A latex immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis coronavirus and its preparation method

Abstract

This invention belongs to the field of biotechnology and relates to a latex immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis coronavirus (TGEV) and its preparation method. The test strip comprises a PVC base, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane is provided with a control line C and a test line T. The conjugate pad is sprayed with latex microspheres-labeled monoclonal antibody A against the TGEV N protein. The test line T is coated with a monoclonal antibody B against the TGEV N protein, and the control line C is coated with goat anti-mouse IgG. The test strip of this invention specifically recognizes TGEV and does not react with other porcine enteric coronaviruses with similar clinical symptoms, such as porcine acute diarrhea syndrome virus (SADS-CoV), porcine epidemic diarrhea virus (PEDV), and porcine delta coronavirus (PDCoV). This test strip offers excellent specificity, high sensitivity, simple operation, and readout within 10 minutes. Compared to traditional colloidal gold detection technology, its stability and color development are significantly improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a latex immunochromatographic test strip for rapidly detecting porcine transmissible gastroenteritis coronavirus and a preparation method thereof. Background Art

[0002] Transmissible gastroenteritis (TGE) is an acute, highly contagious disease caused by the transmissible gastroenteritis virus (TGEV). Clinically, it often presents with diarrhea, vomiting, and dehydration. Rapid and accurate diagnosis is crucial for its prevention and control.

[0003] Commonly used pathogen detection methods include virus isolation and identification, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) and real-time quantitative PCR.

[0004] However, these methods are time-consuming, complex, and require specialized technicians and specialized instruments, making them difficult to meet the requirements for rapid, cost-effective, and efficient monitoring of clinical samples. Compared to these methods, immunochromatographic detection technology can maintain high sensitivity and strong specificity while strictly controlling the overall reaction time to within 10-15 minutes, significantly shortening the operation time and improving detection efficiency.

[0005] The surface of latex microspheres is linked to carboxyl groups, which are activated by water-soluble diimide (EDC) and can therefore covalently bind to the amino groups of proteins or antibodies to form stable and strong chemical bonds. Therefore, the stability of latex microspheres is stronger than that of colloidal gold. At the same time, latex microspheres can tolerate a wide range of pH changes and resist high concentrations of salt ions, making them significantly better than colloidal gold particles. Compared to colloidal gold, 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] Based on the characteristics of latex microspheres and combined with TGEV N protein-specific antibodies, the present invention provides a latex immunochromatographic test strip that can quickly detect TGEV. The test strip is simple to operate, has obvious color development, strong sensitivity, and short detection time. It can be used for the detection of TGEV clinical samples, providing new technical support for the prevention and control of TGEV.

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

[0008] In a first aspect, the present invention provides a monoclonal antibody against the N protein of porcine transmissible gastroenteritis coronavirus, wherein the monoclonal antibody is 1B7 or 2A9.

[0009] The heavy chain variable region of the monoclonal antibody 1B7 of the present invention includes a CDR1 with an amino acid sequence of GFNIKDTY, a CDR2 with an amino acid sequence of IDPADGYT, and a CDR3 with an amino acid sequence of ARPGTLDY. The heavy chain variable region of the monoclonal antibody 2A9 of the present invention includes a CDR1 with an amino acid sequence of GYTFTDFN, a CDR2 with an amino acid sequence of INPNNGRS, and a CDR3 with an amino acid sequence of ARRHWDWYFDV.

[0010] The light chain variable region of the monoclonal antibody 1B7 of the present invention includes a CDR1 with an amino acid sequence of KSVSTSGYSY, a CDR2 with an amino acid sequence of LVS, and a CDR3 with an amino acid sequence of QHIRELTR. The light chain variable region of the monoclonal antibody 2A9 of the present invention includes a CDR1 with an amino acid sequence of KSLLHSN, a CDR2 with an amino acid sequence of QMS, and a CDR3 with an amino acid sequence of AQNLEFPWT.

[0011] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1B7 of the present invention is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2A9 of the present invention is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0012] The second aspect of the present invention provides a DNA sequence encoding the heavy chain variable region and light chain variable region of the monoclonal antibody against the N protein of porcine transmissible gastroenteritis coronavirus.

[0013] The DNA sequence of the heavy chain variable region of the monoclonal antibody 1B7 of the present invention is shown in SEQ ID NO: 5, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 6; the DNA sequence of the heavy chain variable region of the monoclonal antibody 2A9 of the present invention is shown in SEQ ID NO: 7, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0014] The heavy chain constant region of the monoclonal antibody against the N protein of porcine transmissible gastroenteritis coronavirus of the present invention is of IgG1 or IgG2b type, and the light chain constant region of the monoclonal antibody against the N protein of porcine transmissible gastroenteritis coronavirus of the present invention is of Kappa type.

[0015] In a third aspect, the present invention provides a latex immunochromatographic test strip for detecting porcine transmissible gastroenteritis coronavirus, the latex immunochromatographic test strip comprising a PVC base, a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad, wherein the sample pad, conjugation pad, nitrocellulose membrane and water-absorbing pad are sequentially overlapped on the PVC base; a detection line T and a quality control line C are provided on the nitrocellulose membrane, 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 latex microsphere-labeled anti-TGEV N protein monoclonal antibody 1B7; the detection line T is coated with anti-TGEV N protein monoclonal antibody 2A9; the quality control line C is coated with goat anti-mouse IgG.

[0016] Preferably, the sample pad is immersed in a sample pad blocking solution, which is a Tris-HCl solution containing 2% BSA, 0.5% S17, 2% PEG20000 and 0.5% sodium caseinate, wherein S17 is a surfactant.

[0017] Preferably, the preparation method of the conjugate pad is:

[0018] The conjugate pad was blocked with a conjugate pad blocking solution; the conjugate pad blocking solution was an ultrapure aqueous solution containing 5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20 and 1.5% m / v PVP-40.

[0019] The latex microspheres were activated with N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), centrifuged, resuspended in MES buffer, and fully dispersed by sonication. Anti-TGEV N protein monoclonal antibody 1B7 was then added and incubated at room temperature. The latex microspheres were then blocked with a latex microsphere marker blocking solution, incubated at room temperature, and centrifuged. The precipitate was resuspended in a latex microsphere marker protective agent to obtain an anti-TGEV N protein monoclonal antibody 1B7 latex microsphere labeling solution.

[0020] The prepared anti-TGEV N protein monoclonal antibody A latex microsphere labeling solution was evenly sprayed on the blocked conjugate pad;

[0021] The latex microsphere marker blocking solution is an ultrapure aqueous solution containing 10% m / v BSA;

[0022] 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.

[0023] The concentration of the anti-TGEV N protein monoclonal antibody 1B7 latex microsphere labeling solution is: 80 μg TGEV N protein monoclonal antibody 1B7 per 1 ml of latex microsphere solution.

[0024] Preferably, the detection line T is coated with 1 mg / ml of TGEV N protein monoclonal antibody 2A9.

[0025] Preferably, the quality control line C is coated with 0.5 mg / ml goat anti-mouse IgG.

[0026] In a fourth aspect, the present invention provides a method for preparing the latex microsphere immunochromatographic test strip for detecting porcine transmissible gastroenteritis coronavirus, the method comprising the following steps:

[0027] (1) preparing a sample pad and a conjugate pad coated with latex microspheres labeled with antibodies;

[0028] (2) Anti-TGEV N protein monoclonal antibody 2A9 (1 mg / ml) and goat anti-mouse IgG antibody (0.5 mg / ml) were sprayed onto nitrocellulose membrane at intervals of 5–8 mm to serve as the test line T and the quality control line C, respectively;

[0029] (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 transmissible gastroenteritis coronavirus.

[0030] In a fifth aspect, a method for using the test strip of the present invention comprises the following steps:

[0031] (1) Prepare samples to be tested.

[0032] (2) Detection: Take out the test strip and equilibrate it to room temperature. Dilute the sample to be tested with sample diluent at a ratio of 1:5. At room temperature, drop 100 μl of the sample on the sample pad and observe the result within 10 minutes.

[0033] (3) Result determination: After the reaction is completed, the positive and negative properties are qualitatively determined by the naked eye.

[0034] The beneficial effects of the present invention are:

[0035] The latex microsphere immunochromatographic test strip for detecting TGEV provided by the present invention can be used for the specific detection of TGEV pathogens, and the test strip can detect the virus in the culture at a minimum of 10 3.6 TCID 50The test strips exhibited a high specificity, sensitivity, ease of use, and short detection time, providing a rapid method for on-site or laboratory detection of TGEV. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 PCR amplification results of the TGEV N gene. M: DL2000 relative molecular mass standard; 1: control water; 2: N gene, approximately 1000 bp in size.

[0037] Figure 2 The expression and purification results of the pET32a-N recombinant protein are shown. (A) M: Protein marker; 1: pET32a-N before induction; 2: pET32a-N after induction; 3: pET32a-N lysis supernatant; 4: pET32a-N lysis precipitate. (B) M: Protein marker; 1: Purified pET32a-N protein.

[0038] Figure 3 ELISA test of mouse antibody titers after N protein immunization. 1-3: immunized mice 1, 2, and 3, NC: negative control.

[0039] Figure 4 Identification of monoclonal antibody reactivity for IFA. (A) 1B7 monoclonal antibody; (B) 2A9 monoclonal antibody; (C) SP2 / 0 cell culture medium.

[0040] Figure 5 Western blot analysis of monoclonal antibody reactivity. (A) 1B7 monoclonal antibody; (B) 2A9 monoclonal antibody; (C) internal control. M: protein marker, Mock: PK cell control; TGEV: TGEV-infected PK cells.

[0041] Figure 6 The results of subclass identification.

[0042] Figure 7 The results of antibody purification SDS-PAGE identification are shown. M: protein marker, 1: 1B7 monoclonal antibody; 2: 2A9 monoclonal antibody.

[0043] Figure 8Variable region PCR amplification results. (A) Heavy chain variable region PCR amplification results. M: DL2000 marker; 1: Water control; 2: 1B7 monoclonal antibody; 3: 2A9 monoclonal antibody; (B) Light chain kappa variable region PCR amplification results. M: DL2000 marker; 1: Water control; 2: 1B7 monoclonal antibody; 3: 2A9 monoclonal antibody.

[0044] Figure 9 Determination of the optimal amount of antibody labeling on latex microspheres.

[0045] Figure 10 Determination of the optimal antibody labeling concentration at the T line.

[0046] Figure 11 Schematic diagram of test strip assembly.

[0047] Figure 12 Diagram of the sensitivity test results of the test strips.

[0048] Figure 13 Specificity test results of the test strip. DETAILED DESCRIPTION

[0049] The present invention is described in more detail below through specific implementation methods to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of protection of the present invention.

[0050] Example 1 Preparation of monoclonal antibodies against the N protein of porcine infectious diarrhea coronavirus

[0051] 1. Construction, expression, and purification of TGEV N protein recombinant plasmid

[0052] 1.1 Construction of pET32a-N recombinant plasmid

[0053] The TGEV strain was HLJ-17 (Genbank accession number: MT522161.1). The N protein of this strain was used as a reference to design specific primers, upstream P1: 5′-CGC GGATCC ATGGCCAACCAGGGACAACG-3′, downstream P2: 5′-CCG CTCGAGTTAGTTCGTTACCTCATCAATTATC-3′, BamHI and XhoI restriction sites (underlined parts) were introduced into the 5′ ends of the upstream and downstream primers. After primer synthesis, the supernatant RNA of TGEV cells infected with PK15 cells was extracted and reverse transcribed into cDNA, which was then used as a template for PCR amplification. The PCR reaction system is: PrimeSTAR Max Premix (2×) 25μL, 1μL each of P1 and P2, 1μL cDNA, and ddH2O is added to 50μL. 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; 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, the size of the TGEV N fragment is approximately 1000 bp. Gel extraction was then performed according to the instructions of the EZNAGel Extraction Kit from Omega.

[0054] The TGEV N gene PCR gel-recovered product was digested with the pET32a vector using BamHI and XhoI, ligated, and transformed into DH5α competent cells. The constructed recombinant plasmid was sequenced and identified, and the positive recombinant plasmid was designated pET32a-N.

[0055] 1.2N recombinant protein induction and purification

[0056] 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 medium. 600 When the concentration was about 0.8, IPTG was added to a final concentration of 1 mmol / L to induce expression. 5 hours after induction, the precipitate was collected by centrifugation at 12000 rpm for 2 minutes. The precipitate was resuspended in an appropriate amount of PBS and sonicated for 5 minutes (supersonic for 3 seconds, stop for 3 seconds). After sonication, the supernatant and precipitate were centrifuged at 4°C for 10 minutes, and the supernatant and precipitate were collected separately. The supernatant and precipitate were added to 5× loading buffer, boiled in boiling water for 10 minutes, and identified by SDS-PAGE. The results showed that pET32a-N was expressed in both the supernatant and the precipitate ( Figure 2 A), indicating that part of the N protein is soluble.

[0057] The pET32a-N protein was induced to express in amplified according to the above method. The supernatant was collected after sonication and purified by nickel column. The specific purification steps are as follows:

[0058] (1) Resin loading: Take an empty column and add 2 mL of nickel column NTA resin. When the preservation 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);

[0059] (2) Sample loading: When the equilibrium solution reaches 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. Rinse the column once with 5 column volumes of equilibrium solution.

[0060] (3) Protein elution: The protein sample was eluted with different concentrations of imidazole (25 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM and 300 mM) using the equilibrium solution, 1 mL each time, and each concentration was repeated twice. The purpose was to determine the optimal concentration of imidazole for washing and protein elution;

[0061] (4) Column cleaning and storage: Rinse the column once with 5 volumes of 0.5M NaOH, then rinse once with distilled water, then add an appropriate amount of 70% anhydrous ethanol and store in a refrigerator at 4°C. Each collected sample was identified by SDS-PAGE. Figure 2 As shown in B, a high-purity N protein was obtained.

[0062] 2. Animal immunization

[0063] Three 6-week-old female BALB / c mice were immunized with 30 μg of purified N recombinant protein per mouse. For the first immunization, equal volumes of N protein were mixed with complete Freund's adjuvant, emulsified, and injected subcutaneously at multiple sites (one on the back and two on the abdomen). Booster immunizations were performed every two weeks for a total of four immunizations. Booster immunizations consisted of equal volumes of N recombinant protein mixed with incomplete Freund's adjuvant, emulsified, using the same method as the first immunization. Seven days after the fourth immunization, tail blood was collected to determine antibody titers.

[0064] 3. ELISA method to detect polyclonal antibody titer

[0065] The TGEV virus solution and coating solution were diluted 1:1 and coated on the ELISA plate, 50 μL / well, coated overnight at 4°C, washed four times with PBST, and blocked with 2% trehalose at 4°C for 10 hours. The positive serum and negative serum were diluted with PBST in a series ratio, with a dilution gradient from 1:100 to 1:12800, with 8 dilution gradients, at 37°C for 1 hour, washed four times with PBST, and HRP-labeled goat anti-mouse IgG (1:20000 dilution) was added. After washing four times with PBST, TMB was used for color development and the OD was measured on a microplate reader. 450 .

[0066] The results are as follows Figure 3 As shown, the serum of non-immunized mice was used as negative control (NC). When the serum was diluted 1:102400, the OD values ​​of immunized mice No. 1, 2 and 3 were 450 It was still greater than that of the NC group, indicating that the antibody titer could reach above 1:102400.

[0067] 4. Preparation of Monoclonal Antibodies

[0068] The specific steps are as follows:

[0069] (1) Preparation of feeder layer cells: Inject HAT culture medium into the peritoneal cavity of female BALB / c mice, slowly and repeatedly aspirate the liquid, and then plate it into a 96-well plate.

[0070] (2) Cell fusion: Splenocytes and an appropriate amount of SP20 cells were fused with the fusion agent PEG. The fused cells were plated in a 96-well plate containing feeder cells.

[0071] (3) Screening of positive clones:

[0072] a) Indirect ELISA detection method: the purified N protein is coated on an ELISA plate to detect the secretion of antibodies by the fused cells.

[0073] b) Indirect immunofluorescence (IFA) detection method: the supernatant from the ELISA antibody-positive cell wells was added to TGEV-infected PK15 cells, and then FITC-labeled goat anti-mouse IgG was added. After the reaction was completed, the results were observed under a fluorescence microscope.

[0074] (4) Subcloning of positive hybridoma cells: Select ELISA and IFA positive wells and subclone the screened positive hybridoma cells using the limiting dilution method. Observe under an inverted microscope, mark the wells where only a single clone has grown, collect the supernatant, and perform antibody detection using the above ELISA and IFA methods. Positive cells will enter the next round of subcloning, and this will be repeated three times.

[0075] (5) Preparation of ascites: 10-12 week old Balb / c mice were taken and 0.5 mL of Freund's incomplete adjuvant was injected intraperitoneally into each mouse. 1 week later, 5 × 10 5 After 7 to 10 days, the abdominal cavity of the mouse was obviously bulging, and the ascites was collected. The titer was detected by ELISA, and the cells were aliquoted and stored at -80℃.

[0076] (6) Purification of ascites: Affinity chromatography was performed using the PIERCE NAb™ Protein G Spin Purification Kit, and the purity was determined by SDS-PAGE electrophoresis. The heavy and light chains of the purified 1B7 and 2A9 mAbs were approximately 55 kDa and 25 kDa, respectively. Figure 7 ) This indicates that the antibody bands after purification by this method are correct and the purity is high.

[0077] 5. Identification of Monoclonal Antibodies

[0078] (1) Specificity identification: including IFA and Western blot verification.

[0079] a) IFA verification: PK15 cells were infected with TGEV. 36 hours after virus infection, the cells were fixed with paraformaldehyde. After cell fixation, ascites was diluted with PBS (500 times) and added to the virus-infected cells. Then, FITC-anti-mouse secondary antibody (100 times dilution) was incubated and the cells were observed under a fluorescence microscope after the reaction was completed. The results are shown in Figure 2. Figure 4 As shown in A and 4B, monoclonal antibodies 1B7 and 2A9 can detect specific green fluorescence signals in PK15 cells infected with TGEV, while no fluorescence signals were observed in TGEV-infected cells using the supernatant of SP2 / 0 cells ( Figure 4 C).

[0080] b) Western blot verification: Purified N protein or virus-infected and uninfected PK15 cells were collected for SDS-PAGE, and then the protein gel was transferred to NC membrane for western blot verification. The primary antibody was monoclonal antibody 1B7 or 2A9, and the secondary antibody was HRP-anti-mouse IgG. The results are shown in Figure 2. Figure 5 As shown in A and 5B, monoclonal antibodies 1B7 and 2A9 can both recognize the N protein in virus-infected cells. Figure 5 C is the detection of the intracellular reference β-actin.

[0081] (2) Subclass identification: The obtained monoclonal antibodies were identified according to the instructions of the Southern Biotech SBA Clonotyping™ System / HRP Antibody Subclass Identification Kit. The results showed that Figure 6 The heavy chain constant regions of 1B7 monoclonal antibody are of IgG1 type, and those of 2A9 are of IgG2b type, and their light chain constant regions are of Kappa type.

[0082] 6. PCR amplification and sequence determination of monoclonal antibody variable region genes

[0083] First, RNA of monoclonal antibody hybridoma cells was extracted and reverse transcribed into cDNA using Oligo-dt or random primers (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A).

[0084] The antibody variable region genes were amplified using nested PCR. First, the variable region genes were amplified using the aforementioned cDNA as a template using the first round of mouse IgG1 and κ light chain primers. Then, the first round product was used as a template for the second round of mouse IgG1 and κ light chain primers. The PCR reaction system consisted of 25 μL of PrimeSTAR Max Premix (2×), 1 μL each of P1 and P2, 1 μL of cDNA, and ddH2O to 50 μL. The reaction procedure was as follows: pre-denaturation at 98°C for 2 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and extension at 72°C for 10 min. Primers for antibody variable region gene amplification were referenced from the literature (von Boehmer, 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.).

[0085] After amplification, 1% agarose gel electrophoresis was performed and the gene sizes of the heavy chain and κ light chain variable regions were approximately 300 bp ( Figure 8 A and 8B), the target fragment was recovered by gel excision and inserted into the pMD-19T vector for sequence determination.

[0086] The DNA sequence of the heavy chain variable region of monoclonal antibody 1B7 is shown in SEQ ID NO: 5, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 6; the DNA sequence of the heavy chain variable region of monoclonal antibody 2A9 is shown in SEQ ID NO: 7, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0087] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1B7 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of monoclonal antibody 2A9 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0088] The heavy chain variable region of monoclonal antibody 1B7 includes CDR1 with the amino acid sequence of GFNIKDTY, CDR2 with the amino acid sequence of IDPADGYT, and CDR3 with the amino acid sequence of ARPGTLDY; the heavy chain variable region of monoclonal antibody 2A9 includes CDR1 with the amino acid sequence of GYTFTDFN, CDR2 with the amino acid sequence of INPNNGRS, and CDR3 with the amino acid sequence of ARRHWDWYFDV.

[0089] The light chain variable region of monoclonal antibody 1B7 includes CDR1 with the amino acid sequence of KSVSTSGYSY, CDR2 with the amino acid sequence of LVS, and CDR3 with the amino acid sequence of QHIRELTR; the light chain variable region of monoclonal antibody 2A9 includes CDR1 with the amino acid sequence of KSLLHSN, CDR2 with the amino acid sequence of QMS, and CDR3 with the amino acid sequence of AQNLEFPWT.

[0090] Example 2 Preparation of Latex Microsphere Immunochromatographic Test Strips for Detecting Porcine Infectious Diarrhea Coronavirus

[0091] 1. Preparation of the Conjugate Pad

[0092] 1.1 Determination of the optimal amount of latex microsphere-labeled TGEV N protein monoclonal antibody 1B7

[0093] The prepared TGEV N protein monoclonal antibody 1B7 was conjugated to latex microspheres to achieve final concentrations of 1B7 of 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, and 120 μg / mL, respectively. After successful conjugation, the conjugate was stored at 4°C overnight and found to be significantly aggregated at a final concentration of 40 μg / mL, indicating that the conjugation efficiency was too low and unsuccessful. The other four successfully labeled conjugates were used to test the positive and negative controls, such as Figure 9 When the final concentration of the antibody shown is 60 μg / mL, the positive and negative reactions are weaker than those of the other three groups, which will affect the later sensitivity of the test strip; when it is 120 μg / mL, although the positive reaction is the strongest, obvious non-specific bands appear in the negative reaction; when the concentration is 80 μg / mL and 100 μg / mL, the positive and negative reactions are almost the same. Based on cost considerations, 80 μg / mL was finally selected as the optimal latex microsphere-labeled antibody amount for this test strip.

[0094] Figure 9 Middle, 1 and 5: Results of positive and negative controls when the final antibody concentration was 60 μg / mL;

[0095] 2 and 6: Results of positive and negative controls when the final antibody concentration was 80 μg / mL;

[0096] 3 and 7: Results of positive and negative controls when the final antibody concentration was 100 μg / mL;

[0097] 4 and 8: Results of positive and negative controls when the final antibody concentration was 120 μg / mL.

[0098] 1.2 Latex microsphere-labeled TGEV N protein monoclonal antibody 1B7

[0099] 975 μL of 0.1 M pH 5.6 MES buffer was added to a 2 mL centrifuge tube, and 25 μL of latex microspheres with a solid content of 4% (Suzhou Weidu Biotechnology Co., Ltd.) was added and gently mixed; 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) were added, mixed thoroughly, activated at room temperature for 20 minutes, and centrifuged at 17000 r for 20 minutes; the supernatant solution was removed, and the suspension was fully resuspended with 1 mL MES buffer and centrifuged again at 17000 r for 20 minutes; 80 μg of TGEV N protein monoclonal antibody 1B7 was added and incubated at room temperature for 2 hours; 100 μL of 10 w / v% BSA solution was added for blocking, incubated at room temperature for 1 hour, and centrifuged at 17000 r After 10 minutes of centrifugation, 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, then 3 seconds, for a total of 1 minute, and store at 4°C until use.

[0100] 1.3 Preparation of latex microsphere binding pad

[0101] Completely immerse the conjugate pad in a blocking solution (an ultrapure solution containing 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) at 37°C–38°C for 2 hours. Then, remove the pad and dry it in an oven for 2–4 hours. Evenly spray the prepared latex microspheres labeled with the anti-TGEV N protein monoclonal antibody 1B7 onto the conjugate pad using a film sprayer. Dry the pad in a 37°C oven for 2–3 hours and seal for storage.

[0102] 2. Preparation of Sample Pad

[0103] At 37°C to 38°C, soak the sample pad in 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 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.

[0104] 3. Preparation of Nitrocellulose Membrane

[0105] To determine the optimal coating concentration of the antibody at the T line, the monoclonal antibody 2A9 against TGEV N protein was diluted to 0.5, 1.0, 1.5, and 2.0 mg / ml and sprayed on the T line. TGEV cytotoxicity (positive control) and uninfected healthy cell supernatant (negative control) were added and the results were observed after 12 minutes of reaction. The test results are shown in Figure 2. Figure 10 As shown in the figure, 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 mg / ml and 1.5 mg / ml, the reaction effects are almost the same. Considering the saving of raw materials, 1 mg / ml is finally selected as the optimal coating concentration at the T line.

[0106] Figure 10 In the middle, 1 and 5: the results of the positive and negative controls when the T line concentration was 0.5 mg / mL;

[0107] 2 and 6: Results of the positive and negative controls when the T-line concentration was 1.0 mg / mL;

[0108] 3 and 7: Results of positive and negative controls when the T-line concentration was 1.5 mg / mL;

[0109] 4 and 8: Results of the positive and negative controls when the T-line concentration was 2.0 mg / mL.

[0110] 1 mg / ml anti-TGEV protein monoclonal antibody 2A9 and 0.5 mg / ml 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.

[0111] 4. Assembly of test strips

[0112] The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are connected on the PVC base plate in sequence to obtain a test strip. The test strip is cut into strips according to the size of the test strip card, and then the cut test strips are loaded into the test strip card. The structural diagram of the test strip is shown in FIG. Figure 11 shown.

[0113] Example 3 Application of Latex Immunochromatographic Test Strips for Detecting Porcine Infectious Diarrhea Coronavirus

[0114] 1. How to use the test strips

[0115] (1) Detection: Take out the test strip and equilibrate it to room temperature. Dilute the sample to be tested with sample diluent at a ratio of 1:5. At room temperature, drop 100 μl of the sample on the sample pad and observe the result within 10 minutes.

[0116] (2) Result determination: 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 and the quality control line (C line) shows color, it is negative; if the quality control line (C line) does not show color, it can be determined that the test strip is invalid.

[0117] 4. Sensitivity of the test strip

[0118] TGEV TCID 50 is 10 7.8 After a 1:5 dilution of the virus / 0.1 mL, a 2-fold dilution was performed to test the sensitivity of the established test strip.

[0119] The results showed that when the virus was 1:2 12 The test value is still positive after dilution, 1:2 13 The test value after dilution is negative. Therefore, the sensitivity of the test strip prepared by the present invention can reach 1:2 12 , that is, the minimum virus detection amount of this method is 10 3.6 TCID 50 / 0.1mL, such as Figure 12 shown.

[0120] 5. Specificity of the test strip

[0121] TGEV, SADS-CoV, PEDV, and PDCoV were detected by test strips. It was found that only TGEV was positive, while the others did not react, indicating that the method established by the present invention can specifically identify TGEV. Figure 13 shown.

[0122] 6. Stability test

[0123] 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 1, the test strips can be stably stored for 9 months at room temperature and for 12 months at 4°C.

[0124] Table 1 Test strip stability test results

[0125]

[0126] Note: -: negative; +, ++, +++: the positive degree increases in sequence.

[0127] The embodiments described above 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, characteristics 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 transmissible gastroenteritis coronavirus, characterized in that: A: The heavy chain variable region of a monoclonal antibody includes a CDR1 with the amino acid sequence GFNIKDTY, a CDR2 with the amino acid sequence IDPADGYT, and a CDR3 with the amino acid sequence ARPGTLDY; the light chain variable region of a monoclonal antibody includes a CDR1 with the amino acid sequence KSVSTSGYSY, a CDR2 with the amino acid sequence LVS, and a CDR3 with the amino acid sequence QHIRELTR; B: The heavy chain variable region of the monoclonal antibody includes CDR1 with the amino acid sequence of GYTFTDFN, CDR2 with the amino acid sequence of INPNNGRS, and CDR3 with the amino acid sequence of ARRHWDWYFDV; the light chain variable region of the monoclonal antibody includes CDR1 with the amino acid sequence of KSLLHSN, CDR2 with the amino acid sequence of QMS, and CDR3 with the amino acid sequence of AQNLEFPWT.

2. The anti-transmissible gastroenteritis coronavirus N protein monoclonal antibody according to claim 1, characterized in that: A: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; B: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

3. DNA encoding the heavy chain variable region and light chain variable region of the monoclonal antibody against the N protein of porcine transmissible gastroenteritis coronavirus according to claim 1 or 2.

4. The DNA according to claim 3, characterized in that A: The DNA sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 5, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 6; B: The DNA sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 7, and the DNA sequence of the light chain variable region is shown in SEQ ID NO:

8.

5. A recombinant vector, characterized in that A recombinant vector containing the DNA according to claim 3 or 4.

6. A host cell, characterized in that The host cell contains the recombinant vector according to claim 5.

7. Use of the monoclonal antibody against the N protein of porcine transmissible gastroenteritis coronavirus according to claim 1 in the preparation of a drug for detecting porcine transmissible gastroenteritis infection.

8. A latex immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis coronavirus, comprising a PVC base, a sample pad, a conjugation pad, a nitrocellulose membrane, and a water-absorbing pad, wherein the sample pad, conjugation pad, nitrocellulose membrane, and water-absorbing pad are sequentially overlapped on the PVC base; a detection line T and a quality control line C are provided on the nitrocellulose membrane, wherein the detection line T is provided at one end near the conjugation pad, and the quality control line C is provided at one end near the water-absorbing pad; characterized in that: The conjugate pad is sprayed with latex microsphere-labeled monoclonal antibody A against TGEV N protein; the detection line T is coated with monoclonal antibody B against TGEV N protein; the quality control line C is coated with goat anti-mouse IgG; the monoclonal antibody A and monoclonal antibody B are the monoclonal antibodies shown in A or B in claim 1 or 2, respectively.

9. The latex immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis coronavirus according to claim 8, characterized in that: The preparation method of the conjugate pad is as follows: Block the conjugate pad with conjugate pad blocking solution; The latex microspheres were activated with N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, centrifuged, resuspended in MES buffer, and fully dispersed by sonication. Anti-TGEV N protein monoclonal antibody A was then added and incubated at room temperature. The latex microsphere marker blocking solution was then added for blocking, incubated at room temperature, and centrifuged. The precipitate was resuspended in a latex microsphere marker protective agent to obtain an anti-TGEV N protein monoclonal antibody A latex microsphere labeling solution. The prepared anti-TGEV N protein monoclonal antibody A latex microsphere labeling solution was evenly sprayed on the blocked conjugate pad.

10. The latex immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis coronavirus according to claim 9, characterized in that: The conjugate pad blocking solution is an ultrapure aqueous solution containing 5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20 and 1.5% m / v PVP-40.

11. The latex immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis coronavirus according to claim 9, characterized in that: The latex microsphere marker blocking solution is an ultrapure aqueous solution containing 10% m / v BSA.

12. The latex immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis coronavirus according to claim 9, characterized in that: 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.

13. The latex immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis coronavirus according to claim 9, characterized in that: The sample pad was immersed in a sample pad blocking solution; the sample pad blocking solution was a Tris-HCl solution containing 2% BSA, 0.5% S17, 2% PEG20000 and 0.5% sodium caseinate.

14. The method for preparing the latex microsphere immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis coronavirus according to claim 8 or 9, characterized in that: The following steps are involved: (1) Prepare a sample pad and a conjugate pad coated with latex microspheres labeled with antibodies; (2) 1 mg / ml anti-TGEV protein monoclonal antibody B and 0.5 mg / ml goat anti-mouse IgG antibody were sprayed onto the nitrocellulose membrane with an interval of 5 to 8 mm, serving as the test line T and the quality control line C, respectively; (3) The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad were overlapped on the PVC base plate in sequence to obtain a latex microsphere immunochromatographic test strip for detecting porcine transmissible gastroenteritis coronavirus.

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