Cell line for stably expressing porcine delta coronavirus S protein, porcine delta coronavirus IgA antibody detection antigen and kit

Abstract

The invention discloses a cell line for stably expressing porcine delta coronavirus S protein, a porcine delta coronavirus IgA antibody detection antigen and a kit. The method comprises the following steps: based on an S protein sequence of a CH / XJYN / 2016 strain, selecting an extracellular domain part D20-N1077, adding a human source septoglobin family 1D meber 1 signal peptide sequence MRLSVCLLLTLALCCYRANA to an amino terminal of the S protein, adding a 6 * His tag sequence to a carboxyl terminal, carrying out codon optimization according to a CHO expression system, and constructing into a pCDNA3.1 (+) eukaryotic expression vector to obtain a recombinant eukaryotic expression plasmid pcDNA3.1 (+)-PDCoV-S; then transfecting CHO cells with the plasmid, and carrying out two rounds of heredity mycin G418 pressurized screening, limited dilution cloning and continuous subculture to obtain an expiCHO-PDCoV-S stably transfected cell line through screening; and finally, constructing the PDCoV IgA antibody detection kit based on the cell line. The kit has no cross reaction with other common porcine disease virus positive serum, has intra-batch and inter-batch variation coefficients of less than 10%, has good specificity, repeatability and sensitivity, and can be used for early prevention and control of PDCoV.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a cell line that stably expresses porcine delta coronavirus S protein, a detection antigen of porcine delta coronavirus IgA antibody expressed by the cell line, and a kit containing the antigen. Background Technology

[0002] Porcine deltacoronavirus (PDCoV) is an emerging porcine enteric coronavirus that primarily infects piglets, causing acute diarrhea, vomiting, dehydration, and growth retardation, and in severe cases, death, posing a significant threat to the swine industry. In recent years, PDCoV has continued to circulate in pig herds in many countries and can co-infect with other enteroviruses such as porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), leading to more complex clinical symptoms, further complicating immunomolecular detection techniques, and exacerbating economic losses in the pig farming industry.

[0003] PDCoV, a porcine enteric pathogen, is transmitted via the fecal-oral route. After infection, it replicates in intestinal epithelial cells and induces a mucosal immune response. The pathogen initially reacts with the mucosal immune system, and IgA antibodies, as the main effector molecules of mucosal immunity, effectively inhibit further PDCoV infection by preventing viral adhesion to host cell receptors and neutralizing the virus. In the early stages of PDCoV infection, the level of IgA antibodies can be detected to determine the infection status, which is of great significance for early disease control and vaccine immunization assessment. The S protein, a key target protein of porcine delta coronaviruses for recognizing host cell receptors and invading, possesses excellent immunogenicity and reactivity. The natural S protein is a glycosylated protein with a transmembrane structure, and its glycosylation sites are concentrated in the extracellular domain, making it an ideal antigen for establishing ELISA detection methods. Preparing PEDV S protein with its native conformation and high immunoreactivity is crucial for reducing erroneous results. The ExpiCHO eukaryotic expression system can generate stable recombinant cell lines by integrating the target protein gene into host cells, enabling the stable production of homogeneous recombinant proteins with a natural-looking structure. Therefore, constructing an expiCHO-PDCoV-S cell line that stably expresses the porcine delta coronavirus S protein, and developing an antigen for rapid detection of porcine delta coronavirus IgA antibody and an indirect ELISA kit containing the antigen, can provide technical support for the early prevention and control of PDCoV. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for preparing a cell line that stably expresses porcine delta coronavirus S protein.

[0005] Another objective of this invention is to provide an expiCHO-PDCoV-S cell line that stably expresses porcine delta coronavirus S protein, prepared using the above method.

[0006] Another object of the present invention is to provide a detection antigen using a porcine delta coronavirus IgA antibody obtained from the above-mentioned expiCHO-PDCoV-S cell line.

[0007] Another object of the present invention is to provide a detection kit for porcine delta coronavirus IgA antibody containing the above-mentioned detection antigen.

[0008] To achieve its purpose, the present invention adopts the following technical solution: The present invention provides a method for preparing a cell line stably expressing porcine delta coronavirus S protein. The method involves transfecting CHO cells with the eukaryotic expression vector pcDNA3.1(+)-PDCoV-S encoding the extracellular domain portion D20-N1077 of the S protein of the prevalent PDCoV CH / XJYN / 2016 strain, introducing the PDCoV S gene, and then selecting the expiCHO-PDCoV-S stably expressed cell line through two rounds of genetic mycin G418 pressure selection, limiting dilution cloning, and continuous passage culture.

[0009] The above preparation method can be used to obtain a cell line that stably expresses porcine delta coronavirus S protein. The nucleotide sequence of the introduced PDCoV S gene in the genome of the cell line is shown in SEQ ID NO.1, and the amino acid sequence of the PDCoVS protein encoded by the PDCoV S gene is shown in SEQ ID NO.2.

[0010] The present invention provides a porcine delta coronavirus IgA antibody detection antigen, which is a recombinant PDCoV S protein. The recombinant PDCoV S protein is obtained by collecting the culture supernatant of the expiCHO-PDCoV-S stable cell line and purifying it using Ni affinity chromatography.

[0011] The present invention provides a detection kit for porcine delta coronavirus IgA antibody, comprising an enzyme-labeled plate coated with an antigen; wherein the antigen is the detection antigen described above, namely recombinant PDCoV S protein; The preparation process of the enzyme-labeled plate coated with the antigen includes the following steps: (1) Dilute the PDCoV S protein with coating buffer, add it to each well of the ELISA plate, and incubate at 4°C overnight to complete the coating; (2) Discard the residual protein solution in the wells of the ELISA plate, wash with PBST, and pat the ELISA plate dry; (3) Add sealing solution to each well and incubate at 37°C for 3 hours to complete the sealing.

[0012] Preferably, the concentration of the diluted PDCoV S protein is 2 μg / mL; the coating solution is a carbonate buffer at pH 9.6, and the blocking solution is a PBST solution containing 1% BSA.

[0013] Furthermore, the kit also includes a negative control, a positive control, a horseradish peroxidase-labeled secondary antibody, a colorimetric solution, a stop solution, and a washing solution.

[0014] Preferably, the secondary antibody is a goat anti-pig IgA antibody; the washing solution is a PBS solution containing 0.05% Tween-20 with a pH of 7.4; and the diluent is a 0.01M PBST buffer with a pH of 7.4 containing 1% BSA and 0.1% liquid biological preservative Proclin-300.

[0015] The present invention provides a method for using a porcine delta coronavirus IgA antibody detection kit for non-diagnostic purposes, comprising the following steps: (1) Dilute the negative control, positive control and the serum or milk sample to be tested, add them to the ELISA plate coated with PDCoV S protein, and incubate at 37°C for 1 h; after incubation, wash the ELISA plate with PBST and pat dry; (2) Add diluted horseradish peroxidase-labeled secondary antibody to each well and incubate at 37°C for 40 min; after incubation, wash the microplate with washing solution and pat dry; (3) Add the colorimetric solution to each well and incubate at 37°C for 20 min; (4) Add the stop solution to each well and measure the absorbance of the reaction solution at 450 nm to determine whether the sample contains PDCoV-specific IgA antibody.

[0016] Result Interpretation: For serum samples, when OD 450 nm A value ≥0.14396 is considered positive (OD). 450 nm A value <0.14396 is considered negative; for milk samples, when OD... 450 nm A value ≥0.33381 is considered positive (OD). 450 nm A value less than 0.33381 is considered negative.

[0017] By adopting the above technical solution, the beneficial effects of the present invention are as follows: This invention is based on the S protein sequence of the CH / XJYN / 2016 strain, selecting its extracellular domain portion D20-N1077. A human secretoglobin family 1D member 1 signal peptide sequence MRLSVCLLLLTLALCCYRANA was added to the N-terminus of the S protein, and a 6×His tag sequence was added to the C-terminus. Codon optimization was performed according to the CHO expression system, and the resulting plasmid was constructed into the pCDNA3.1(+) eukaryotic expression vector to obtain the recombinant eukaryotic expression plasmid pcDNA3.1(+)-PDCoV-S. The pcDNA3.1(+)-PDCoV-S plasmid was then transfected into CHO cells. After two rounds of selection using genimycin G418 pressure, limiting dilution cloning, and continuous passage culture, a stable expiCHO-PDCoV-S cell line was obtained. Finally, based on this stable expiCHO-PDCoV-S cell line, a PDCoVIgA antibody detection kit was constructed. This kit shows no cross-reactivity with other common swine virus positive sera, and the intra- and inter-batch coefficients of variation are both less than 10%. It has good specificity, repeatability, and sensitivity, and can be used for the early prevention and control of PDCoV. Attached Figure Description

[0018] Figure 1 This is a diagram of the linearized nucleic acids for the plasmid in Example 1; Figure 2 Results of determination of the optimal screening concentration of G418 for CHO cells; Figure 3 The results of RT-PCR and Western blot identification of the PDCoV S monoclonal cell line; Figure 4 Results of stability assessment of PDCoV S monoclonal cell lines; Figure 5 Results of PDCoV S protein purification; Figure 6 The results of the determination of the critical value of the ELISA detection method; Figure 7 The results are specific to the ELISA detection method. Figure 8 This represents the results of the sensitivity determination of the ELISA detection method. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0020] Example 1 1. Construction and screening of cell lines stably expressing porcine delta coronavirus S protein Based on the S protein sequence of the CH / XJYN / 2016 strain (GenBank accession number: MN064712), the extracellular domain portion D20-N1077 was selected for expression. A human secretoglobin family 1 D member 1 signal peptide sequence MRLSVCLLLLTLALCCYRANA was fused to the N-terminus of the S protein, and a 6×His tag sequence was added to the C-terminus. The amino acid sequence of the resulting fusion protein is shown in SEQ ID NO.2. The fusion protein sequence was then sent to Wuhan Jinkairui Biotechnology Co., Ltd. for codon optimization and synthesis using the CHO expression system. The optimized nucleotide sequence is shown in SEQ ID NO.1, and it was cloned into the pCDNA3.1(+) eukaryotic expression vector to obtain the recombinant eukaryotic expression plasmid pcDNA3.1(+)-PDCoV-S.

[0021] Linearization and recovery of plasmids: The plasmids were linearized by digestion with MfeI restriction endonuclease. The digestion system contained: 12.5 μg plasmid, 5 μL 10× Buffer, 5 μL MfeI enzyme, and ddH2O was added to bring the volume to 50 μL. The mixture was incubated at 37°C for 3 h.

[0022] After enzyme digestion, the digestion products were analyzed and identified using 0.8% agarose gel electrophoresis, such as... Figure 1 As shown, the enzyme digestion product bands are single and the fragment size is as expected, indicating that the plasmid linearization was successful.

[0023] Purify and recover the correctly identified target bands using the PureLink PCR Purification Kit according to the instructions, and store at -20°C for later use.

[0024] Determination of the optimal G418 selection concentration for CHO cells: ExpiCHO cells were loaded at 1×10 6 Cells were seeded at a density of 10 cells / mL in shake flasks containing 30 mL of ExpiCHO™ expression medium, with 12 replicates. Gradient concentrations of G418 (0, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, and 700 μg / mL) were added to each flask, and cell viability was monitored daily for 7 consecutive days. The optimal selection concentration was determined to be the G418 concentration that caused approximately 50% cell death 3–5 days post-seeding. Based on the results, 650 μg / mL was ultimately determined to be the optimal G418 selection concentration for constructing stable cell lines. (See attached results). Figure 2 .

[0025] Cell transfection and screening of monoclonal cell lines One day in advance, dilute ExpiCHO cells in the logarithmic growth phase and in good condition to 1×10⁻⁶. 6 Cells were cultured at 5 × 10⁶ viable cells / mL overnight. The plasmid was diluted and the transfection complex was prepared according to the transfection kit instructions. The complex was then slowly added to the culture flask and placed in suspension on a rocker at 37°C, 8% CO₂, and 125 rpm. After two days, cell viability and density were assessed using a Countstar cell counter, followed by the first round of pressure screening. Cells were cultured at 5 × 10⁶ cells / mL. 5 Initially seeded at a density of 100 viable cells / mL into 125 mL shake flasks, and 650 μg / mL of G418 was added to initiate pressure selection. Subsequently, cells were seeded every 3-4 days at a rate of 3 × 10⁻⁶ cells / mL. 5 Passage cells at a viable cell / mL rate until cell viability exceeds 85%, completing the first round of selection. Resuscitate the cells from the first round of selection in 30 mL of ExpiCHO™ stable medium pre-warmed to 37 °C for the second round of pressurized selection. The G418 concentration in the second round is 5 times the initial concentration from the first round, injected at 3 × 10⁻⁶ every 3-4 days. 5 Passage at 1 viable cell / mL. The second round of selection ends when cell viability reaches 90% or higher. After pressure selection, cells are seeded at 0.5 cells / well into 96-well plates and cultured statically at 37°C and 8% CO2 for 17 days. Monoclonal cells showing growth are selected for initial screening. The monoclonal cells with the highest expression levels are then serially expanded into 24-well plates, 6-well plates, and 125mL shake flasks, and subsequently cryopreserved.

[0026] Total RNA was extracted from the PDCoV S monoclonal cell line, and PCR amplification was performed using ExpiCHO cell genomic RNA as a negative control. The results showed a single, clear, specific band at 3200 bp, while no band was detected in the negative control, indicating that the PDCoV S gene had been successfully integrated into the CHO cell genome (Figure 3A). Simultaneously, the culture supernatant of the PDCoV-S-CHO cell line was collected, and Western blot analysis was performed using PDCoV-positive serum as the primary antibody and HRP-labeled goat anti-pig IgG as the secondary antibody. The results showed a single, specific band at approximately 160 kDa, larger than the theoretical value and consistent with expectations, indicating that this PDCoV S monoclonal cell line can stably secrete and express the glycosylated S protein with good immunoreactivity (Figure 3B).

[0027] Stability study of PDCoV S protein expression cell lines: The selected monoclonal cell lines were passaged continuously, and the cells were cryopreserved every 5 passages. The cell lines at passages 0, 5, 10, 15, and 20 were then revived, and the expression levels of the cell lines at different passages were detected using SDS-PAGE. Figure 4 The expression level was quantitatively detected by grayscale analysis. The results are shown in Table 1. The expression level of 20 cells was only 8.82% lower than that of 0 cells, indicating that the cell line has good stability.

[0028] Table 1. Quantitative analysis of PDCoV monoclonal cell lines by grayscale Preparation, purification and identification of recombinant PDCoV S protein: The culture supernatant of S protein monoclonal cells was collected by centrifugation at 3000×g for 30 min and filtered through a 0.22 μm filter membrane. The filtered supernatant was loaded onto a pre-equilibrated Ni-TED gravity column. After collecting the flow-through, unbound contaminating proteins were eluted with buffer containing 25 mmol / L imidazole, followed by elution of the target protein with buffer containing 500 mmol / L imidazole. The purity of the eluted product was determined by SDS-PAGE, and a single band was observed at approximately 160 kDa, indicating a purity exceeding 90%. Figure 5 The protein concentration was determined to be 0.8 mg / mL using a BCA protein concentration assay kit. After aliquoting, it was stored at −80°C.

[0029] Example 2 Preparation of enzyme-labeled plates coated with antigens: The purified PDCoV S recombinant protein was diluted to 2 μg / mL with carbonate buffer (pH 9.6), and 100 μL / well was added to each well. The plates were incubated overnight at 4°C (12 h). After incubation, the residual protein solution was discarded, and the plates were washed five times with PBST. The plates were then blotted dry. 200 μL / well of PBST containing 1% BSA was added for blocking, and the plates were incubated at 37°C for 3 h. The residual blocking solution was discarded, and the plates were washed five times with PBST. The plates were then blotted dry. This yielded PDCoV S protein-coated microplates.

[0030] Example 3 An indirect ELISA kit for rapid detection of porcine delta coronavirus IgA antibodies comprises the following components: ELISA plate: The ELISA plate coated with PDCoV S protein prepared in Example 2, with a coating antigen concentration of 2 μg / mL; Enzyme-labeled secondary antibody: HRP-labeled goat anti-porcine IgA; Diluent: 0.01M pH 7.4 containing 1% BSA and 0.1% liquid biological preservative Proclin-300. PBST buffer; Washing solution: 0.01M PBS solution with pH 7.4 containing 0.05% Tween-20; Colorimetric solution: TMB solution; Termination solution: 2 mol / L H2SO4 solution; Negative control: negative control serum containing antibodies against PDCoV infection; Positive control: PDCoV antibody-positive quality control serum.

[0031] Example 4 The method for detecting PDCoV IgA antibodies using the indirect ELISA kit of Example 2 includes the following steps: (1) Add the test samples: Dilute the negative control, positive control and test samples to the optimal concentration (serum 1:160; milk 1:400) with diluent, 100 μL / well, as negative control group, positive control group and experimental group, and perform replicate experiments. Incubate at 37°C for 1 h, discard the residual test sample solution, wash 5 times with PBST, and pat dry the ELISA plate.

[0032] (2) Add enzyme-labeled antibody: Dilute horseradish peroxidase-labeled goat anti-pig IgA antibody to 1:20000 with diluent, 100 μL / well, incubate at 37°C for 40 min, discard residual enzyme-labeled antibody solution, wash 5 times with PBST, and pat dry the enzyme-labeled plate.

[0033] (3) Color development and termination: Add TMB color development solution, 100 μL / well, incubate at 37°C in the dark for 20 min, add termination solution, 100 μL / well, and measure the absorbance value at 450 nm.

[0034] (4) Result Interpretation: The absorbance values ​​(OD) at 450 nm of 133 PDCoV-negative serum samples and 14 PDCoV-negative breast milk samples were measured respectively. 450 The mean (X) and standard deviation (SD) of the sample (nm) are calculated. According to statistical principles, X + 3SD is used as the threshold for determining positive or negative: OD 450 Samples with nm ≥ X + 3SD are considered positive, and those with nm ≥ X + 3SD are considered negative.

[0035] Optimize the different conditions in the above detection method: Determination of optimal antigen coating concentration and sample dilution The optimal concentration was determined using checkerboard titration. PDCoV S protein was diluted to 2 μg / mL, 1 μg / mL, 0.5 μg / mL and 0.25 μg / mL, 100 μL / well, and coated overnight at 4°C (12 h).

[0036] Add 200 μL of PBST solution containing 5% skim milk powder to each well, and block at 37°C for 2 h. After blocking, discard the blocking solution and wash 5 times with PBST. Dilute positive and negative serum samples from 1:20, 1:40 to 1:640; dilute positive and negative milk samples from 1:200, 1:400 to 1:1000, and add 100 μL of each solution to the above ELISA plate, incubating at 37°C for 45 min. After incubation, wash 5 times with PBST, add 100 μL of horseradish peroxidase-labeled goat anti-pig IgA antibody diluted 1:20000 to each well, and incubate at 37°C for 40 min. Wash 5 times with PBST. Add 100 μL of TMB chromogenic solution to each well and incubate at 37°C in the dark for 10 min. Add 100 μL of stop solution to each well and measure the absorbance at 450 nm. The optimal coating concentration and optimal sample dilution are determined according to the OD of the positive sample. 450 nm / Negative sample OD 450 nm The maximum choice of value (P / N).

[0037] Table 2 Determination of Optimal Coating Concentration and Serum Dilution Table 3 Determination of Milk Dilution The P / N ratio was calculated, and the optimal antigen coating concentration was found to be 2 μg / mL, the optimal serum dilution was 1:160, and the optimal milk dilution was 1:400.

[0038] Example 5 Screening and determination of antigen coating conditions Based on the optimal antigen coating concentration and optimal sample dilution ratio determined in Example 3, three coating conditions were set up: incubation at 37°C for 1 hour, incubation at 37°C for 2 hours, and overnight at 4°C, to screen for the optimal antigen coating method. Other experimental conditions were performed as in Example 3. OD values ​​for each group were measured. 450 nm The values ​​(Table 4) were used to determine the optimal coating conditions based on the principle of maximizing the P / N value. Based on the results, overnight coating at 4°C was ultimately determined as the optimal antigen coating condition.

[0039] Table 4 Screening and Determination of Antigen Coating Conditions Example 6 Selection of the optimal sealing solution and screening and determination of the sealing time The microplates were coated according to the optimal coating conditions determined in the above embodiments. Screening was performed using 5% BSA, 3% BSA, 1% BSA, and 0.5% BSA solutions as blocking solutions, respectively. All other experimental steps were performed as described in Example 3. The OD values ​​of each group were measured. 450 nmThe values ​​(Table 5) were compared, and the blocking effect was comprehensively analyzed. Finally, PBST solution containing 1% BSA was determined to be the best blocking solution.

[0040] Table 5 Screening and Determination of Optimal Conditions for Sealing Fluid ELISA plates were coated with 2 μg / mL PDCoV S protein as the coating antigen, and PBST solution containing 1% BSA was used as the blocking solution. Screening was performed by incubating at 37°C for 1 h, 2 h, and 3 h, respectively. All other experimental procedures followed those in Example 3. The OD values ​​of each group were measured. 450 nm Based on the values ​​(Table 6), after comprehensive analysis, the optimal sealing time was determined to be 37°C incubation for 3 hours.

[0041] Table 6 Determination of Optimal Closure Conditions Example 7 Screening and determination of the optimal reaction time for samples ELISA plates were coated with 2 μg / mL PDCoV S protein as the coating antigen and blocked with PBST solution containing 1% BSA at 37°C for 3 h. Incubation times of 37°C for 30 min, 45 min, and 60 min were set as selection criteria for sample incubation time, and the remaining experimental steps were performed according to Example 3. The OD values ​​of each group were measured. 450 nm Based on the values ​​(Table 7), the optimal incubation time for the samples was finally determined to be 37°C for 1 hour.

[0042] Table 7 Screening and Determination of Optimal Reaction Time for Samples Example 8 Screening and determination of optimal dilution and reaction time for enzyme-labeled secondary antibodies ELISA plates were coated with 2 μg / mL PDCoV S protein as the coating antigen, and blocked with PBST solution containing 1% BSA at 37°C for 3 h. Secondary antibody dilutions of 1:10000, 1:20000, and 1:30000 were used as selection criteria; all other experimental steps were performed according to Example 3. The OD values ​​of each group were measured. 450 nm The values ​​(Table 8) were used to determine that 1:20000 was the optimal dilution for the secondary antibody.

[0043] Table 8 Screening and Determination of Optimal Dilution Ratio for Secondary Antibodies The enzyme-labeled secondary antibody was diluted 1:20000 and added to the ELISA plate at a volume of 100 μL / well. The plates were incubated at 37°C for 20 min, 40 min, and 60 min, respectively, to compare the ratio of OD values ​​of positive to negative samples (P / N). The optimal reaction time was determined based on maximizing the P / N value. The experimental results (Table 9) show that incubation at 37°C for 40 min is the optimal incubation time for the enzyme-labeled secondary antibody.

[0044] Table 9 Screening and identification of optimal reaction time for secondary antibodies Example 9 Determining the optimal color development time The ELISA plate was coated with 2 μg / mL PDCoV S protein as the coating antigen, blocked with PBST solution containing 1% BSA at 37°C for 3 h, and then incubated with 1:20000 diluted enzyme-labeled antibody at 37°C for 40 min. The reaction time was set at 37°C in the dark for 10, 20, and 30 min as screening conditions. All other experimental steps were performed according to Example 2. The OD values ​​of each group were measured. 450nm Based on the values ​​(Table 10), the optimal color development time was finally determined to be 20 min of reaction in the dark at 37°C.

[0045] Table 10 Selection and Determination of Optimal Color Development Time Example 10 Determination of critical values Based on the optimized reaction conditions, 133 negative serum samples with clear backgrounds and 14 negative breast milk samples were tested, and their OD values ​​were measured. 450 nm The values ​​were calculated, and the mean (X) and standard deviation (SD) of each sample group were determined. See the attached table for details. Figure 6 When the test sample is serum, if OD 450 nm A value ≥ 0.14396 indicates a positive result for the serum sample, signifying the presence of porcine delta coronavirus IgA antibodies; if the OD value is ≥ 0.14396, the serum sample is considered positive. 450 nm A value < 0.14396 indicates that the serum sample is negative, meaning it does not contain porcine delta coronavirus IgA antibodies. For milk samples, if the OD value is < 0.14396, the serum sample is considered negative, indicating the absence of porcine delta coronavirus IgA antibodies. 450 nm A value ≥0.33381 indicates that the milk sample is positive, meaning it contains porcine delta coronavirus IgA antibodies; if the OD value is ≥0.33381, the milk sample is considered positive. 450 nm If the value is <0.33381, the milk sample is considered negative, indicating that it does not contain porcine delta coronavirus IgA antibodies.

[0046] Example 11 Specific experiments Based on the optimized reaction conditions, standard positive sera for porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), porcine foot-and-mouth disease virus type O (FMDV), and porcine rotavirus (PoRV) were tested. PDCoV positive controls, PDCoV negative controls, and a blank control group were established, and OD was measured. 450 nm value.

[0047] The results showed that ( Figure 7 An indirect ELISA method established using recombinant PDCoV S protein as the coating antigen can specifically bind only to PDCoV positive controls and has no cross-reactivity with other swine virus positive sera, indicating that the detection method has good specificity.

[0048] Example 12 Repeatable experiments Based on the optimized reaction conditions, 5 positive samples and 5 negative samples were randomly selected and tested using ELISA plates coated with the same batch and plates coated with different batches, respectively. Each sample was tested three times, and the OD was measured. 450 nm The values ​​were calculated, and the mean, standard deviation, and coefficient of variation for each group were determined. The results are shown in Table 11-12.

[0049] Table 11 Determination of intra-batch variation rate Table 12 Determination of inter-batch variation rate Repeatability experiments showed that the inter-batch and intra-batch variability rates of serum and milk were both less than 10%, indicating that the method has good repeatability and stability.

[0050] Example 13 Sensitivity Measurement Experiment Based on the optimized reaction conditions, positive serum and positive milk samples were randomly selected and their OD values ​​were measured at different dilutions (1:100, 1:200, 1:400, 1:800, 1:1600, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, and 1:5000). 450 nm The results (Figure 8) show that IgA antibody signals can still be detected when the sample is diluted to 1:4500, indicating that the detection method has high sensitivity and can detect trace amounts of antibodies in the sample.

Claims

1. A method for preparing a cell line stably expressing porcine delta coronavirus S protein, characterized in that, The preparation method involves first transfecting CHO cells with the eukaryotic expression vector pcDNA3.1(+)-PDCoV-S, which encodes the extracellular domain portion D20-N1077 of the S protein of the prevalent PDCoV CH / XJYN / 2016 strain, and then introducing the PDCoV S gene. After two rounds of selection under pressure with genimycin G418, limiting dilution cloning, and continuous passage culture, a stable expiCHO-PDCoV-S cell line was obtained.

2. A cell line stably expressing porcine delta coronavirus S protein, prepared by the method of claim 1, characterized in that, The nucleotide sequence of the introduced PDCoV S gene in the cell line genome is shown in SEQ ID NO.1, and the amino acid sequence of the PDCoV S protein encoded by the PDCoVS gene is shown in SEQ ID NO.

2.

3. A detection antigen for porcine delta coronavirus IgA antibodies, characterized in that, The antigen is a recombinant PDCoV S protein, which is obtained by purifying the culture supernatant of the cell line described in claim 2 using Ni affinity chromatography.

4. A detection kit for porcine delta coronavirus IgA antibodies, characterized in that, The kit includes an enzyme-labeled plate coated with an antigen; the antigen is the detection antigen described in claim 3. The preparation process of the enzyme-labeled plate coated with the antigen includes the following steps: (1) Dilute the PDCoV S protein with coating buffer, add it to each well of the ELISA plate, and incubate at 4°C overnight to complete the coating; (2) Discard the residual protein solution in the wells of the ELISA plate, wash with PBST, and pat the ELISA plate dry; (3) Add sealing solution to each well and incubate at 37°C for 3 hours to complete the sealing.

5. The porcine delta coronavirus IgA antibody detection kit as described in claim 4, characterized in that, The concentration of the diluted PDCoVS protein was 2 μg / mL.

6. The porcine delta coronavirus IgA antibody detection kit as described in claim 5, characterized in that, The coating solution was a carbonate buffer solution with a pH of 9.6, and the blocking solution was a PBST solution containing 1% BSA.

7. The porcine delta coronavirus IgA antibody detection kit as described in claim 6, characterized in that, The kit also includes a negative control, a positive control, a horseradish peroxidase-labeled secondary antibody, a chromogenic solution, a stop solution, a washing solution, and a diluent.

8. The porcine delta coronavirus IgA antibody detection kit as described in claim 7, characterized in that, The secondary antibody is a goat anti-pig IgA antibody.

9. The porcine delta coronavirus IgA antibody detection kit as described in claim 7, characterized in that, The washing solution is a PBS solution containing 0.05% Tween-20 at pH 7.4; the diluent is a 0.01M PBST buffer at pH 7.4 containing 1% BSA and 0.1% liquid biological preservative Proclin-300.

10. The method of using the porcine delta coronavirus IgA antibody detection kit as described in any one of claims 7-9 for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Dilute the negative control, positive control and the serum or milk sample to be tested, add them to the ELISA plate coated with PDCoV S protein, and incubate at 37°C for 1 h; after incubation, wash the ELISA plate with PBST and pat dry; (2) Add diluted horseradish peroxidase-labeled secondary antibody to each well and incubate at 37°C for 40 min; after incubation, wash the microplate with washing solution and pat dry; (3) Add the colorimetric solution to each well and incubate at 37°C for 20 min; (4) Add the stop solution to each well and measure the absorbance of the reaction solution at 450 nm to determine whether the sample contains PDCoV-specific IgA antibody; Result Interpretation: For serum samples, when OD 450 nm A value ≥0.14396 is considered positive (OD). 450 nm A value <0.14396 is considered negative; for milk samples, when OD... 450 nm A value ≥0.33381 is considered positive (OD). 450 nm A value less than 0.33381 is considered negative.

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