Porcine epidemic diarrhea virus antigen, ELISA detection reagent, kit, application and nucleic acid molecule

Abstract

The invention provides an antigen of a porcine epidemic diarrhea virus, an ELISA detection reagent, a kit, application and a nucleic acid molecule. The antigen comprises a PEDV S protein mutant, and the PEDV S protein mutant has an amino acid sequence as shown in SEQ ID NO.3; the ELISA detection reagent comprises the antigen; the ELISA detection kit comprises the ELISA detection reagent, and the ELISA detection kit comprises the ELISA detection reagent. According to the invention, the mutated PEDV S protein is used as an antigen for ELISA detection, so that the mutant PEDV S protein has excellent effects in the aspects of specificity, repeatability and the like, and the sensitivity of the mutant PEDV S protein is obviously superior to that of a wild type PEDV S protein.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an antigen, an ELISA detection reagent, a kit, an application and a nucleic acid molecule of porcine epidemic diarrhea virus. Background Art

[0002] Porcine Epidemic Diarrhea Virus (PEDV) is one of the main pathogens causing diarrhea in piglets. Its clinical manifestations are mainly diarrhea, vomiting and dehydration. PEDV is susceptible to pigs of all ages, especially newborn piglets. The morbidity and mortality rates of PEDV after infection are extremely high. In recent years, the morbidity and mortality rates of this disease in my country's pig farming industry have been on the rise, causing huge economic losses to the pig farming industry.

[0003] PEDV S (Spike protein, S) protein is a type I transmembrane glycoprotein embedded in the surface of virus particles. It is responsible for the binding of the virus to specific receptors on the cell surface and mediates the cell invasion of the virus. It has good immunogenicity. The antibody detection kit developed for this protein can be used for the monitoring and diagnosis of PEDV antibodies in pig herds. The preparation of S protein with a conformation similar to that of natural virus particles is the key to the development of antibody detection kits.

[0004] The Chinese invention patent application with publication number CN104155454A discloses an ELISA kit for detecting antibodies to the epidemic strain of porcine epidemic diarrhea virus, which uses PEDV S recombinant protein as an antigen to perform ELISA detection on porcine epidemic diarrhea virus antibodies. Although the patent application can detect porcine epidemic diarrhea virus antibodies, the S protein antigen coated therein is prepared by a prokaryotic Escherichia coli expression system and is a truncated form of the S protein, and its detection effect needs to be further improved.

[0005] In view of this, it is necessary to provide an antigen, ELISA detection reagent, kit, application and nucleic acid molecule of porcine epidemic diarrhea virus to solve or at least alleviate the technical defects of how to improve the ELISA detection effect of antibodies against epidemic strains of porcine epidemic diarrhea virus. Summary of the invention

[0006] The main purpose of the present invention is to provide an antigen, ELISA detection reagent, kit, application and nucleic acid molecule of porcine epidemic diarrhea virus to solve the technical problem of how to improve the ELISA detection effect of antibodies against epidemic strains of porcine epidemic diarrhea virus.

[0007] To achieve the above object, the present invention provides an antigen of porcine epidemic diarrhea virus, wherein the antigen comprises a PEDV S protein mutant, and the PEDV S protein mutant has an amino acid sequence as shown in SEQ ID NO.3.

[0008] The present invention also provides a nucleic acid molecule encoding a PEDV S protein mutant, wherein the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.4.

[0009] The present invention also provides a use of any of the above-mentioned antigens in the preparation of an ELISA detection reagent for porcine epidemic diarrhea virus.

[0010] The present invention also provides a use of any of the above-mentioned antigens in the preparation of an ELISA detection kit for porcine epidemic diarrhea virus.

[0011] The present invention also provides a use of any of the above-mentioned antigens in establishing an ELISA detection method for porcine epidemic diarrhea virus antibodies.

[0012] The present invention also provides an ELISA detection reagent for porcine epidemic diarrhea virus, wherein the ELISA detection reagent comprises any of the antigens described above.

[0013] The present invention also provides an ELISA detection kit for porcine epidemic diarrhea virus, and the ELISA detection kit comprises: an enzyme labeling plate coated with any of the above-mentioned antigens.

[0014] Furthermore, the coating concentration of the antigen is 3-5 ng / μL.

[0015] Furthermore, the ELISA detection kit also includes one or more of negative control serum, positive control serum, enzyme-labeled secondary antibody, washing solution, diluent, color developing solution, and stop solution.

[0016] Furthermore, the positive and negative critical value of the ELISA detection kit is 0.424.

[0017] The present invention also provides a use of any of the above-described ELISA detection kits in detecting porcine epidemic diarrhea virus antibodies for non-diagnostic purposes.

[0018] The beneficial effects of the present invention include at least:

[0019] The present invention analyzes the three-dimensional structure of PEDV S protein and finds that the isoleucine (I) and leucine (L) at positions 1076-1077 of PEDV S protein are key amino acid sites for connecting HR1 (Helix region 1) and CH (Central helix) of S2 subunit. 1076 IL 1077 Mutation to 2 prolines ( 1076 PP 1077 ), which can destroy the formation of the secondary structure and obtain the mutant protein PEDV S2P; the S protein prepared by the present invention has a natural trimer similar to the S protein on the surface of the virus particle, and is close to the conformation of the S protein on the surface of the virus; the present invention uses the mutant protein PEDV S2P as an antigen for ELISA detection, which not only has excellent effects in terms of specificity and repeatability, but also has significantly better sensitivity than the wild-type PEDV S protein; therefore, the antigen obtained by the present invention based on the mutation of the PEDV S protein has a better detection effect on porcine epidemic diarrhea virus antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0021] Figure 1 The results of the analysis of the transmembrane region of the PEDV S protein in Example 1 of the present invention; in the figure, transmembrane refers to the transmembrane region; inside refers to the intracellular region; outside refers to the extracellular region;

[0022] Figure 2 This is the plasmid map of pFastBac 1-PEDV-SWT in Example 1 of the present invention;

[0023] Figure 3 The three-dimensional structure diagram of the PEDV S2P protein in Example 1 of the present invention;

[0024] Figure 4 is the plasmid map of pFastBac 1-PEDV-S2P in Example 1 of the present invention;

[0025] Figure 5 The SDS-PAGE results of the PEDV S2P protein in Example 2 of the present invention; in the figure, M is the molecular weight of the standard protein; E1-E4 are the eluates collected after imidazole elution;

[0026] Figure 6 These are the results of the serum antibody recognition sensitivity test of the PEDV S2P and PEDV SWT proteins in Example 4 of the present invention; in the figure, 12 (S2P) is the test result of the serum antibody level corresponding to the coated mutant S2P protein, and 12 (SWT) is the test result of the serum antibody level corresponding to the coated SWT protein (where 12 is the number of the serum during the test).

[0027] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with the implementation methods and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Furthermore, the technical solutions between the various embodiments of the present invention may be combined with each other, but this must be based on the fact that they can be implemented by ordinary technicians in the field. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0030] The present invention provides an antigen of porcine epidemic diarrhea virus, the antigen comprising a PEDV S protein mutant, the PEDV S protein mutant having an amino acid sequence as shown in SEQ ID NO.3. In the present invention, the PEDV S protein mutant corresponds to the PEDV S2P protein in the embodiment. It should be noted that the isoleucine (I) and leucine (L) at positions 1076-1077 of the PEDV S protein are key amino acid sites for connecting the S2 subunit HR1 (Helix region 1) and CH (Central helix). 1076 IL 1077 Mutation to 2 prolines ( 1076 PP 1077 ), which can destroy the formation of secondary structure; the present invention will 1076 IL 1077 Mutation to 2 prolines ( 1076 PP 1077 ), and the mutant protein obtained after modification is used for ELISA detection, and has excellent detection performance. In the present invention, the PEDV S protein mutant is expressed in Sf9 cells.

[0031] In the present invention, the nucleic acid molecule encoding the PEDV S protein mutant has the nucleotide sequence shown in SEQ ID NO. 4; therefore, the present invention also provides a nucleic acid molecule encoding the PEDV S protein mutant, the nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO. 4. In the present invention, the nucleic acid molecule is cloned into the pFastBac 1 vector and expressed in Sf9 cells.

[0032] The present invention also provides a use of any of the above-mentioned antigens in preparing an ELISA detection reagent for porcine epidemic diarrhea virus. The present invention also provides a use of any of the above-mentioned antigens in preparing an ELISA detection kit for porcine epidemic diarrhea virus.

[0033] The present invention also provides an application of any of the above-mentioned antigens in establishing an ELISA detection method for porcine epidemic diarrhea virus antibodies; the application belongs to the establishment of a detection standard and does not belong to a disease detection method.

[0034] The present invention also provides an ELISA detection reagent for porcine epidemic diarrhea virus, wherein the ELISA detection reagent comprises any of the antigens described above.

[0035] The present invention also provides an ELISA detection kit for porcine epidemic diarrhea virus, the ELISA detection kit comprising: an ELISA plate coated with any of the above-mentioned antigens; that is, the kit can also be understood as comprising any of the above-mentioned ELISA detection reagents.

[0036] In the present invention, the coating concentration of the antigen can be 3-5 ng / μL; the method for obtaining the ELISA plate coated with the antigen can include: using a coating solution to dilute the antigen to 3-5 ng / μL to obtain a diluent; then adding the diluent to the reaction wells of the ELISA plate respectively, and incubating; after the incubation is completed, the reaction wells of the ELISA plate are washed to obtain the ELISA plate coated with the antigen; the coating solution contains sodium carbonate and sodium bicarbonate, the concentration of sodium carbonate can be 14-16 mM, and the concentration of sodium bicarbonate can be 33.5-35.5 mM; the incubation can be performed at 2-8°C, and the incubation time can be 10-14h.

[0037] In the present invention, the ELISA detection kit further comprises one or more of negative control serum, positive control serum, enzyme-labeled secondary antibody, washing solution, diluent, color developing solution, and stop solution.

[0038] In the present invention, the enzyme-labeled secondary antibody is goat anti-pig HRP-labeled IgG, and the dilution of the enzyme-labeled secondary antibody can be 1:15000-1:25000; the washing solution can include PBST buffer; the diluent can include PBS solution; the color developing solution can include TMB color developing solution; the stop solution can include a sulfuric acid solution with a concentration of 1.8-2.2M.

[0039] In the present invention, the positive and negative critical value of the ELISA detection kit is 0.424; that is, when the ELISA detection kit is used to perform ELISA detection on the test serum, if the OD value is ≥ 0.424, it is determined to be a positive serum; otherwise, it is determined to be a negative serum.

[0040] The present invention also provides an application of any of the above-described ELISA test kits in detecting porcine epidemic diarrhea virus antibodies for non-diagnostic purposes. In the application of the present invention, the dilution multiple of the serum to be tested is 1:150-1:250, the dilution multiple of the enzyme-labeled secondary antibody is 1:15000-1:25000, the incubation time of the color developing solution is 13-18 minutes, the incubation time of the serum is 27-33 minutes, and the incubation time of the enzyme-labeled secondary antibody is 27-33 minutes.

[0041] The following are specific examples of the present invention:

[0042] Example 1

[0043] Design of PEDV S2P protein:

[0044] 1. The domestic variant strain (GenBank: KC210145) was selected as the reference strain sequence, and the transmembrane region and intracellular region were predicted using TMHMM2.0 software. The extracellular region of the S protein was selected, and the IGκ signal peptide sequence, T4 foldon trimerization motif and His Tag sequence were introduced at the amino and carboxyl termini. The codon sequence was optimized according to the preference of insect cell host codons. The S protein gene sequence was cloned into the pFastBac 1 vector using the EcoRⅠ and XbaⅠ restriction sites and named pFastBac 1-PEDV-SWT. The PEDV S protein obtained after expression and purification was named PEDV SWT protein.

[0045] In this example, TMHMM software was used to predict and analyze the transmembrane region of the PEDV S protein. The results showed that 1-1327aa of the S protein was the extracellular region, 1328-1350aa was the transmembrane region, and 1351-1386aa was the intracellular region ( Figure 1 ); SignalP-5.0 software prediction results showed that 1-25aa was a signal peptide.

[0046] In this example, the PEDV S protein gene was connected to the pFastBac 1 expression vector through the EcoRⅠ and XbaⅠ restriction sites; the pFastBac 1-PEDV-SWT recombinant plasmid expressing the PEDV SWT protein is shown in the following figure: Figure 2 As shown, the plasmid sequencing results were consistent with the target gene sequence.

[0047] The amino acid sequence of the PEDV SWT protein is as follows (SEQ ID NO.1):

[0048]

[0049] The nucleotide sequence encoding the PEDV SWT protein is as follows (SEQ ID NO.2):

[0050]

[0051] 2. The resolved three-dimensional structure of PEDV S protein was downloaded from the PDB database (PDB ID: 6u7k). The amino acid sequences of S proteins of other coronaviruses were compared and the three-dimensional structure of S protein was analyzed by Pymol software to screen out the key amino acid sites that maintain the stability of S protein structure. The multi-site proline (P) mutation strategy was used to design the S protein with stable conformation before fusion. Primers were designed and the amino acid sites were mutated using the rapid site-directed mutagenesis kit to construct the recombinant expression plasmid of PEDV S protein mutant, named pFastBac 1-PEDV-S2P.

[0052] In this example, by analyzing the three-dimensional structure of the PEDV S protein, it was found that the isoleucine (I) and leucine (L) at positions 1076-1077 of the PEDV S protein are key amino acid sites for connecting the S2 subunit HR1 (Helix region 1) and CH (Central helix). 1076 IL 1077 Mutation to 2 prolines ( 1076 PP 1077 ), which can destroy the formation of secondary structure; the protein mutant of PEDV S protein in the present invention is named PEDV S2P protein, and its three-dimensional structure is as follows Figure 3 In this example, the plasmid pFastBac1-PEDV-S2P was successfully constructed by using a site-directed mutagenesis kit; the pFastBac 1-PEDV-S2P recombinant plasmid expressing the PEDV S2P protein is shown in Figure 4 As shown, the plasmid sequencing results were consistent with the target gene sequence.

[0053] The amino acid sequence of PEDV S2P protein is as follows (SEQ ID NO.3):

[0054]

[0055] The nucleotide sequence encoding the PEDV S2P protein is as follows (SEQ ID NO.4):

[0056]

[0057] Example 2

[0058] Expression and purification of PEDV S2P protein:

[0059] 1. Construction of recombinant baculovirus

[0060] The pFastBac 1-PEDV-S2P recombinant plasmid (1 μL) with correct sequencing was transposed into DH10 Bac competent cells, and then plated onto LB agar plates containing Kan (50 μg / mL), Gen (7 μg / mL), Tet (10 μg / mL), IPTG (40 μg / mL), and X-gal (100 μg / mL). After culturing at 37°C for 16 h, a monoclonal white colony was picked, shaken and cultured, and the recombinant Bacmid DNA was extracted using the alkaline lysis method. The positively identified recombinant Bacmid DNA was extracted using Cellfectin TM II transfection reagent was transfected into Sf9 cells. 4 days after transfection, the P1 recombinant baculovirus was harvested when the cells became larger and showed obvious pathological changes. The P1 virus was amplified with an MOI (multiplicity of infection, MOI) of 0.05 to the P2 virus for 2 days. The cell status was observed at all times. When the cells became significantly larger, they were centrifuged at 2,000g for 10 minutes, the P2 virus was harvested, and 2% FBS was added to the P2 virus for aliquoting and storage for protein expression.

[0061] 2. Expression and purification of recombinant proteins

[0062] The amplified P2 virus was inoculated at an MOI of 0.5 and the infection density was 2.0×10 6 cell / mL of Sf9 cells, after expression for 72 hours, the culture product was centrifuged at 2,000g for 15 minutes to collect the culture supernatant; the protein was purified by nickel column affinity chromatography, the supernatant was combined with Ni-NTA filler at room temperature for 30 minutes, and the impurities were eluted with Wash buffer (50mM Tris-HCl, 200mM NaCl, 20mM Imidazole, pH 8.0) with an imidazole concentration of 20mM, and finally the target protein was eluted with Elution buffer (50mM Tris-HCl, 200mM NaCl, 250mM Imidazole, pH 6.0) with an imidazole concentration of 250mM, and the purified protein was identified by SDS-PAGE.

[0063] In this example, the PEDV S2P protein was purified by nickel column affinity chromatography; see Figure 5As shown, the SDS-PAGE results showed that the PEDV S2P protein was successfully purified and the protein molecular weight was consistent with the expected size.

[0064] Example 3

[0065] Antibody ELISA test:

[0066] 1. In this embodiment, the operation process of antibody ELISA detection is as follows:

[0067] 1. Protein coating

[0068] The purified PEDV S2P protein was diluted to the specified concentration using coating solution (15 mM sodium carbonate, 34.5 mM sodium bicarbonate), 100 μL was added to each well of the ELISA plate, sealed with tin foil, and incubated at 4 °C for 12 h; washed 5 times with PBST (0.05% Tween-20), adding 200 μL to each well each time, and washed on a shaker at room temperature for 5 min.

[0069] 2. Closed

[0070] Use PBS solution to dilute BSA powder according to (W / V=3%), dissolve it completely and mix it evenly, add 100 μL to each well, seal with tin foil, and incubate at 37°C for 3 h; wash with PBST (0.05% Tween-20) 5 times, adding 200 μL to each well each time, and wash on a shaker at room temperature for 5 min.

[0071] 3. Incubate with primary antibody

[0072] Use PBST to dilute the serum according to the specified dilution multiple, mix it evenly and centrifuge it, add 100 μL to each well, seal it with tin foil, and incubate it at 37°C; use PBST (0.05% Tween-20) to wash 5 times, adding 200 μL to each well each time, and wash on a shaker at room temperature for 5 minutes.

[0073] 4. Incubate with secondary antibody

[0074] Use PBST to dilute the enzyme-labeled secondary antibody according to the specified dilution multiple, mix it evenly and spin it off, add 100 μL to each well, seal it with tin foil, and incubate it at 37°C; use PBST (0.05% Tween-20) to wash 5 times, adding 200 μL to each well each time, and wash on a shaker at room temperature for 5 minutes.

[0075] 5. TMB incubation

[0076] After TMB returned to room temperature, add 50 μL of TMB to each well, seal with tin foil, and incubate at 37°C in the dark.

[0077] 6. Color development and termination

[0078] Add 50 μL 2M HO 2 SO 4 Solution, stop the reaction, and quickly read the OD of each well of the ELISA plate using a microplate reader 450 Numeric value.

[0079] 2. Determination of the optimal coating concentration of PEDV S2P protein

[0080] Use coating solution to dilute PEDV S2P protein of known concentration to 0.1, 0.25, 0.5, 1, 2, 4 ng / μL, respectively. Add the diluted PEDV S2P protein to the ELISA plate, 100 μL per well, seal with tin foil and incubate at 4°C for 12 h. Keep other conditions of ELISA operation unchanged and measure the OD of positive serum and negative serum. 450 The P / N value of positive serum and negative serum was calculated to determine the optimal coating concentration of PEDV S protein.

[0081] In this example, the protein coating concentration was changed, and other conditions were kept the same. 450 The values ​​are shown in Table 1, and the P / N values ​​of each serum were calculated. The results showed that the P / N value was the highest when the concentration was 4 ng / μL. In this example, P1 and P2 refer to PEDV positive serum, and NC refers to PEDV negative serum.

[0082] Table 1 Determination of the optimal protein coating concentration

[0083]

[0084] 3. Determination of the optimal serum dilution

[0085] The serum was diluted at 1:25, 1:50, 1:100, and 1:200 and mixed thoroughly. After centrifugation, it was added to the ELISA plate coated with PEDVS2P protein, 100 μL per well, sealed with tin foil, and incubated at 37°C. Keeping other conditions of ELISA operation unchanged, the OD of positive serum and negative serum was measured. 450 The P / N value of positive serum and negative serum was calculated to determine the optimal serum dilution.

[0086] In this example, the serum dilution factor was changed, and other conditions were kept the same. 450 The values ​​are shown in Table 2. The P / N values ​​of each serum were calculated. The results showed that the P / N value was the highest when the serum dilution factor was 1:200.

[0087] Table 2 Determination of the optimal dilution factor of serum

[0088]

[0089] 4. Determination of the optimal dilution of enzyme-labeled secondary antibody

[0090] Dilute the enzyme-labeled secondary antibody at 1:5000, 1:10000, 1:20000, and 1:30000 and mix thoroughly. After centrifugation, add 100 μL to each well of the ELISA plate coated with PEDV S2P protein, seal with tin foil, and incubate at 37°C. Keep other conditions of ELISA operation unchanged and measure the OD of positive and negative serum. 450 The P / N value of positive serum and negative serum was calculated to determine the optimal dilution of enzyme-labeled secondary antibody.

[0091] In this example, the dilution multiple of the enzyme-labeled secondary antibody was changed, and other conditions were kept the same. 450 The values ​​are shown in Table 3. The P / N values ​​of each serum were calculated. Through comprehensive comparative analysis of the data, the optimal condition was when the enzyme-labeled secondary antibody dilution factor was 1:20000.

[0092] Table 3 Determination of the optimal dilution factor of enzyme-labeled secondary antibody

[0093]

[0094] 5. Determination of the optimal incubation time for TMB

[0095] Add TMB restored to room temperature to the ELISA plate coated with PEDV S2P protein, 50 μL per well, seal with tin foil and place at 37°C for 5 min, 10 min, and 15 min, respectively. Keep other ELISA operation conditions unchanged and measure the OD of positive and negative serum 450 The P / N value of positive serum and negative serum was calculated to determine the optimal incubation time for TMB.

[0096] In this example, the TMB incubation time was changed, and other conditions were kept the same. 450 The values ​​are shown in Table 4, and the P / N values ​​of each serum were calculated. The results showed that the P / N value was the highest when the TMB incubation time was 15 min.

[0097] Table 4 Determination of optimal incubation time for TMB

[0098]

[0099] 6. Determination of the optimal serum incubation time

[0100] The diluted serum was thoroughly mixed, and after a brief centrifugation, it was added to the ELISA plate coated with PEDV S2P protein, 100 μL per well, sealed with tin foil, and incubated at 37°C for 30 min, 45 min, 60 min, and 90 min, respectively. Keeping other conditions of ELISA operation unchanged, the OD values ​​of positive and negative serum were measured. 450 The P / N value of positive serum and negative serum was calculated to determine the optimal incubation time of serum.

[0101] In this example, the serum incubation time was changed, and other conditions were kept the same. 450 The values ​​are shown in Table 5, and the P / N values ​​of each serum were calculated. Through comprehensive comparative analysis of the data, the best condition was when the serum incubation time was 30 min.

[0102] Table 5 Determination of the optimal incubation time for serum

[0103]

[0104] VII. Determination of the optimal incubation time for enzyme-labeled secondary antibodies

[0105] Mix the diluted enzyme-labeled secondary antibody thoroughly, add it to the ELISA plate coated with PEDV S2P protein after a short spin, 100 μL per well, seal with tin foil and place at 37°C for 30 min, 45 min, and 60 min respectively. Keep other ELISA operation conditions unchanged and measure the OD of positive and negative serum 450 The P / N value of positive serum and negative serum was calculated to determine the optimal incubation time of enzyme-labeled secondary antibody.

[0106] In this example, the incubation time of the enzyme-labeled secondary antibody was changed, and other conditions were kept the same. 450 The values ​​are shown in Table 6, and the P / N values ​​of each serum were calculated. Through comprehensive comparative analysis of the data, the optimal condition was when the enzyme-labeled secondary antibody incubation time was 30 min.

[0107] Table 6 Determination of the optimal incubation time for enzyme-labeled secondary antibodies

[0108]

[0109] 8. Determination of positive and negative critical values

[0110] According to the antibody ELISA detection method established in this example, 72 PEDV-negative pig sera were tested to determine their OD 450 The values ​​were calculated and the OD values ​​of 72 negative sera were obtained. 450 The average value (AV) and standard deviation (SD) of the values ​​are used to calculate the positive and negative critical values ​​according to the statistical formula: average value (AV) + 3*standard deviation (SD).

[0111] The antibody ELISA detection method established in this embodiment adopts the operating process of the first part of this embodiment; and, in the antibody ELISA detection method established in this embodiment, the protein coating concentration is 4 ng / μL, the serum dilution factor is 1:200, the dilution factor of the enzyme-labeled secondary antibody is 1:20000, the TMB incubation time is 15 min, the serum incubation time is 30 min, and the enzyme-labeled secondary antibody incubation time is 30 min.

[0112] According to the antibody ELISA detection method established in this example, 72 PEDV-negative pig sera were tested to determine their OD 450 As shown in Table 7, the OD values ​​of 72 negative pig sera were calculated. 450 The average value (AV) and standard deviation (SD) of the values ​​are calculated according to the statistical formula: average value (AV) + 3*standard deviation (SD), and the critical value is 0.424. If the OD value of the test sample is ≥ 0.424, it is judged as a positive serum; otherwise, it is judged as a negative serum.

[0113] Table 7 Critical value determination

[0114]

[0115] 9. Specificity Test

[0116] Two positive pig sera of PRV, PCV2, CSFV, and PRRSV were selected, for a total of 8 sera. The antibody ELISA detection method established in this example was used for detection, and the OD 450 The specificity of the method was determined by comparing the values ​​with the critical value.

[0117] Two positive sera of PRV, PCV2, CSFV, and PRRSV were selected, for a total of eight sera. The antibody ELISA detection method established in the above example was used for detection, and the OD 450 The S / P value was calculated. As shown in Table 8, the OD values ​​of the positive sera of pseudorabies (PRV), porcine circovirus type 2 (PCV2), classical swine fever (CSFV), and porcine blue ear (PRRSV) were all less than 0.424, so they were judged to be PEDV negative. The test results showed that the established ELISA antibody detection method had good specificity. In this embodiment, PC is PEDV positive pig serum, and NC is PEDV negative pig serum.

[0118] Table 8 Specificity test

[0119]

[0120] 10. Repeatability test

[0121] 1. Intra-batch replication

[0122] 15 porcine serum samples (7 PEDV positive sera and 8 negative sera) were selected and tested using the same batch of ELISA plates coated with PEDVS2P protein and the antibody ELISA detection method established in this example. Duplicate wells were set and the OD values ​​were measured. 450 The average value (AV) and standard deviation (SD) were calculated, and the coefficient of variation (CV) was calculated according to the formula: coefficient of variation CV (%) = standard deviation (SD) / average value (AV) * 100%.

[0123] In this embodiment, according to OD 450 The values ​​were calculated, the average value (AV) and the standard deviation (SD) were calculated, and the coefficient of variation (CV) was calculated according to the formula: coefficient of variation CV (%) = standard deviation (SD) / average value (AV) * 100%. The calculated intra-batch coefficient of variation was 0.000%-0.8929%, which was less than 10%, indicating that the established antibody ELISA method had good intra-batch repeatability and stability; the specific test results are shown in Table 9.

[0124] Table 9 PEDV indirect ELISA intra-batch repeat test

[0125]

[0126] 2. Batch-to-batch replication

[0127] Fifteen porcine serum samples (7 PEDV positive sera and 8 negative sera) were selected and tested using ELISA plates coated with PEDV S2P protein from three different batches using the antibody ELISA detection method established in this example to determine the OD 450 The average value (AV) and standard deviation (SD) were calculated, and the coefficient of variation (CV) was calculated according to the formula: coefficient of variation CV (%) = standard deviation (SD) / average value (AV) * 100%.

[0128] In this embodiment, according to OD 450 The values ​​were calculated, the average value (AV) and the standard deviation (SD) were calculated, and the coefficient of variation (CV) was calculated according to the formula: coefficient of variation CV (%) = standard deviation (SD) / average value (AV) * 100%. The calculated inter-batch coefficient of variation was 0.6692%-7.7462%, which was less than 10%, indicating that the established antibody ELISA method had good inter-batch repeatability and stability; the specific test results are shown in Table 10.

[0129] Table 10 PEDV indirect ELISA intra-batch repeat test

[0130]

[0131] 11. ELISA sample testing

[0132] 146 porcine serum samples tested by the Ingena kit were selected and tested using the antibody ELISA detection method established in the above example to determine their OD 450 The value is obtained and the positive or negative is judged, and the consistency rate with the results tested by the Ingena test kit is analyzed based on the judgment results.

[0133] In this example, the total coincidence rate was 99.32% (145 / 146), of which the positive coincidence rate was 98.61% (71 / 72); and the negative coincidence rate was 100% (74 / 74).

[0134] Example 4

[0135] Sensitivity comparison test:

[0136] In this example, PEDV S2P protein and PEDV SWT protein were coated respectively to test the sensitivity of serum antibody recognition; and PEDV positive serum was diluted 1:100, 1:300, 1:500, 1:1000, 1:2000, 1:5000, 1:10000, and 1:100000 respectively.

[0137] See also Figure 6 As shown, the results of ELISA test showed that the dilution multiple of PEDV positive serum was between 1:100-1:1000, and the OD of S2P protein group was 450 The values ​​were higher than those of the SWT group, indicating that the S2P protein was more sensitive to serum antibodies than the SWT protein.

[0138] The operation process of ELISA detection in this embodiment is the same as that in Example 3; in this embodiment, except for the dilution multiple of serum, the experimental conditions of ELISA are: protein coating concentration is 4 ng / μL, serum incubation time is 30 min, secondary antibody is diluted 1:20000 and incubated for 30 min, and TMB incubation time is 15 min.

[0139] The above technical solutions of the present invention are only preferred embodiments of the present invention, and the patent scope of the present invention is not limited thereto. All equivalent structural changes made by using the contents of the present invention specification and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. An antigen of porcine epidemic diarrhea virus, characterized in that: The antigen includes a PEDV S protein mutant, and the PEDV S protein mutant has an amino acid sequence as shown in SEQ ID NO.

3.

2. A nucleic acid molecule encoding a PEDV S protein mutant, characterized in that: The nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.

4.

3. Use of the antigen as claimed in claim 1 in the preparation of an ELISA detection reagent or an ELISA detection kit for porcine epidemic diarrhea virus.

4. Use of the antigen as claimed in claim 1 in establishing an ELISA detection method for porcine epidemic diarrhea virus antibodies.

5. An ELISA detection reagent for porcine epidemic diarrhea virus, characterized in that: The ELISA detection reagent comprises the antigen as claimed in claim 1.

6. An ELISA detection kit for porcine epidemic diarrhea virus, characterized in that: The ELISA detection kit comprises: an ELISA plate coated with the antigen as claimed in claim 1.

7. The ELISA detection kit according to claim 6, characterized in that The coating concentration of the antigen was 3-5 ng / μL.

8. The ELISA detection kit according to claim 6, characterized in that The ELISA detection kit also includes one or more of negative control serum, positive control serum, enzyme-labeled secondary antibody, washing solution, diluent, color developing solution, and stop solution.

9. The ELISA detection kit according to claim 6, characterized in that The positive / negative critical value of the ELISA detection kit is 0.

424.

10. Use of the ELISA test kit according to any one of claims 6 to 9 for detecting antibodies against porcine epidemic diarrhea virus for non-diagnostic purposes.

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