made up to 1 L with water, pH 7.4). Then the cells were permeabilized with 0.1% Triton X-100 at room temperature for 10 min and washed three times with PBS. Subsequently, the hybridoma cell supernatant and anti-mouse FITC-labeled goat anti-mouse IgG were incubated. After the reaction, the results were observed under a fluorescence microscope.

[0087] 2.4 Identification of monoclonal antibody specificity

[0088] After three rounds of subcloning, the cell supernatants secreted by the obtained single cell lines were detected by IFA and western blot. The results of IFA identification showed that specific green fluorescence signals ( Figure 4 A and 4B) could be detected when the cell supernatants of 1F10 and 1E1 were used to treat PEDV-infected Vero E6 cells, while no fluorescence signal was seen when the SP2 / 0 cell supernatant was used to treat PEDV-infected cells ( Figure 4 C). Vero E6 cells with and without virus infection were collected. After cell lysis, SDS-PAGE was performed, and then the protein gel was transferred to an NC membrane for western blot verification. The cell supernatants of 1F10 and 1E1 were used as the primary antibody and incubated at room temperature for 1 h, and anti-mouse HRP-IgG was used as the secondary antibody and incubated at room temperature for 1 h. After each incubation, the cells were washed four times with PBST, and then developed by exposure. The western blot results showed that 1F10 and 1E1 could recognize the PEDV N protein in virus-infected cells ( Figure 5 ).

[0089] 2.5 Identification of monoclonal antibody subclass

[0090] The antibody subclass of the obtained monoclonal antibodies was identified according to the operation instructions of the SBA Clonotyping TM System / HRP antibody subclass identification kit from Southern Biotech. The results were as Figure 6 shown. The heavy chain constant regions of both 1F10 and 1E1 monoclonal antibodies were of the IgG1 type, and the light chain constant regions were both of the Kappa type.

[0091] 2.6 Preparation and purification of ascites

[0092] Ten- to twelve-week-old Balb / c mice were taken. Each mouse was intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. One week later, each mouse was intraperitoneally injected with 5×10 5Hybridoma cells (0.2 mL). After 7 - 10 days, the abdominal cavity of the mouse swells significantly. Ascites is collected and aliquoted. The purification of ascites is carried out by affinity chromatography using the NAb TM Protein G Spin Purification Kit from PIERCE. After purification, the purity is identified by SDS - PAGE electrophoresis ( Figure 7 ).

[0093] 2.7 PCR Amplification and Sequencing of Monoclonal Antibody Variable Region Genes

[0094] Extract the RNA of 1F10 and 1E1 hybridoma cells, and reverse - transcribe them into cDNA using Oligo - dt or random primers respectively (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A).

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

[0096] After amplification, 1% agarose gel electrophoresis is carried out. The sizes of the heavy - chain and κ - light chain variable region genes are approximately 300 bp ( Figure 8 A and 8B). Cut the gel to recover the target fragment. Insert the recovered target fragment into the pMD - 18T vector for sequence determination.

[0097] The sequencing results were aligned and analyzed with the antibody gene library (IMGT). The sequencing results confirmed that the amplified sequences were the DNA sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody. Specifically, the DNA sequences encoding the heavy chain variable regions of 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 regions of 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 regions of 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 regions of 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 CDR1, CDR2, and CDR3 in the heavy and light chain variable regions of monoclonal antibody 1F10 are shown in Table 1 below.

[0099] Table 1 Amino acid sequences of CDR1, CDR2, and CDR3 in the heavy and light chain variable regions 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 CDR1, CDR2, and CDR3 in the heavy and light chain variable regions of monoclonal antibody 1E1 are shown in Table 2 below.

[0102] Table 2 Amino acid sequences of CDR1, CDR2, and CDR3 in the heavy and light chain variable regions 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 a latex microsphere immunochromatographic test strip for detecting porcine epidemic diarrhea virus

[0105] 3.1 Preparation of the conjugate pad

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

[0107] The prepared PEDV N protein monoclonal antibody 1F10 was conjugated with 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 4 successfully labeled conjugates were used to detect the positive control and the negative control respectively, as Figure 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 the sample pad blocking solution (Tris-HCl solution containing 2% BSA, 0.5% S17, 2% PEG200000, 0.5% sodium caseinate, and 0.05% Krovin 300) for 2 h, take it out and dry it in an oven for 2 - 4 h, then place it in an aluminum foil bag for drying and standby.

[0114] 3.3 Preparation of nitrocellulose membrane

[0115] To determine the optimal coating concentration of the antibody at the T line, dilute the monoclonal antibody 1E1 of PEDV N protein to 0.5, 1.0, 1.5, 2.0 mg / mL, spray it on the T line, add PEDV cytotoxin (positive control) and the supernatant of healthy cells without virus inoculation (negative control), and observe the results after reacting for 12 min. The detection results are as Figure 10 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, non-specificity appears in the negative control; when the concentrations are 1.0 and 1.5 mg / mL, the reaction effects are almost the same. Considering the saving of raw materials, finally select 1 mg / mL as the optimal coating concentration at the T line.

[0116] Spray the monoclonal antibody 1E1 of PEDV N protein at 1 mg / mL and the goat anti-mouse IgG antibody at 0.5 mg / mL on the nitrocellulose membrane at intervals of 5 - 8 mm, as the test line (T) and the quality control line (C) respectively. After the lines are drawn, place them at 37 °C - 38 °C for drying for 2 - 3 h.

[0117] 3.4 Assembly of the test strip

[0118] Connect the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad in sequence on the PVC bottom plate to obtain the test strip. Cut the test strip according to the size of the test strip cartridge, and then put the cut test strip into the test strip cartridge. The structural schematic diagram of the test strip is as Figure 11 shown.

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

[0120] 4.1 Usage method of the test strip

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

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

[0123] 4.2 Sensitivity of the test strip

[0124] Dilute the PEDV TCID 50 with a value of 10 6.2 / 0.1 mL of the virus 1:5 serially diluted and then further diluted 2-fold to detect the sensitivity of the established test strip. The results show that when the virus is diluted 1:2 10 times and the detection value is still positive, and when diluted 1:2 11 times, the detection value is negative. Therefore, the sensitivity of the test strip prepared in this example can reach 1:2 10 , that is, the lowest virus detection amount of the method of the present invention is 10 3.2 TCID 50 / 0.1 mL, as Figure 12 shown.

[0125] 4.3 Specificity of the test strip

[0126] Use the test strip to detect PEDV, TGEV, SADS-CoV, and PDCoV. It is found that only PEDV shows a positive result, and the others do not react, indicating that the method established in the present invention can specifically identify PEDV, as Figure 13 shown.

[0127] 4.4 Stability test

[0128] Stability is an important factor for on-site application. Store the test strips at room temperature (18 - 25 °C) and 4 °C respectively, and conduct stability tests 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 at room temperature for 9 months and at 4 °C for 12 months.

[0129] Table 3 Stability test results of the test strip

[0130]

[0131]

[0132] Note: -: Negative; +, ++, +++: The degree of positivity increases in turn.

[0133] The above-described embodiments are only preferred embodiments of the present invention and do not limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structure, features, and principles described in the scope of the present invention patent shall be included within the scope of the present invention's patent application.

Claims

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

2. The monoclonal antibody against the N protein of porcine epidemic diarrhea virus according to claim 1, wherein The amino acid sequences of the heavy chain variable region and 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 monoclonal antibody against the N protein of porcine epidemic diarrhea virus as claimed in claim 1.

4. The gene according to claim 3, wherein 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 as claimed in claim 3.

6. A host cell comprising the recombinant expression vector as claimed in claim 5.

7. Use of the monoclonal antibody as claimed in claim 1 or 2, the gene as claimed in claim 3 or 4, the recombinant expression vector as claimed in claim 5, and the host cell as claimed in claim 6 in the preparation of a detection reagent or kit for porcine epidemic diarrhea virus.

8. A latex immunochromatographic test strip for rapid detection of porcine epidemic diarrhea virus, characterized in that, The test strip comprises a PVC bottom plate, a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad, and the sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent pad are sequentially overlapped on the PVC bottom plate; a test line T and a quality control line C are provided on the nitrocellulose membrane, the test line T is arranged at one end close to the conjugate pad, and the quality control line C is arranged at one end close to the absorbent pad; the conjugate pad is sprayed with a monoclonal capture antibody against the N protein of porcine epidemic diarrhea virus labeled with latex microspheres; the test line T is coated with the monoclonal antibody against the N protein of porcine epidemic diarrhea virus as claimed in 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 against the N protein of porcine epidemic diarrhea virus comprises 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 comprises 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 the latex microsphere immunochromatographic test strip for rapid detection of porcine epidemic diarrhea virus as claimed in claim 8 or 9, the method comprising the following steps: (1) Preparing a conjugate pad coated with a monoclonal capture antibody against the N protein of porcine epidemic diarrhea virus labeled with latex microspheres; (2) Spraying the monoclonal antibody against the N protein of porcine epidemic diarrhea virus as claimed in claim 1 and the goat anti-mouse IgG antibody on the nitrocellulose membrane at an interval of 5-8 mm, respectively serving as the test line T and the quality control line C; (3) The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are successively lapped on the PVC bottom plate to obtain a latex microsphere immunochromatographic test strip for detecting porcine epidemic diarrhea virus.

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