Indirect ELISA kit for rapidly detecting porcine epidemic diarrhea virus IgA antibody and application thereof

Abstract

The invention discloses an indirect ELISA kit for rapidly detecting a porcine epidemic diarrhea virus IgA antibody and application of the indirect ELISA kit, and belongs to the technical field of immunology. The invention provides a recombinant PEDV-RBD protein. The amino acid sequence of the recombinant PEDV-RBD protein is shown as SEQ ID NO. 3. The indirect ELISA kit for detecting the porcine epidemic diarrhea virus IgA antibody is constructed based on the recombinant PEDV-RBD protein, the PEDV IgA antibody in pig breast milk or serum can be accurately captured for detection, the kit has the advantages of high sensitivity and strong specificity, and a technical means is provided for prevention and control of porcine epidemic diarrhea.

Description

Technical Field

[0001] The invention relates to the technical field of immunology, and in particular to an indirect ELISA kit for rapidly detecting porcine epidemic diarrhea virus IgA antibodies and an application thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDV), posing a serious threat to the global pig industry. PEDV is mainly transmitted through the fecal-oral route. After infection, it can cause severe watery diarrhea, vomiting, dehydration and other symptoms in piglets, with a very high mortality rate. Especially in the cold winter and spring seasons, PEDV spreads very quickly, and pigs of different ages are susceptible to infection, especially young suckling piglets, whose morbidity and mortality rates are extremely high, reaching 100%. Therefore, rapid and accurate detection of IgA antibodies to PEDV in pigs is of great significance for disease prevention and control, immune effect evaluation and vaccine development.

[0003] Currently, the main means of preventing and controlling PED include vaccination and strengthening feeding management. However, the effectiveness of the vaccine depends largely on the antibody level in the pig herd, especially the IgA antibodies in colostrum, which play a vital role in protecting newborn piglets. Therefore, the development of a rapid and accurate PEDVIgA antibody detection method is of great significance for evaluating vaccine effectiveness, formulating immunization programs, and conducting epidemiological surveys. Summary of the invention

[0004] The purpose of the present invention is to provide an indirect ELISA kit for rapid detection of porcine epidemic diarrhea virus IgA antibodies and its application to solve the problems existing in the above-mentioned prior art. The present invention constructs an indirect ELISA kit for detecting porcine epidemic diarrhea virus IgA antibodies based on recombinant PEDV-RBD protein, which can accurately capture PEDVIgA antibodies in pig breast milk or serum for detection, has the advantages of high sensitivity and strong specificity, and provides a technical means for the prevention and control of porcine epidemic diarrhea.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a recombinant PEDV-RBD protein, whose amino acid sequence is shown in SEQ ID NO.3.

[0007] The present invention also provides a gene encoding the above-mentioned recombinant PEDV-RBD protein, and the nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0008] In the present invention, the coding gene of the recombinant PEDV-RBD protein is obtained by PCR amplification using the cDNA of the PEDV HN2021 strain as a template.

[0009] The primer pair for PCR amplification is shown in SEQ ID NO.1-2.

[0010] The reaction system of the PCR amplification is: KOD OneTM PCR Master Mix 25 μL, template DNA 2 μL, primer F 1 μL, primer R 1 μL, ddH2O 21 μL.

[0011] The reaction procedure of the PCR amplification was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 15 s, a total of 32 cycles, complete extension at 72°C for 5 min, and termination at 4°C.

[0012] The present invention also provides the use of the recombinant PEDV-RBD protein in preparing a reagent or a kit for detecting porcine epidemic diarrhea virus IgA antibodies.

[0013] Preferably, the reagent or kit is used to detect porcine epidemic diarrhea virus IgA antibodies in breast milk or serum.

[0014] The present invention also provides an indirect ELISA kit for detecting porcine epidemic diarrhea virus IgA antibodies, wherein the kit contains an ELISA plate coated with the recombinant PEDV-RBD protein.

[0015] Preferably, the coating concentration of the recombinant PEDV-RBD protein is 0.5-2 μg / mL.

[0016] The coating time of the recombinant PEDV-RBD protein is 2 hours, and the coating temperature is 37°C.

[0017] Preferably, the blocking solution used when coating the recombinant PEDV-RBD protein is 2.5% BSA.

[0018] The temperature of the blocking solution during blocking is 4°C and the time is 14-16 hours.

[0019] Preferably, the kit further comprises an HRP-labeled goat anti-swine IgA solution, a diluent, a washing solution, a TMB substrate solution and a stop solution.

[0020] The diluent is a 0.01 M PBS buffer at pH 7.4 containing 10% bovine serum albumin and 0.1% liquid biological preservative Proclin-300.

[0021] The washing solution is 0.01 M PBST buffer with a pH of 7.4 and containing 0.5% Tween 20.

[0022] The stop solution is a H2SO4 solution with a concentration of 2 mol / L.

[0023] Preferably, the concentration of the HRP-labeled goat anti-swine IgA solution is 12.5-50 ng / mL.

[0024] Preferably, when the kit is used, the TMB substrate solution is added and the reaction is carried out for 15-30 minutes, and the stop solution is added to terminate the reaction.

[0025] The detection principle of the kit is as follows: the sample to be tested is co-incubated with an ELISA plate coated with a recombinant PEDV-RBD protein in the kit, the specific antibodies in the serum are accurately captured by the PEDV-RBD protein, and the HRP-labeled sheep anti-swine IgA antibody is used as a detection antibody to combine with the specific antibodies captured by the PEDV-RBD protein in the ELISA plate to form a "PEDV-RBD protein-PEDV-RBD specific IgA antibody-enzyme-labeled antibody" trimer complex, and a color reaction occurs after adding TMB substrate solution, and the result can be directly determined by measuring the absorbance value.

[0026] The present invention discloses the following technical effects:

[0027] The present invention uses porcine epidemic diarrhea virus HN2021 strain to prepare recombinant PEDV-RBD protein, and establishes an indirect ELISA kit for detecting porcine epidemic diarrhea virus IgA antibodies based on the recombinant protein. The overall reaction time is strictly controlled within 1.5-2h under the condition of ensuring high sensitivity and strong specificity, which greatly shortens the operation time and improves the detection efficiency. The present invention improves the sensitivity of the kit by optimizing the parameter conditions of different reagents in the kit. When OD 450nm ≥0.3195, the sample to be tested is judged to be positive, that is, the sample to be tested contains porcine epidemic diarrhea virus IgA antibody; when OD 450nm When the value is less than 0.3195, it is judged as negative, that is, the sample to be tested does not contain porcine epidemic diarrhea virus IgA antibody. The invention is convenient and easy to operate, efficient and low in cost, and the detection specificity reaches 100%, providing a technical means for the prevention and control of porcine epidemic diarrhea. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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 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 these drawings without paying creative work.

[0029] Figure 1 The results of double enzyme digestion verification of the recombinant plasmid pET-32a-PEDV-RBD are shown in Figure 1. Lane M: Marker; Lane 1: Undigested plasmid; Lane 2: pET32-PEDV-RBD plasmid identified by double enzyme digestion with BamH I and EcoR I;

[0030] Figure 2 The results of SDS-PAGE analysis of purified PEDV-RBD protein, wherein lane M: Marker; lane 1: purified RBD protein;

[0031] Figure 3 The results of Western blotting of purified PEDV-RBD protein, where lane M: Marker; lane 1: purified RBD protein;

[0032] Figure 4 This is the optimization result of the coating concentration of recombinant PEDV RBD protein;

[0033] Figure 5 This is the optimization result of the blocking solution type;

[0034] Figure 6 This is the optimization result of the dilution of enzyme-labeled secondary antibody;

[0035] Figure 7 This is the result of optimizing the color development time;

[0036] Figure 8 ROC curve diagram for determining the critical value;

[0037] Fig. 9 It is the specific test result of the indirect ELISA test method;

[0038] Fig.10 This is the sensitivity test result of the indirect ELISA detection method. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] The technical solution of the present invention is as follows:

[0045] Primers were designed based on the RBD gene sequence of the PEDV HN2021 strain published on GenBank, and the RBD gene was amplified, and the RBD gene was connected to a vector to obtain a recombinant vector; the obtained recombinant vector was transduced into Escherichia coli to construct a recombinant bacterium; after the recombinant bacterium was expanded, IPTG was induced for expression, and the recombinant bacterium after induced expression was crushed and purified to isolate the recombinant PEDV-RBD protein.

[0046] The recombinant PEDV-RBD protein was used as the antigen for coating and an indirect ELISA detection method was established. The indirect ELISA detection method was established by optimizing the antigen coating amount, blocking solution type, secondary antibody dilution and color development time in the detection method. Finally, the established indirect ELISA detection method was verified in terms of specificity, sensitivity, repeatability and other aspects.

[0047] "Primer" refers to a macromolecule with a specific nucleotide sequence that is stimulated to synthesize at the initiation of nucleotide polymerization, and is usually connected to the reactant in the form of hydrogen bonds. Primers are usually two artificially synthesized oligonucleotide sequences. In some embodiments of the present invention, the primers are designed using Primmer5.0 software.

[0048] "Vector" refers to a self-replicating DNA molecule that transfers DNA fragments (target genes) to recipient cells in genetic engineering recombinant DNA technology. In some embodiments of the present invention, the vector can be selected from bacterial plasmids, bacteriophages, and animal and plant viruses; in other embodiments of the present invention, the vector is selected from bacterial plasmids, and further selected from pET-32a vectors.

[0049] "Recombinant vector" refers to a DNA sequence that can be inserted into an exogenous DNA sequence and replicate autonomously, usually composed of two or more DNA fragments from different sources. In some embodiments of the present invention, the recombinant vector is obtained by recombining the RBD gene with the vector, and the recombinant connection method can be selected from the enzyme ligation method, homologous recombination method and overlap extension PCR method; in other embodiments of the present invention, the recombinant connection method is selected from the enzyme ligation method, and is further selected from the method of using EcoI and HindIII as restriction sites and T4 ligase to connect.

[0050] "Transduction" refers to the process of transferring DNA or RNA from one cell to another cell. It is one of the ways to transfer genetic material between bacteria. In some embodiments of the present invention, the transduction method can be selected from heat shock method, electrotransformation method, electroporation method, microinjection, gene gun and virus-mediated method; in other embodiments of the present invention, the transduction method is selected from heat shock method.

[0051] "Induced expression" refers to the process in which exogenous genes are activated or enhanced in expression under certain conditions. This expression mode usually depends on specific inducers, such as metabolites, chemicals or other environmental signals. In some embodiments of the present invention, the inducer can be selected from chemical substances; in other embodiments of the present invention, the inducer is selected from IPTG (isopropyl-β-D-thiogalactopyranoside).

[0052] The principle of the "indirect ELISA detection method" is to connect the antigen to a solid phase carrier, and the antibody to be tested in the sample is combined with it to form a solid phase antigen-test antibody complex, and then an enzyme-labeled secondary antibody (antibody against the antibody to be tested) is combined with the antibody in the solid phase immune complex to form a solid phase antigen-test antibody-enzyme-labeled secondary antibody complex. The color development degree after adding the substrate can be quantitatively measured by visual inspection or spectrophotometer to determine the content of the antibody to be tested. In some embodiments of the present invention, the enzyme-labeled secondary antibody can be selected from enzyme-labeled sheep anti-swine IgA antibodies, and further selected from HRP-labeled sheep anti-swine IgA antibodies.

[0053] The source information of some reagents and biological materials involved in the embodiments of the present invention has been stated, but it is not intended to limit the technical solution of the present invention and they can also be purchased through conventional channels; the reagents and biological materials without source information are all commercially available products and can be purchased through conventional channels.

[0054] Example 1 Preparation of recombinant PEDV-RBD protein

[0055] 1. Preparation of PEDV-RBD protein

[0056] a. According to the RBD gene sequence of PEDV HN2021 strain (GenBank accession number: OR707084.1), a pair of primers was designed using Primmer5.0 software. The primer information is shown in Table 1. The cDNA of the correctly sequenced PEDV HN2021 strain preserved by the Animal Molecular Pathogens Team of Henan Agricultural University was used as a template, and the primers PEDV RBD F and PEDVRBD R were used to PCR amplify the RBD gene. The PCR reaction system is shown in Table 2. After the mixed system is centrifuged and mixed, it is placed in the PCR instrument for amplification. The reaction procedure is: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 15s, a total of 32 cycles, 72℃ thorough extension for 5min, and 4℃ end. After the amplification is completed, 6μL of PCR product is taken for detection by 1% agarose gel electrophoresis and product gel recovery is performed. The purified double-enzyme PCR product and the pET-32a empty vector were double-enzyme digested with restriction endonucleases EcoR I and Hind III. Then, the recombinant plasmid pET-32a-PEDV-RBD was obtained by ligating with T4 ligase. The obtained recombinant plasmid pET-32a-PEDV-RBD was double-enzyme digested with BamH I and EcoR I, and the digestion product was verified by gel electrophoresis. The verification results are shown in Figure 1 As shown, it shows that the recombinant plasmid was successfully constructed.

[0057] The recombinant plasmid pET-32a-PEDV-RBD was transferred into the DH5α strain, and the recombinant strain BL21-N was screened using LB medium agar plates containing ampicillin (50 μg / mL), and the bacteria were enriched in LB liquid medium. After the enrichment, the strains were freeze-dried and stored at -80°C for future use.

[0058] Table 1 Primer information

[0059]

[0060]

[0061] Table 2 PCR reaction system

[0062] KODOneTMPCRMasterMix 25μL Template DNA 2μL Primer F 1μL Primer R 1μL <![CDATA[ddH2O]]> 21μL Total volume 50μL

[0063] b. Take the recombinant E. coli BL21-N expressing PEDV-RBD protein and streak it on LB agar plate (containing 50 μg / mL ampicillin), culture it at 37℃ for 16-20h, pick a single colony and inoculate it in LB liquid culture medium (containing 50 μg / mL ampicillin), and culture it at 37℃, 180r / min until OD 600nm After reaching 0.7-0.8, add IPTG with a final concentration of 0.1mmol / L aseptically, and induce at 180r / min in a shaker at 37℃ for 6-8 hours. Then, ultrasonic lysis is performed in an ice bath to obtain PEDV-RBD protein. SDS-PAGE detection shows a band of about 55-70KDa, indicating successful induced expression.

[0064] c. Collect the successfully induced bacterial solution and centrifuge it to collect the bacteria. Wash them three times with pre-cooled PBS or Buffer A solution and wash them with an appropriate amount of PBS or Buffer A solution (50 mmol L -1 Tris, 0.5 mmol L -1 EDTA, 50 mmol L -1 Resuspend the cells with NaCl. Use a pressure crusher to crush 4-5 times, centrifuge, and remove the supernatant. In an ice water bath, use an appropriate amount of inclusion body washing solution (1 mol L -1 Tris-HCL, 1% Triton X-100, 1mmol L -1Wash the precipitate with EDTA (concentrated to 500mL), stir and centrifuge thoroughly, discard the supernatant, repeat 3 times; wash the precipitate with 2mol / L urea in an ice water bath, stir thoroughly for 10min, discard the supernatant; wash the precipitate with 1mol / L sodium chloride solution in an ice water bath, stir thoroughly for 10min, centrifuge and discard the supernatant; protein denaturation treatment: denature the inclusion body protein with 8mol / L urea and 0.5M DTT at 4℃ for 2h; protein renaturation treatment: retain the supernatant after centrifugation, add 1mL of oxidized glutathione to the denatured protein solution, then add 1mL of reduced glutathione, and then renature in an ice bath for 2h. The above renatured protein solution was dialyzed with 4M urea at 4℃ overnight, and the dialysate (4M urea) was replaced once the next day for 12h, and then replaced with 2M urea for overnight dialysis.

[0065] d. After purification, a highly pure recombinant PEDV-RBD protein was obtained, which was observed as a single clear band by SDS-PAGE. Figure 2 The results of Western blotting are shown in Figure 3 As shown, the protein concentration was 2 mg / mL as determined by the Bradford method.

[0066] The amino acid sequence of the recombinant PEDV-RBD protein is shown in SEQ ID NO.3, and the coding gene sequence of the protein is shown in SEQ ID NO.4.

[0067] SEQ ID NO.3:

[0068] GISYQPCTANCIGYAANVFATEPNGHIPEGFSFNNWFLLSNDSTLVHGKVVSNQPLLVNCLLAIPKIYGLGQFFSFNQTIDGVCNGAAVQRAPEALRFNINDSSVILAEGSIVLHTALGTNFSFVCSNSSNPHLATFAIPLGATQVPYYCFFKVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYLHLGLLDAVTINFTGHGTDDDVSGFWTIASTNFVDALIEVQRTTIQRILYCDDPVSQLKCSQVAFDLDDGFYTISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGGHSGANLIASATTINGFSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSFLAGVYYTSDS。

[0069] SEQ ID NO.4:

[0070]

[0071] Example 2 Preparation of PEDV Antibody Positive Quality Control Serum

[0072] Healthy pigs aged 3 to 5 weeks with negative PEDV antigen and antibody tests were used and recombinant PEDV HN2021 inactivated virus (TCID 50 =10 7 / 0.1mL) was mixed with Seppic 206 adjuvant and then emulsified to prepare the vaccine. The pigs were immunized twice in the neck muscle, and the serum was collected after the test was qualified. The separated positive serum was prepared to obtain the positive control serum. The prepared positive control serum was tested, and the results showed that the prepared positive control serum had aseptic growth, and the titer of the PEDV neutralization test was repeated 5 times and was ≥1:8, indicating that the prepared positive control serum was of qualified quality and could be used for subsequent research.

[0073] Example 2 Establishment of indirect ELISA detection method

[0074] 1. Preliminary establishment of detection methods

[0075] (1) Add the diluted 0.25-4μg / mL PEDV-RBD protein suspension to the ELISA wells, incubate in a 37°C incubator for 2 hours, remove the supernatant, wash with PBST three times and discard the supernatant. Add a certain amount of blocking solution (2.5g BSA dissolved in 95mL PBS, dilute to 100mL with purified water) and block at 4°C for 14-16 hours to occupy the gaps on the surface of the ELISA wells that are not bound to the PEDV-RBD protein, and prepare the ELISA plate.

[0076] (2) Dilute the milk or serum sample 64 times with a diluent and mix thoroughly to obtain a sample solution; the diluent is a 0.01M pH 7.4 PBS buffer containing 10% bovine serum albumin and 0.1% liquid biological preservative Proclin-300.

[0077] (3) Add 100 μL of diluent, standard solution, negative standard, positive standard, and sample solution to the wells containing the enzyme label, respectively, as the blank group, standard group, negative control group, positive control group, and experimental group, and perform a duplicate well experiment, and then incubate at 37°C for 60 min.

[0078] The above-mentioned positive standard is a PEDV antibody positive quality control serum (obtained after infecting pigs with PEDV HN2021 strain, with a titer of 1 / 400), which is dissolved in sample diluent before use.

[0079] The above negative standard is a negative control serum containing uninfected PEDV antibodies, which is dissolved in sample diluent before use.

[0080] (4) Discard the unbound sample solution and rinse three times with 200 μL of washing solution; the washing solution is 0.01 M PBST buffer with pH 7.4 containing 0.5% Tween 20.

[0081] (5) Use diluent to dilute the enzyme-labeled secondary antibody (HRP-labeled goat anti-swine IgA antibody, purchased from abcam) with an original concentration of 1 mg / mL by 10,000-80,000 times, add 100 μL of the diluted enzyme-labeled secondary antibody to each well, and repeat step (4) for washing.

[0082] (6) Add 100 μL of TMB substrate solution (Sangon Biotechnology (Shanghai) Co., Ltd.) to each well and incubate at 37°C in the dark for 15 min.

[0083] (7) Add 50 μL of stop solution (2 mol / L H2SO4 solution) to each well and measure the absorbance of each centrifuge tube at a wavelength of 450 nm using an ELISA reader within 30 min.

[0084] (8) Result determination: 150 samples of PEDV serum were tested and the ROC curve was drawn based on the results. The largest Youden index was selected as the basis. The corresponding sensitivity and specificity were 92.2% and 98.0%, respectively. The corresponding absorbance value was 0.3195. When OD 450nm When OD ≥0.3195, the serum sample is considered positive. 450nm <0.3195 is negative.

[0085] 3. Optimize different conditions in the detection method

[0086] (1) Determination of the optimal antigen coating amount

[0087] Use coating solution to dilute PEDV-RBD protein to the following concentrations: 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL, 0.25μg / mL, add the diluted protein solution of different concentrations to the enzyme-labeled wells, 100μL per well, and then place at 37℃ for 2h. Then perform the routine steps, and after the color development is completed, select the best protein coating concentration by P / N value. The results are as follows: Figure 4 As shown in the figure, the concentration of PEDV-RBD protein is determined to be 2 μg / mL when the P / N value is the highest, so the concentration of the required coating antigen selected in the present invention is 2 μg / mL.

[0088] (2) Determination of the optimal blocking solution

[0089] Since different blocking solutions have different effects and the blocking effects of different blocking solution concentrations are different, the ELISA plate coated with the best protein concentration will be blocked. Use a determined antigen coating concentration, after overnight coating, select the blocking solution: 1% BSA, 2.5% BSA, 5% BSA, 1% skim milk, 2.5% skim milk, 5% skim milk, add 200μL to each well. Place at 4℃ for blocking for 14-16h. After the blocking is completed, perform subsequent routine operations, and finally select the best blocking solution based on the P / N value. The results are as follows Figure 5 As shown, it was found from the calculated P / N that the P / N value of 2.5% BSA was the highest, so 2.5% BSA was selected as the optimal blocking condition.

[0090] (3) Determination of the optimal dilution of the optimal secondary antibody

[0091] The enzyme-labeled antibody binds to the enzyme-labeled wells through the antigen-antibody binding reaction, which affects the test results. Therefore, the dilution of the enzyme-labeled antibody is optimized. The secondary antibody is diluted at a ratio of 1:10000, 1:20000, 1:40000, 1:60000, and 1:80000, and each dilution is repeated for 8 wells. After completing the subsequent experiment, the OD is measured with an enzyme-labeled instrument. 450nm The optimal dilution of the secondary antibody is selected with the maximum P / N value. Figure 6 As shown, the P / N value is the highest when the antibody is diluted 40,000 times, so the enzyme-labeled antibody dilution of 1 / 40,000 is selected as the best reaction condition.

[0092] (4) Determination of the optimal color development time

[0093] Horseradish peroxidase (HRP) can catalyze the oxidation of substrate TMB to oxTMB, which shows a blue reaction. The reaction is terminated by adding a stop solution. Therefore, the color development time is selected to be 5, 10, 15, 20, and 30 minutes, and the OD is measured. 450nm To calculate the P / N value, select the best color development time. Figure 7 As shown, the optimal color development time of the present invention is 15 minutes.

[0094] (5) Determination of critical value

[0095] According to the optimized optimal reaction conditions (the optimal coating antigen concentration is 2 μg / mL, the optimal blocking solution is 2.5% BSA, the blocking time is 2 h, the optimal enzyme-labeled antibody (1 mg / mL) dilution is 1 / 40000, and the optimal color development time is 15 min), an indirect ELISA method was established to detect 150 PEDV clinical sera, and the ROC curve was drawn accordingly, as shown in Figure 2. Figure 8As shown, the largest Youden index was selected as the basis, where the corresponding sensitivity and specificity were 92.2% and 98.0% respectively, and the corresponding absorbance value was 0.3195, the area under the curve was 0.9845, and the 95% confidence interval was 0.9684 to 1.000. Therefore, when OD 450nm When it is greater than 0.3195, it is considered positive, otherwise it is negative.

[0096] Example 3 Validation of the indirect ELISA detection method

[0097] 1. Specificity experiment

[0098] The method established in Example 2 was used to detect the positive sera of PDCoV, TGEV, CSFV, PRV, PEDV, TGEV, PRRSV, PCV2, and PSV (preserved by the Animal Molecular Pathogenesis Team of Henan Agricultural University, with a titer of 1 / 400). At the same time, a standard positive control group and a standard negative control group were set up, and the specificity of the method was determined by measuring the absorbance value.

[0099] The results are as follows Fig. 9 As shown, the detection method can specifically detect PEDV-RBD antibodies and cannot recognize other antibodies. Therefore, the detection method established by the present invention has good specificity.

[0100] 2. Sensitivity test

[0101] The PEDV standard positive sample (original titer is 1 / 400) was tested by multiple dilution, such as 1:2, 1:4, 1:8, ..., until the reaction is negative, and each dilution was repeated 3 times. Fig.10 As shown, when the PEDV positive samples were diluted to 1:1024 (V / V), the results were all positive, while when the positive sample dilution was 1:2048 (V / V), the detection result was negative. Therefore, the detection method established by the present invention has a high sensitivity.

[0102] 3. Intra-batch and inter-batch repeatability test:

[0103] Under the same reaction conditions as above, 10 samples to be tested (5 positive samples and 5 negative samples) were randomly selected, and the same batch of purified recombinant protein was used to coat the ELISA plate. The 10 sera were tested with the established indirect ELISA method for 3 consecutive days for intra-batch repeatability test; 3 batches of purified recombinant protein were used to coat the ELISA plate, and the 10 sera were tested with the established indirect ELISA method for 3 consecutive days. Each serum was repeated 3 wells for inter-batch repeatability test. Calculate OD 450nm The mean, standard deviation and coefficient of variation were calculated to verify the repeatability of the method.

[0104] As shown in Table 3, the coefficients of variation of intra-batch and inter-batch repeated tests were 1.895%-7.637% and 3.233%-9.6367%, respectively, both less than 10%. The detection method of the present invention has good repeatability and stability.

[0105] Table 3 Test results

[0106]

[0107]

[0108] 4. Clinical application

[0109] (1) 92 samples of pig sera randomly collected from the clinic were compared and identified using commercially available kits, and it was determined that 43 of them were positive and 49 were negative.

[0110] The serum samples identified above were tested by the detection method of the present invention, and the results are shown in Table 4, showing that 56 positive sera and 36 negative sera were detected. The detection results of the detection method of the present invention were consistent with the commercially available kit (IDEXXPEDVIgA antibody detection kit) at a rate of 85.9%.

[0111] Table 4 Test results

[0112]

[0113] (2) 92 samples of pig milk randomly collected from the clinic were compared and identified using a commercially available kit, and it was determined that 91 samples were positive and 1 sample was negative.

[0114] The milk samples identified above were tested by the detection method of the present invention, and the results are shown in Table 5, showing that 79 positive milk samples and 13 negative milk samples were detected. The detection results of the detection method of the present invention were consistent with the commercially available kit (IDEXXPEDVIgA antibody detection kit) at a rate of 86.9%.

[0115] Table 5 Test results

[0116]

[0117] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A recombinant PEDV-RBD protein, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

2. The gene encoding the recombinant PEDV-RBD protein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

3. Use of the recombinant PEDV-RBD protein according to claim 1 in the preparation of a reagent or kit for detecting porcine epidemic diarrhea virus IgA antibodies.

4. The use according to claim 3, characterized in that: The reagent or kit is used for detecting porcine epidemic diarrhea virus IgA antibodies in breast milk or serum.

5. An indirect ELISA kit for detecting porcine epidemic diarrhea virus IgA antibodies, characterized in that: The kit contains an ELISA plate coated with the recombinant PEDV-RBD protein according to claim 1.

6. The kit according to claim 5, characterized in that The coating concentration of the recombinant PEDV-RBD protein is 0.5-2 μg / mL.

7. The kit according to claim 5, characterized in that The blocking solution during the coating of the recombinant PEDV-RBD protein is 2.5% BSA.

8. The kit according to claim 5, characterized in that The kit also includes HRP-labeled goat anti-pig IgA solution, diluent, washing solution, TMB substrate solution and stop solution.

9. The kit according to claim 8, characterized in that The concentration of the HRP-labeled goat anti-swine IgA solution is 12.5-50 ng / mL.

10. The kit according to claim 8, characterized in that When the kit is used, the TMB substrate solution is added and reacted for 15-30 minutes, and the stop solution is added to terminate the reaction.

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