Immunomagnetic beads coated with recombinant PEDV S1 protein and application thereof

Abstract

The present invention discloses a porcine epidemic diarrhea virus S1 protein and its application. The present invention also discloses a magnetic particle chemiluminescence method for detecting PEDV IgG antibody and PEDV IgA antibody respectively using PEDV S1 recombinant protein as antigen and assembling a kit, which can effectively perform serological diagnosis and investigation on the currently popular PEDV virus, judge the PEDV antibody level and natural infection status in the pig herd, and provide a rapid and simple detection method for the prevention and treatment of PEDV virus.

Description

Technical Field

[0001] The invention belongs to the field of veterinary biotechnology, and particularly relates to immunomagnetic beads coated with recombinant PEDV S1 protein and application thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease caused by infection with porcine epidemic diarrhea virus (PEDV), characterized by watery diarrhea, vomiting, and dehydration. The mortality rate of suckling piglets infected with PEDV is as high as 90% to 100%, and it can be infected in all age groups. The new and highly virulent PEDV strain that spread rapidly in 2010 caused the death of a large number of piglets, with a mortality rate close to 100%, causing huge economic losses to the pig industry.

[0003] Serological tests for PEDV mainly include neutralization test (SNT), indirect hemagglutination assay (IHA), indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA), of which only ELISA is suitable for high-throughput detection. However, the ELISA method for clinical detection of PEDV antibodies, especially specific IgA antibodies, has low sensitivity and a narrow detection range, that is, it is not capable of distinguishing high, medium and low antibody levels, which is not conducive to diagnosis and evaluation of vaccine immune effects.

[0004] Magnetic particles (MP) are a new type of nanoparticle material. Chemical functional groups such as -OH, -COOH and -NH2 can be synthesized on their surface, which can couple with biologically active proteins. Magnetic particles have a high surface mass ratio and can bind more antigens to participate in immune reactions. Magnetic particles have a small particle size ranging from a few hundred nanometers to a few microns, and can be evenly suspended in the reaction system. Magnetic particles also have superparamagnetism, and gather together under an external magnetic field, and can be evenly dispersed in the medium after the magnetic field is removed. Compared with solid-phase microplates, magnetic particles are more ideal as immunoassay carriers. When combined with chemiluminescent immunoassay technology, they can greatly increase the specificity and sensitivity of the detection system and shorten the detection time. They have the potential to be applied to fully automatic detection systems.

[0005] Magnetic microparticle chemiluminescence detection technology is superior to traditional ELISA in terms of detection time and price. Currently, there are no reports on magnetic microparticle chemiluminescence detection methods for PEDV antibodies. Summary of the invention

[0006] In order to solve one of the above-mentioned technical problems existing in the prior art, the present invention provides an immunomagnetic bead coated with a recombinant PEDVS1 protein and an application thereof.

[0007] In a first aspect, the present invention provides an immunomagnetic bead coated with a recombinant PEDV S1 protein, wherein the recombinant PEDV S1 protein has an amino acid sequence shown in SEQ ID NO:1.

[0008] In a second aspect, the present invention provides a nucleic acid molecule encoding the recombinant PEDV S1 protein, wherein the nucleic acid molecule comprises:

[0009] (1): the nucleotide sequence shown in SEQ ID NO: 2; or

[0010] (2): A sequence complementary to the nucleotide sequence of (1); or

[0011] (3): A sequence having 95% or more homology with the nucleotide sequence of (1).

[0012] In some embodiments, the nucleotide sequence encoding the recombinant PEDV S1 protein has the nucleotide sequence shown in SEQ ID NO:2.

[0013] In some embodiments, the nucleotide sequence encoding the recombinant PEDV S1 protein is shown as SEQ ID NO:2.

[0014] In a third aspect, the present invention provides an expression system, which is an insect rod-shaped expression system.

[0015] In some embodiments, the insect baculovirus expression system is Sf9 insect cells.

[0016] Compared with E. coli expression, the expression system of the present invention enables the recombinant protein to be correctly folded and modified after translation, making the expressed protein more similar to the natural protein and having higher activity (including antigenicity, immunogenicity, function) and stability.

[0017] In a fourth aspect, the present invention provides use of the recombinant PEDV S1 protein in preparing a kit for detecting porcine epidemic diarrhea virus infection.

[0018] In some embodiments, the antibodies or active fragments against PEDV S1 protein include PEDV IgG antibodies and PEDV IgA antibodies.

[0019] In a fifth aspect, the present invention provides use of the recombinant PEDV S1 protein in preparing a diagnostic reagent for detecting porcine epidemic diarrhea virus infection.

[0020] In a sixth aspect, the present invention provides a kit for detecting porcine epidemic diarrhea virus infection, the kit comprising immunomagnetic beads coated with recombinant PEDV S1 protein.

[0021] In a seventh aspect, the present invention provides use of the recombinant PEDV S1 protein in quality control of a detection kit for porcine epidemic diarrhea virus infection.

[0022] In an eighth aspect, the present invention provides use of the recombinant PEDV S1 protein in the preparation of a porcine epidemic diarrhea virus vaccine.

[0023] In a ninth aspect, the present invention provides a kit, comprising an antibody that specifically binds to the recombinant PEDV S1 protein.

[0024] In some embodiments, the kit further comprises immunomagnetic beads, enzyme-labeled secondary antibodies, positive controls and negative controls, sample diluents, color development solutions, washing solutions, and magnetic plates.

[0025] In some embodiments, the enzyme-labeled secondary antibody is a rabbit anti-pig IgG or IgA antibody.

[0026] The beneficial effects of the present invention include at least one of the following:

[0027] The recombinant PEDV S1 protein of the invention has good antigenic reactivity.

[0028] The present invention utilizes an insect rod-shaped expression system to obtain a recombinant PEDV S1 protein, and uses the protein as an antigen to establish a magnetic particle chemiluminescence method for detecting PEDV antibodies (e.g., PEDV IgG antibodies and PEDVIgA antibodies) and assembles the method into a kit. The method can effectively perform serological diagnosis and investigation on the currently prevalent PEDV virus, determine the PEDV antibody level and the status of natural infection in the pig herd, provide a rapid and simple detection method for the prevention and treatment of the PEDV virus, and can also be used for the analysis and evaluation of the immune effect of clinical PEDV vaccines.

[0029] The present invention provides a magnetic particle chemiluminescence kit that is rapid, accurate, simple, and suitable for large-scale detection of porcine epidemic diarrhea virus antibodies. The magnetic particle chemiluminescence detection technology has the advantages of high detection sensitivity, wide detection range, and high detection efficiency. It can be used for porcine epidemic diarrhea virus antibody detection and virus infection diagnosis, as well as vaccine immune effect evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1The SDS-PAGE analysis of PEDV S1 protein is shown, wherein M: protein molecular weight standard; 1: expression product; 2: flow-through; 3: washing solution; 4: eluate.

[0031] Figure 2 The PEDV-IgG kit calibration curve is shown.

[0032] Figure 3 The PEDV-IgA kit calibration curve is shown. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0034] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0035] Example 1. Preparation and identification of recombinant PEDV S1 protein

[0036] 1) Construction of recombinant expression plasmid: Using wild-type PEDV S1 protein as a template, amplify by PCR, and add a histidine tag (6*His) at the N-terminus during gene cloning. Artificially synthesize the PEDV S1 protein gene into the PfastBac1 plasmid and name it pFastBac1-PEDV-S1. The amino acid sequence of the recombinant protein expressed by the recombinant plasmid is:

[0037] SEQ ID NO:1

[0038]

[0039]

[0040] The recombinant PEDV S1 protein of the present invention contains the main neutralizing epitope of the virus, can stimulate the body to produce neutralizing antibodies, and can be used as a target antigen for PED serological diagnosis.

[0041] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 is:

[0042] SEQ ID NO:2

[0043]

[0044] 2) Preparation of recombinant protein bacmid: Transform the pFastBac1-PEDV-S1 recombinant plasmid into MAX DH10Bac TM In competent cells, recombinant bacmids are generated.

[0045] 3) Construction of recombinant baculovirus: The recombinant bacmid DNA was transfected into the Sf9 insect cell line to generate recombinant baculovirus.

[0046] 4) Expression of recombinant protein: refer to Baculovirus expression system operation manual, the recombinant baculovirus was inoculated into Sf9 cells and cultured for two generations. The virus solution was collected and inoculated into High Five cells to express the PEDV-S1 recombinant protein.

[0047] 5) Purification of recombinant protein: The recombinant protein PEDV-S1 was purified by Ni 2+ Purification by affinity chromatography.

[0048] 6) Identification of recombinant protein:

[0049] The purified PEDV S1 protein was subjected to SDS-PAGE electrophoresis, and the results showed that a specific protein band appeared at about 110 kDa. Figure 1 shown.

[0050] Example 2. Preparation of a magnetic microparticle chemiluminescent kit for detecting PEDV antibodies

[0051] 1) Horseradish peroxidase-labeled rabbit anti-swine IgG or IgA antibody: purchased from Seracare (KPL).

[0052] 2) Preparation of immunomagnetic beads: After thoroughly mixing the magnetic beads with a mixer, transfer 2 mg of magnetic beads to a centrifuge tube with a pipette, place the centrifuge tube on a magnetic rack to completely separate the solid and liquid, and remove the supernatant. Mix and wash the magnetic beads 2-3 times with coupling buffer (15mM MES PH=5.5). Add 50μL activation buffer (10mg / mL EDC) to the magnetic beads, mix thoroughly, and react on a rotating mixer at room temperature for 30min. Use a magnetic rack to completely separate the solid and liquid, and mix and wash the magnetic beads 2-3 times with coupling buffer. Add 4ug PEDV S1 protein, mix thoroughly, and rotate at room temperature for 3h. Use a magnetic rack to completely separate the solid and liquid, wash the magnetic beads 2-3 times with washing solution, and disperse the coupled magnetic beads into 600μL blocking buffer (2% BSA aqueous solution (mass volume ratio)). After thorough mixing, rotate at room temperature for 1h to block the immunomagnetic beads. The solid and liquid were completely separated by a magnetic rack, and the magnetic beads were washed 2-3 times with a washing solution. The washed magnetic beads were dispersed into 400 μL storage buffer (0.1% BSA) and stored at 4°C.

[0053] 3) Sample diluent: 1% BSA, dissolve 10 g of bovine serum albumin (BSA) in 800 mL of PBST, and add sterile water to 1000 mL.

[0054] 4) Horseradish peroxidase-labeled secondary antibody: Dilute the horseradish peroxidase-labeled rabbit anti-swine IgG or IgA antibody in "1)" 100,000 times with PBS containing 1% bovine serum albumin (BSA) to obtain the horseradish peroxidase-labeled secondary antibody.

[0055] 5) Washing solution: NaCl 80g, KCl 2g, KH2PO4 2g, Na2HPO4·12H2O 29g, Tween-20 20mL, add distilled water to 1L, adjust the pH to 7.4 to make a 10-fold (10×) concentration of washing solution, dilute 10 times with distilled water before use to the direct use concentration.

[0056] 6) Substrate colorimetric solution: contains solution A and solution B, which are mixed in a 1:1 ratio and used immediately after preparation. Solution A contains luminol and a luminescence enhancer, while the main components of solution B are peroxide and an enhancer.

[0057] 7) Preparation of negative control serum and positive control serum:

[0058] a. Negative control serum: serum from healthy piglets that have not been vaccinated and are PEDV negative in both fluorescent PCR and ELISA tests;

[0059] b. Positive control serum: Dilute the PEDV antibody positive serum (inactivated) to the working concentration in proportion with diluent.

[0060] 8) Calibration curve drawing: Use the calibration sample concentration and the corresponding luminescence value to do logistic curve fitting to obtain a fitting curve, namely the calibration curve, see Figure 2 and Figure 3 ,in Figure 2 The calibration curve of the PEDV-IgG kit is shown. Figure 3 The PEDV-IgA kit calibration curve is shown.

[0061] 9) Composition of PEDV-IgG or PEDV-IgA test kit:

[0062] a. Immunomagnetic beads: 4mL;

[0063] b. Washing solution: 10-fold concentrated 1M PBS (pH 7.4) containing 0.5% Tween-20 75 mL;

[0064] c. Sample diluent: 30mL;

[0065] d. Enzyme-labeled secondary antibody: 30mL;

[0066] e. Substrate color developing solution A: 15mL;

[0067] f. Substrate colorimetric solution B: 15 mL;

[0068] g. PEDV negative and positive control serum: 1.5 mL each;

[0069] h. Magnetic plate;

[0070] i. Sample tube.

[0071] Example 3. Use of PEDV Antibody Detection Kit

[0072] a. Dilute the serum sample to be tested 20 times with sample diluent, add 100 μL of diluted serum and 20 μL of immunomagnetic beads to the sample tube, react at room temperature for 15 minutes, separate the immunomagnetic beads from the reaction system using a magnetic plate, and wash 3 times with 250 μL to 300 μL of washing solution;

[0073] b. Add 100 μL of enzyme-labeled secondary antibody, react at room temperature for 15 min, and wash three times with 250 μL to 300 μL of washing solution;

[0074] c. Mix solution A and solution B in a ratio of 1:1 to prepare a substrate colorimetric solution, add 100 μL of the substrate colorimetric solution, and react at room temperature in the dark for 2 minutes;

[0075] d. The microplate chemiluminescence detection instrument detects the luminescence value and determines the negative or positive result of the sample to be tested according to the judgment criteria.

[0076] Example 4. Determination of the judgment criteria of the PEDV magnetic microparticle chemiluminescent antibody detection kit

[0077] 20 SPF pig sera (Table 1, Table 2) were tested with PEDV-IgG or PEDV-IgA magnetic microparticle chemiluminescent antibody detection kits, and the test data were substituted into the standard curve to obtain the titer of negative serum measured 20 times. The average values ​​of negative serum measured by the two methods were calculated to be 0.48RU / mL and 0.69RU / mL, respectively, and the standard deviations SD were 0.38RU / mL and 0.43RU / mL, respectively. According to statistical principles, the critical value of the PEDV-IgG kit was determined to be X+3SD≈1.6RU / mL. Similarly, the critical value of the negative and positive results of the PEDV-IgA kit was determined to be X+3SD≈2.0RU / mL.

[0078] Table 1. Luminescence values ​​of 20 SPF pig sera detected by PEDV-IgG kit

[0079]

[0080]

[0081] Table 2. Luminescence values ​​of 20 SPF pig sera detected by PEDV-IgA kit

[0082] Sample No. Luminous value Sample No. Luminous value 1 2739.86 11 2206.39 2 3019.54 12 1973.32 3 958.17 13 6653.813 4 704.39 14 393.63 5 1709.18 15 1388.06 6 2201.99 16 1367.34 7 4464.575 17 5536.694 8 6588.097 18 2387.205 9 1248.22 19 2126.19 10 5206.00 20 8162.176

[0083] From the data in Table 1 and Table 2, it can be seen that the luminescence values ​​of PEDV-IgG and PEDV-IgA in detecting 20 SPF pig sera ranged from 324.44 to 9164.343 and 393.63 to 8162.176, respectively, and both methods had low luminescence background values. Among them, the luminescence value of PEDV-IgG can be as low as 324.44, and the luminescence value of PEDV-IgA can be as low as 393.63. The method of the present invention has high detection sensitivity and a wide detection range.

[0084] Example 5. Specific identification of PEDV magnetic microparticle chemiluminescent antibody detection kit

[0085] The established PEDV-IgG or PEDV-IgA magnetic microparticle chemiluminescence method was used to detect the positive sera of PCV2, CSFV, PRV, and PRRSV, respectively. The results showed that the detection values ​​were all less than the critical value of 1.6RU / mL or 2.0RU / mL, indicating that the recombinant protein of the present invention had no cross-reaction with other common virus positive sera and had good specificity.

[0086] Example 6. PEDV magnetic microparticle chemiluminescent antibody detection kit for determining PEDV antibody levels and natural infection status in pigs

[0087] The established PEDV-IgA magnetic microparticle chemiluminescence detection method was used to detect 66 clinical samples of pig serum, including 22 serum samples of PEDV-negative pigs, 22 serum samples of pigs naturally infected with PEDV, and 22 serum samples of pigs vaccinated with PEDV vaccine but not infected. The PEDV antibody level in the pig herd was analyzed (Table 3). When the detection value is ≥2.0RU / mL, the sample is PEDV antibody positive, and when the detection value is <2.0RU / mL, the sample is PEDV antibody negative. The test results showed that the positive rate of the naturally infected group was 100%, and the average detection concentration was 68.77RU / mL. The positive rate of the vaccinated but uninfected group was 54.5%, and the average detection concentration was 2.72RU / mL. The antibody level of natural infection is significantly higher than that of vaccination. The highest antibody level of natural infection can reach 155.69RU / mL, while the highest level of vaccine immunity is only 10.29RU / mL. This can be used to preliminarily evaluate the infection of PEDV in the pig herd.

[0088] Table 3. PEDV-IgA kit clinical pig serum sample test results

[0089] Sample No. Negative pig (RU / mL) Naturally infected pigs (RU / mL) Vaccinated but uninfected pigs (RU / mL) 1 0.63 77.00 0.77 2 0.28 93.89 0.43 3 0.23 136.38 1.23 4 0.43 36.55 5.83 5 0.14 76.63 0.98 6 0.97 21.41 0.60 7 1.38 14.72 2.39 8 0.43 46.22 2.95 9 0.34 115.61 0.55 10 0.72 137.35 10.29 11 0.53 38.33 2.16 12 0.13 44.12 0.82 13 0.17 42.14 7.84 14 0.37 36.76 2.11 15 0.36 66.48 8.12 16 1.18 35.83 1.75 17 0.46 59.71 1.19 18 0.56 67.38 2.44 19 0.51 50.38 0.28 20 1.69 155.69 2.71 21 1.40 52.57 2.87 22 0.13 107.69 1.41 average value 0.59 68.77 2.72

[0090] From the data in Table 3, it can be seen that the PEDV-IgA magnetic microparticle chemiluminescence detection method can distinguish naturally infected pigs from vaccinated but uninfected pigs. According to the analysis of the results, it can be seen that the detection value of naturally infected pigs is much higher than that of vaccinated but uninfected pigs. The highest detection value of naturally infected pigs can reach 137.35RU / mL, while the highest detection value of vaccinated but uninfected pigs is only 10.29RU / mL.

[0091] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. An immunomagnetic bead coated with recombinant PEDV S1 protein, characterized in that: The amino acid sequence of the recombinant PEDV S1 protein is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding a recombinant PEDV S1 protein, characterized in that: The amino acid sequence of the recombinant PEDV S1 protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2, or is a sequence having more than 95% homology with SEQ ID NO:

2.

3. An expression system, characterized in that The expression system expresses recombinant PEDV S1 protein, the amino acid sequence of the recombinant PEDV S1 protein is shown in SEQ ID NO: 1, the expression system is an insect bacillus expression system, and the expression system comprises the nucleic acid molecule encoding the recombinant PEDV S1 protein according to claim 2.

4. The expression system according to claim 3, characterized in that The insect rod expression system is Sf9 insect cells.

5. Use of a recombinant PEDV S1 protein in the preparation of a kit for detecting porcine epidemic diarrhea virus infection, wherein the kit is used to distinguish between pigs naturally infected with porcine epidemic diarrhea virus and vaccinated pigs, and the amino acid sequence of the recombinant PEDV S1 protein is shown in SEQ ID NO:

1.

6. The use according to claim 5, characterized in that The kit comprises an enzyme-labeled secondary antibody, which is an antibody against porcine IgA.

7. Use of a recombinant PEDV S1 protein in the preparation of a diagnostic reagent for detecting porcine epidemic diarrhea virus infection, wherein the amino acid sequence of the recombinant PEDV S1 protein is shown in SEQ ID NO:

1.

8. A kit for detecting porcine epidemic diarrhea virus infection, characterized in that: The kit comprises the immunomagnetic beads coated with the recombinant PEDV S1 protein according to claim 1.

9. The kit according to claim 8, characterized in that The kit comprises an enzyme-labeled secondary antibody, which is an antibody against porcine IgA.

10. The kit according to claim 8 or 9, characterized in that The kit also includes a positive control and a negative control, a sample diluent, a color developing solution, a washing solution and a magnetic plate.

Images