Recombinant antigen and ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for detecting porcine respiratory and reproductive disorder virus antibody based on recombinant antigen

Abstract

The invention relates to the related technical field of detection of a porcine respiratory and reproductive disorder virus antibody, and particularly provides a recombinant antigen and an ELISA (enzyme-linked immunosorbent assay) detection kit for detecting the porcine respiratory and reproductive disorder virus antibody based on the recombinant antigen. The invention provides a recombinant antigen. The recombinant antigen is selected from a structure as shown in a formula (I), wherein the AAS1 and the AAS2 independently represent an amino acid sequence as shown in SEQ ID NO.8 or an amino acid sequence as shown in SEQ ID NO.9, and the AAS1 and the AAS2 represent different amino acid sequences; l represents an amino acid sequence # imgabs0 # in the direction from the N terminal to the C terminal, and n is selected from integers from 1 to 5. The recombinant antigen disclosed by the invention can be specifically combined with a porcine respiratory and reproductive disorder virus antibody, so that false positive detection results caused by interfering substances are greatly reduced. (1) AAS1-L-AAS2.

Description

Technical Field

[0001] The present application relates to the technical field related to porcine respiratory and reproductive virus antibody detection, and specifically provides a recombinant antigen and an ELISA detection kit for detecting porcine respiratory and reproductive virus antibodies based on the recombinant antigen. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). The disease can cause reproductive disorders in sows and severe respiratory diseases in piglets, posing a huge threat to the pig industry.

[0003] PRRSV is an enveloped, single-stranded, positive-strand RNA virus that is extremely prone to mutation and recombination to form new strains during evolution, which greatly increases the difficulty of its diagnosis. Among them, the structural protein N encoded by PRRSV is highly conserved in type I and type II viruses, and can quickly stimulate the body to produce an immune response after viral infection or immunization. The antibodies produced can maintain a high level for a long period of time. The disadvantage is that the structural protein N cannot induce the body to produce neutralizing antibodies. PRRSV encodes a glycoprotein GP5 with multiple transmembrane structures, which can induce the production of partial neutralizing antibodies, which is of great significance for the clinical diagnosis of PRRSV; however, the neutralizing antibody is generally produced late, and the early detection rate is relatively low, which cannot timely reflect the overall antibody level of the pig herd, and thus cannot provide effective guidance for the prevention and control of the disease.

[0004] At present, there are many methods for detecting viruses in clinical practice, among which enzyme-linked immunosorbent assay (ELISA) is a commonly used serological antibody detection method in clinical practice. However, non-specific interfering substances contained in serum, such as heterophilic antibodies HA and rheumatoid factor RF, can interfere with the detection accuracy of ELISA and may result in more false positive test results. Summary of the invention

[0005] In view of this, the present application provides a recombinant antigen and an ELISA test kit for detecting porcine respiratory and reproductive virus antibodies based on the recombinant antigen. The recombinant antigen of the present application can specifically bind to porcine respiratory and reproductive virus antibodies, greatly reducing false positive test results caused by interfering substances.

[0006] In the first aspect of the present application, the present application provides a recombinant antigen, wherein the recombinant antigen is selected from the structure shown in formula (I):

[0007] AAS1-L-AAS2

[0008] (I)

[0009] Wherein, AAS1 and AAS2 each independently represent the amino acid sequence shown in SEQ ID NO.8 or the amino acid sequence shown in SEQ ID NO.9, and AAS1 and AAS2 represent different amino acid sequences;

[0010] L represents a flexible connecting peptide, and L is selected from the amino acid sequence from the N-terminus to the C-terminus n is selected from an integer from 1 to 5 (eg, 1, 2, 3, 4 or 5).

[0011] In some specific embodiments, AAS1 represents the amino acid sequence shown in SEQ ID NO. 8, and AAS2 represents the amino acid sequence shown in SEQ ID NO. 9. In some optional embodiments, AAS1 represents the amino acid sequence shown in SEQ ID NO. 9, and AAS2 represents the amino acid sequence shown in SEQ ID NO. 8.

[0012] In some optional embodiments, n is selected from integers from 2 to 4. In some specific embodiments, n is selected from 3; correspondingly, L is selected from the amino acid sequence shown in SEQ ID NO.10.

[0013] In some specific embodiments, the recombinant antigen is selected from the amino acid sequence shown in SEQ ID NO.7.

[0014] In the second aspect of the present application, the present application provides a gene encoding the recombinant antigen described in the first aspect of the present application, wherein the encoding gene is selected from the structure shown in formula (II):

[0015] E1-NAS1-L'-NAS2-E2

[0016] (II)

[0017] Wherein, NAS1 and NAS2 each independently represent the nucleotide sequence shown in SEQ ID NO.2 or the nucleotide sequence shown in SEQ ID NO.3, and NAS1 and NAS2 represent different nucleotide sequences;

[0018] The nucleotide sequence shown in SEQ ID NO.2 encodes the amino acid sequence shown in SEQ ID NO.8;

[0019] The nucleotide sequence shown in SEQ ID NO.3 encodes the amino acid sequence shown in SEQ ID NO.9;

[0020] L' represents a nucleotide sequence encoding a flexible connecting peptide, and L' is selected from the nucleotide sequence from the 5' end to the 3' end n' is selected from an integer from 1 to 5 (eg, 1, 2, 3, 4 or 5);

[0021] E1 and E2 are each independently selected from restriction enzyme cleavage sites.

[0022] In some specific embodiments, NAS1 represents the nucleotide sequence shown in SEQ ID NO. 2, and NAS2 represents the nucleotide sequence shown in SEQ ID NO. 3. In some optional embodiments, NAS1 represents the nucleotide sequence shown in SEQ ID NO. 3, and NAS2 represents the nucleotide sequence shown in SEQ ID NO. 2.

[0023] In some optional embodiments, n' is selected from integers from 2 to 4. In some specific embodiments, n' is selected from 3; equivalently, L' is selected from the nucleotide sequence shown in SEQ ID NO.4.

[0024] In some optional embodiments, E1 and E2 are each independently selected from Nde I restriction site and Xho I restriction site. In some specific embodiments, E1 is selected from Nde I restriction site. In some specific embodiments, E2 is selected from Xho I restriction site. In other specific embodiments, E1 is selected from Nde I restriction site, and E2 is selected from Xho I restriction site. In some specific embodiments, the Nde I restriction site is selected from the nucleotide sequence shown in SEQ ID NO.5. In some specific embodiments, the Xho I restriction site is selected from the nucleotide sequence shown in SEQ ID NO.6.

[0025] In some specific embodiments, the encoding gene is selected from the nucleotide sequence shown in SEQ ID NO.1.

[0026] In the third aspect of the present application, the present application provides a recombinant expression vector, which includes the coding gene described in the second aspect of the present application.

[0027] In some optional embodiments, the recombinant expression vector also includes an initial vector.

[0028] In some optional embodiments, the initial vector is selected from at least one of pGEX series vectors, pET series vectors, pMAL series vectors, and pUC series vectors. In some specific embodiments, the initial vector is a pUC57 vector.

[0029] In the fourth aspect of the present application, the present application provides a use of any of the following items in the preparation of a product for detecting antibodies to porcine respiratory and reproductive disorder virus:

[0030] (I) the recombinant antigen described in the first aspect of the present application; and / or

[0031] (II) the coding gene described in the second aspect of the present application; and / or

[0032] (III) The recombinant expression vector described in the third aspect of the present application.

[0033] In some optional embodiments, the product comprises at least one of an ELISA detection kit and a magnetic microparticle chemiluminescence detection kit. In some specific embodiments, the product is an ELISA detection kit.

[0034] In the fifth aspect of the present application, the present application provides an ELISA detection kit for detecting porcine respiratory and reproductive virus antibodies based on the recombinant antigen described in the first aspect of the present application.

[0035] In some optional embodiments, the ELISA detection kit includes a coating antigen and a competing antigen;

[0036] Wherein, the coating antigen is selected from the recombinant antigen described in the first aspect of the present application.

[0037] In some optional embodiments, the competing antigen is selected from goat anti-pig IgG labeled with horseradish peroxidase.

[0038] In some optional embodiments, the ELISA detection kit further comprises a sample diluent; wherein the sample diluent is selected from a buffer solution or a buffer solution containing an immunoblocker.

[0039] In some embodiments, the buffer solution is phosphate buffer.

[0040] In some embodiments, the sample diluent is selected from a buffer solution.

[0041] In other specific embodiments, the sample diluent is selected from a buffer solution containing an immunoblocking agent.

[0042] In some optional embodiments, the immunoblocking agent includes at least one of fetal bovine serum, casein and rabbit IgG antibody.

[0043] In some embodiments, the immunoblocking agent comprises at least one of component 1, component 2 and component 3;

[0044] Wherein, the component 1 comprises at least one of bovine serum albumin, casein, gelatin, skim milk, and whey protein;

[0045] The component 2 includes at least one of pig serum, fetal bovine serum, and mouse serum;

[0046] The component 3 includes at least one of mouse IgG antibody, human IgG antibody, goat IgG antibody, sheep IgG antibody and rabbit IgG antibody.

[0047] In some optional embodiments, the concentration of component 1 is (1-10)% (W / V); for example, (1-9)%, (1-8)%, (1-7)%, (1-6)%, (1-5)%, (1-4)%, (1-3)%, (1-2)%, (1.5-10)%, (1.5-9)%, (1.5-8)%, (1.5-7)%, (1.5-6)%, (1.5-5)%, (1.5-4)%, (1.5-3)%, (1.5-2)% or 2%, etc.

[0048] In some optional embodiments, the concentration of component 2 is (0.1-5)% (V / V); for example, (0.1-4)%, (0.1-3)%, (0.1-2)%, (0.1-1)%, (0.1-0.9)%, (0.1-0.8)%, (0.1-0.7)%, (0.1-0.6)%, (0.1-0.5)%, (0.1-0.4)%, (0.1-0.3)%, (0.1-0.2)% or 0.2%, etc.

[0049] In some optional embodiments, the concentration of component 3 is (5-100) μg / mL; for example, (5-90) μg / mL, (5-80) μg / mL, (5-70) μg / mL, (5-60) μg / mL, (5-50) μg / mL, (5-40) μg / mL, (5-30) μg / mL, (5-20) μg / mL, (5-10) μg / mL, (10-100) μg / mL, (10-90) μg / mL, (10-80) μg / mL, (10-70) μg / mL, (10-60) μg / mL, (10-50) μg / mL, (10-40) μg / mL, (10-30) μg / mL, (10-20) μg / mL or 10 μg / mL, etc.

[0050] In some optional embodiments, the immunoblocker includes component 1 and component 3. In other optional embodiments, the immunoblocker includes casein and rabbit IgG antibody. In some specific embodiments, the immunoblocker includes casein and rabbit IgG antibody; wherein the concentration of casein is 1.5% (W / V) and the concentration of rabbit IgG antibody is 50ug / mL.

[0051] In some optional embodiments, the immunoblocker includes component 1, component 2 and component 3. In other optional embodiments, the immunoblocker includes casein, fetal bovine serum and rabbit IgG antibody. In some specific embodiments, the immunoblocker includes casein, fetal bovine serum and rabbit IgG antibody; wherein the concentration of casein is 1.5% (W / V), the concentration of fetal bovine serum is 2% (V / V), and the concentration of rabbit IgG antibody is 50ug / mL. In other specific embodiments, the immunoblocker includes casein, fetal bovine serum and rabbit IgG antibody; wherein the concentration of casein is 2% (W / V), the concentration of fetal bovine serum is 0.2% (V / V), and the concentration of rabbit IgG antibody is 10ug / mL.

[0052] This application has the following beneficial effects:

[0053] First, the recombinant antigen of the present application can specifically bind to porcine respiratory and reproductive virus antibodies, greatly reducing the false positive test results caused by interfering substances.

[0054] Second, further, the immunoblockers used in the ELISA detection kit of the present application, especially immunoblocker 3, can effectively reduce the detection background value of negative serum samples while ensuring the positive detection rate, thereby improving the negative detection rate and the overall compliance rate. DETAILED DESCRIPTION

[0055] The present application discloses a recombinant antigen and an ELISA test kit for detecting porcine respiratory and reproductive disorder virus antibodies based on the recombinant antigen. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application.

[0056] Terminology explanation:

[0057] The term "Porcine reproductive and respiratory syndrome virus (PRRSV)" is an enveloped, single-stranded, positive-sense RNA virus that is extremely prone to mutation and recombination to form new strains during the evolution process. It can cause abortion, stillbirth, weak fetuses in sows, and respiratory difficulties and high mortality in piglets as its main characteristics.

[0058] The term "flexible connecting peptide" refers to a peptide segment used to connect two or more adjacent proteins. The flexible connecting peptide used in the present application is a relatively long flexible peptide segment that can reduce possible steric hindrance.

[0059] The term "immunoblocker" refers to a preparation that can effectively block the influence of nonspecific interfering substances in the serum to be tested under specific conditions. The composition or ratio of the immunoblocker used in this application is different from any type of existing commercial immunoblocker or any publicly available immunoblocker.

[0060] The operations of "coating", "sealing", "washing", "drying", "patting dry", "incubation" and the like mentioned in this application are all routine experimental operations in the field, and relevant technical personnel in the field should understand the specific contents, implementation processes and methods of the operations.

[0061] Unless otherwise stated, all chemical reagents used in this application were of analytical grade and obtained from commercial sources.

[0062] Unless otherwise stated, the biological materials used in this application were obtained from commercial sources.

[0063] Unless otherwise stated, the experimental methods used in this application are conventional methods.

[0064] Unless otherwise specified, the clinical serum samples involved in this application were prepared by centrifuging whole blood at 4°C, 4000 r / min for 5 min to separate serum and adding Proclin 300 (48914-U, SIGMA) with a final concentration of 2% (W / W).

[0065] Unless otherwise specified, the preparation process of PBS buffer used in this application is: 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and water to 1 L.

[0066] Unless otherwise specified, the preparation process of the PBST buffer used in this application is as follows: 0.05 volume % Tween-20 is added to the PBS buffer.

[0067] Unless otherwise specified, the preparation process of the skim milk blocking solution used in the present application is as follows: skim milk powder is diluted with PBST buffer, and the concentration of the skim milk blocking solution is 5% (mass volume ratio w / v).

[0068] In order to make the objectives, technical solutions and advantages of the present application clearer, the implementation scheme of the present application will be further described in detail below in conjunction with examples.

[0069] Example 1 Gene encoding recombinant antigen:

[0070] In this example, the nucleotide sequence of the gene encoding the recombinant antigen (denoted as rNG5 gene) is shown in SEQ ID NO.1.

[0071] Table 1 Nucleotide sequence information of coding genes:

[0072]

[0073]

[0074] Unless otherwise stated, this application commissioned Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the coding gene and connect the coding gene to the pUC57 vector (denoted as pUC57:rNG5).

[0075] Among them, the amino acid sequence of the recombinant antigen expected to be expressed by the coding gene SEQ ID NO.1 is shown in Table 2.

[0076] Table 2 Amino acid sequence information of recombinant antigens

[0077]

[0078] Primer Premier 6.0 software was used to design amplification primers encoding gene SEQ ID NO.1. The nucleotide sequence information of the amplification primers is shown in Table 3.

[0079] Table 3 Nucleotide sequence information of amplification primers

[0080]

[0081]

[0082] Unless otherwise stated, this application commissioned Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize amplification primers.

[0083] Example 2 Construction of recombinant expression vector:

[0084] Using pUC57:rNG5 as a template, the rNG5 gene fragment was amplified using amplification primers rNG5-F-Nde I and rNG5-R-Xho I. The amplified rNG5 gene fragment and the pET28a vector were double-digested using Nde I restriction endonuclease and Xho I restriction endonuclease at the same time, and the double-digested product of the rNG5 gene fragment and the double-digested product of the pET28a vector were connected using T4 DNA ligase. The ligation product was transferred into Escherichia coli DH5α competent cells, and the Escherichia coli DH5α competent cells were spread on kanamycin-resistant LB agar plates (containing 50 μg / mL kanamycin) and cultured in a 37°C constant temperature incubator until single colonies were clear.

[0085] Select a single colony with good growth and obvious characteristics on the kanamycin-resistant LB agar plate, inoculate it into LB liquid medium (containing 50 μg / mL kanamycin) and culture it at 37°C and 220 r / min for 6 hours; then, use a plasmid extraction kit to extract the plasmid. The extracted plasmid is double-digested with Nde I restriction endonuclease and Xho I restriction endonuclease to obtain two fragments of 5300 bp and 702 bp, respectively.

[0086] Among them, the plasmid after double enzyme digestion verification was entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing verification, and the correct recombinant plasmid was named pET28a:rNG5.

[0087] Example 3 Expression and purification of recombinant antigens:

[0088] The recombinant plasmid pET28a:rNG5 was transferred into the competent cells of Escherichia coli BL21 (DE3), and the recombinant strain was named BL21 (DE3) / pET28a: rNG5. The recombinant strain BL21 (DE3) / pET28a: rNG5 was inoculated into LB liquid medium (containing 50 μg / mL of kanamycin) and cultured at 37 ° C, 220 r / min for 10 hours; then inoculated into a 500 mL shake flask (containing 200 mL of LB liquid medium) at a 1% (V / V) inoculation amount, and cultured at 37 ° C, 220 r / min until the bacterial density OD 600 = about 0.6; cool down by 25°C, add isopropyl-β-D-thiogalactopyranoside (IPTG, CAS No. 367-93-1) with a final concentration of 0.5 mmol / L, continue to shake culture at 37°C and 220 r / min for 12 h, and end the fermentation.

[0089] Centrifuge at 8000r / min for 10min and collect the bacteria; resuspend the bacteria in PBS buffer; use an ultrasonic disruptor (800w, ultrasonic for 2s, stop for 5s, cycle until the ultrasonic duration reaches 20min) to disrupt the bacteria, centrifuge at 13000r / min for 30min, separate the supernatant and precipitate, resuspend the precipitate in an equal volume ratio of PBS buffer, and perform SDS-PAGE protein gel electrophoresis on the supernatant. Among them, the SDS-PAGE protein gel electrophoresis results showed that the target protein band of about 25KDa was detected in the supernatant, and the protein expression level was about 0.5mg / mL.

[0090] The supernatant was filtered using a 0.22 μm filter, and the treated sample was purified using a protein purifier (purchased from Taidu Biotechnology Co., Ltd.) to purify the target protein. Ni NTA Beads 6FF protein agarose purification resin (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.) was used as filler, with a column volume of about 10 mL, a sample loading flow rate of 2 mL / min, and a 50 mmol / L imidazole solution (solvent: PBS buffer) was used to elute the impurities, and a 500 mmol / L imidazole solution (solvent: PBS buffer) was used to elute the target protein. The eluted target protein was identified using SDS-PAGE protein gel electrophoresis, and the results showed that the purity of the target protein recombinant antigen was about 90%, which met the use requirements of diagnostic reagent raw materials.

[0091] Reactivity characterization of recombinant antigens:

[0092] The reactivity and specificity of the purified recombinant antigen were analyzed by Western Blot.

[0093] First, the recombinant antigen was subjected to SDS-PAGE protein gel electrophoresis. After the electrophoresis, it was transferred to a nitrocellulose NC membrane by wet transfer, and skim milk blocking solution was added and blocked at room temperature for 1 hour. The PRRSV antibody positive serum and negative serum obtained from clinical isolation were used as primary antibodies (serum was diluted at 1:100 (V / V) using skim milk blocking solution), and the primary antibody was incubated at room temperature for 1 hour; washed 3 times with PBST buffer; horseradish peroxidase-labeled goat anti-swine IgG antibody was used as a secondary antibody (horseradish peroxidase-labeled goat anti-swine IgG antibody was diluted at 1:50000 (V / V) using skim milk blocking solution), and the secondary antibody was incubated at room temperature for 1 hour; washed 3 times with PBST buffer; and DAB staining solution (purchased from Kangwei Century Biotechnology Co., Ltd.) was used for color development. The color development results showed that the recombinant antigen could react specifically with PRRSV antibody positive serum, but not with PRRSV antibody negative serum. This shows that the recombinant protein can specifically bind to the antibodies produced after PRRSV immunization or infection.

[0094] Example 4 ELISA test kit for detecting porcine respiratory and reproductive disorder virus antibodies based on recombinant antigens:

[0095] In this embodiment, the main components of the ELISA detection kit for detecting porcine respiratory and reproductive virus antibodies based on recombinant antigens are: a coating plate coated with a recombinant antigen, a sample diluent, a washing solution, a negative control, a positive control, a horseradish peroxidase-labeled goat anti-swine IgG antibody working solution, a color developing solution, and a stop solution.

[0096] Coating plate coated with recombinant antigen: using carbonate buffer (pH=9.6) to coat the recombinant antigen on the ELISA plate at a coating concentration of (1-5) μg / mL and a coating amount of (0.1-0.5) μg / well, and coating at 2-8°C overnight; discarding the coating solution, washing 2-4 times with phosphate buffer, and patting or draining; adding blocking solution (phosphate buffer with 1% (mass volume ratio w / v) casein and 0.1% (mass volume ratio w / v) bovine serum albumin), blocking at 2-8°C overnight; discarding the blocking solution, washing 3 times with phosphate buffer, and patting or draining; after drying at 37°C for 2-3h, putting the coated plate coated with the recombinant antigen into an aluminum foil bag containing a desiccant, and storing at 4°C for standby use.

[0097] Sample diluent: phosphate buffer or phosphate buffer containing immunoblockers. If the sample diluent is a phosphate buffer containing immunoblockers, the immunoblockers include at least one of fetal bovine serum, casein, and rabbit IgG antibodies. The concentration of fetal bovine serum is (0.1-5)% (V / V), the concentration of casein is (1-10)% (W / V), and the concentration of rabbit IgG antibodies is (5-100) μg / mL.

[0098] Washing solution: phosphate buffer containing 0.05% (V / V) Tween-20.

[0099] Horseradish peroxidase-labeled goat anti-swine IgG antibody working solution: Use phosphate buffer containing 1% (W / V) casein and 1% (W / V) sucrose to dilute the horseradish peroxidase-labeled goat anti-swine IgG antibody at a ratio of 1: (70000-80000) (V / V) to prepare the horseradish peroxidase-labeled goat anti-swine IgG antibody working solution.

[0100] Color developing solution: Luminescent substrate solution A and luminescent substrate solution B are prepared in a volume ratio of 1:1.

[0101] Among them, luminescent substrate solution A: dissolve 3,3',5,5'-tetramethylbenzidine (20 mg) in anhydrous ethanol (10 mL), dilute to 100 mL with ddH2O, mix well and package aseptically.

[0102] Luminescent substrate solution B: Add citric acid (2.1 g), anhydrous Na2HPO4 (2.82 g), and 0.75% (V / V) urea hydrogen peroxide in anhydrous ethanol (0.64 mL), make up to 100 mL with ddH2O, mix well, and dispense aseptically.

[0103] Stop solution: H2SO4 aqueous solution with a concentration of 2 mol / L.

[0104] Positive control: Pigs were challenged with a virulent strain of PRRSV, and the pig serum tested positive for PRRSV antibodies by polymerase chain reaction (PCR).

[0105] Negative control: serum from pigs with negative PRRSV antibodies detected by PCR or piglets that did not eat colostrum, or replaced by sample diluent. Specifically, the negative control used in this embodiment is serum from pigs with negative PRRSV antibodies detected by PCR.

[0106] In this embodiment, the method for using the ELISA detection kit for detecting porcine respiratory and reproductive disorder virus antibodies based on recombinant antigens mainly includes the following steps:

[0107] Step S401, using a sample diluent to dilute a clinical serum sample or plasma sample at 1:40 (V / V); wherein, if the sample diluent is a phosphate buffer containing an immunoblocker, a blocking reaction is performed (conditions: temperature is (20-37)°C, time is (10-30) min), and then step S402 is performed; if the sample diluent is a phosphate buffer, no blocking reaction is performed, and step S402 is performed directly.

[0108] Step S402: Add the sample, negative control and positive control obtained in step S401 to the coated plate coated with the recombinant antigen, 100 μL / well, and incubate at 20-37° C. for (10-30) min.

[0109] Step S403, wash the coated plate obtained in step S402 with washing solution, 300 μL / well, wash 3 times, 3 min / time.

[0110] Step S404: add horseradish peroxidase-labeled goat anti-swine IgG antibody working solution to the coated plate obtained in step S403, 100 μL / well, and incubate at (20-37)° C. for (10-30) min.

[0111] Step S405, wash the coated plate obtained in step S404 with washing solution, 300 μL / well, 5 times, 3 min / times.

[0112] Step S406, add the color developing solution to the coated plate obtained in step S405, 100 μL / well, and incubate in the dark at (20-37)°C for (5-15) min; then, add the stop solution, 50 μL / well.

[0113] Step S407, placing the coated plate into an ELISA reader, and reading the value at a test wavelength of 450 nm.

[0114] The test result of the sample is determined according to the S / P value; if the S / P value is ≧0.4, the test result of the sample is determined to be positive; if the S / P value is <0.4, the test result of the sample is determined to be negative.

[0115] This embodiment optimizes the conditions of step S401 based on the ELISA test kit for detecting porcine respiratory and reproductive virus antibodies using recombinant antigens. Among them, 12 serum samples that were clinically determined to be negative by the commercial IDEXX porcine blue ear antibody test kit were selected for optimization experiments.

[0116] Table 4 Detection results of optimization experiment (S / P value):

[0117]

[0118]

[0119] It can be seen from Table 4 that the recombinant antigen of the present application can specifically bind to porcine respiratory and reproductive virus antibodies, greatly reducing the false positive test results caused by interfering substances.

[0120] Among them, when the kit of this embodiment uses sample diluent A (phosphate buffer without immunoblocking agent) in step S401, 8 serum samples out of 12 serum samples that were clinically determined to be negative by the commercial IDEXX PRRSV antibody detection ELISA test kit were determined to be positive and 4 serum samples were determined to be negative. It can be seen that the recombinant antigen of the present application can specifically bind to porcine respiratory and reproductive virus antibodies, greatly reducing the false positive test results caused by interfering substances.

[0121] Compared with sample diluent A (phosphate buffer without immunoblocker), when the kit of this embodiment uses sample diluent B (phosphate buffer containing immunoblocker 1) in step S401, immunoblocker 1 can partially reduce the S / P values ​​of negative serum samples 5 to 8, and the blocking effect becomes more significant with the increase of blocking time and blocking temperature; however, the S / P values ​​of positive serum samples 1 to 4 are partially increased, and immunoblocker 1 has no obvious blocking effect on abnormal serum samples 9 to 12 that are prone to false positives.

[0122] Compared with sample diluent A (phosphate buffer without immunoblocker), when the kit of this embodiment uses sample diluent C (phosphate buffer containing immunoblocker 2) in step S401, immunoblocker 2 can partially reduce the S / P value of positive serum samples 1 to 4, and the detection value decreases more significantly with the increase of blocking time and blocking temperature. However, for serum samples 5 to 12, immunoblocker 2 not only fails to play an effective blocking role, but causes the detection value to increase abnormally.

[0123] Compared with sample diluent A (phosphate buffer without immunoblocker), when the kit of this embodiment uses sample diluent D (phosphate buffer containing immunoblocker 3) in step S401, the blocking effect of immunoblocker 3 is better than that of immunoblocker 1 and immunoblocker 2. Specifically, immunoblocker 3 can make the S / P values ​​of positive serum samples 1 to 4 unchanged, and at the same time significantly reduce the S / P values ​​of serum samples 5 to 12, especially for abnormal serum samples 9 to 12 that are prone to false positives, the test results are all negative.

[0124] In summary, the immunoblockers used in the ELISA test kit for detecting porcine respiratory and reproductive virus antibodies based on recombinant antigens, especially immunoblocker 3, can effectively reduce the detection background value of negative serum samples while ensuring the positive detection rate, thereby improving the negative detection rate and the overall compliance rate.

[0125] Example 5 Characteristics of the ELISA test kit for detecting porcine respiratory and reproductive disorder virus antibodies based on recombinant antigens Opposite sex research:

[0126] Porcine respiratory and reproductive virus antibody-positive serum, porcine circovirus type 2 (PCV-2) antibody-positive serum, porcine pseudorabies virus (PRV) antibody-positive serum, African swine fever virus (ASFV) antibody-positive serum, porcine epidemic diarrhea virus (PEDV) antibody-positive serum, and porcine foot-and-mouth disease virus (FMDV) antibody-positive serum were collected from clinical sites. The above-collected clinical positive serum samples were verified by the corresponding commercial ELISA antibody detection kits, and the verification results were all positive.

[0127] The ELISA test kit for detecting porcine respiratory and reproductive virus antibodies based on recombinant antigen in Example 4 (the sample diluent is phosphate buffer containing immunoblocker 3) was used to detect the above-mentioned different virus antibody-positive sera, and the test results are shown in Table 5.

[0128] Table 5 Specificity test results of different virus antibody positive serum:

[0129] Antibody positive serum ELISA test kit of the present application Porcine respiratory and reproductive virus S / P values ​​are all greater than 0.4 (all judged as positive) Porcine circovirus type II S / P values ​​are all less than 0.2 (all judged as negative) Pseudorabies virus S / P values ​​are all less than 0.2 (all judged as negative) African swine fever virus S / P values ​​are all less than 0.2 (all judged as negative) Porcine epidemic diarrhea virus S / P values ​​are all less than 0.2 (all judged as negative) Swine foot-and-mouth disease virus S / P values ​​are all less than 0.2 (all judged as negative)

[0130] It can be seen from Table 5 that the ELISA detection kit of the present application for detecting porcine respiratory and reproductive virus antibodies based on recombinant antigens has good specificity.

[0131] Example 6 Sensitivity of ELISA test kit for detecting porcine respiratory and reproductive disorder virus antibodies based on recombinant antigen Perceptual Research:

[0132] Porcine reproductive and respiratory syndrome virus antibody-positive sera from clinical sources were collected and diluted in multiples using PBS buffer at a dilution multiple of 1:(2-128) (V / V). The ELISA detection kit for detecting porcine respiratory and reproductive virus antibodies based on recombinant antigens in Example 4 (the sample diluent was a phosphate buffer containing immunoblocker 3), the ELISA detection kit for detecting porcine respiratory and reproductive virus antibodies based on recombinant antigens in Example 4 (the sample diluent was a phosphate buffer without immunoblockers), and the commercial IDEXX PRRSV antibody detection ELISA detection kit were used for detection, and the detection results are shown in Table 6.

[0133] Table 6 Sensitivity test results of different kits (S / P values):

[0134]

[0135] As can be seen from Table 6, the sensitivity of the ELISA test kit of the present application (the sample diluent is a phosphate buffer containing immunoblocker 3) is comparable to that of the commercial IDEXX PRRSV antibody detection ELISA test kit.

[0136] Example 7 Application of ELISA test kit for detecting porcine respiratory and reproductive disorder virus antibodies based on recombinant antigen Use evaluation:

[0137] Using the ELISA detection kit for detecting porcine respiratory and reproductive virus antibodies based on recombinant antigen in Example 4 (the sample diluent is a phosphate buffer containing immunoblocker 3), the ELISA detection kit for detecting porcine respiratory and reproductive virus antibodies based on recombinant antigen in Example 4 (the sample diluent is a phosphate buffer without immunoblocker), the commercial IDEXX PRRSV antibody detection ELISA detection kit, the domestic PRRSV antibody detection kit 1 and the domestic PRRSV antibody detection kit 2, 195 serum samples from the clinic were tested, and the positive detection rate, negative detection rate and overall compliance rate were statistically compared. The test results are shown in Table 7.

[0138] Table 7 Test results of different kits for 195 clinical serum samples:

[0139]

[0140] As can be seen from Table 7, compared with the commercialized IDEXX PRRSV antibody detection ELISA test kit, the negative serum detection compliance rate and the positive serum detection compliance rate of the ELISA test kit of the present application are not less than 92%, and the overall compliance rate is above 93%. It can be seen that in terms of detection with the IDEXX PRRSV antibody detection ELISA test kit, the ELISA test kit of the present application has a high consistency in test results. Moreover, compared with the domestic PRRSV antibody detection kit 1 and the domestic PRRSV antibody detection kit 2, the test results of the ELISA test kit of the present application are more credible.

[0141] Furthermore, in terms of consistency with the commercial IDEXX PRRSV antibody detection ELISA test kit, the ELISA test kit of the present application (the sample diluent is a phosphate buffer containing an immunoblocker 3) performs better than the ELISA test kit of the present application (the sample diluent is a phosphate buffer without an immunoblocker). It can be seen that the immunoblocker involved in the ELISA test kit of the present application can effectively block the potential non-specific interferences contained in some serum samples, while maintaining the positive detection rate, it can reduce the negative serum sample detection background and improve the compliance rate of negative serum detection, thereby improving the overall detection compliance rate with the commercial IDEXX PRRSV antibody detection ELISA test kit.

[0142] The above is a detailed introduction to a recombinant antigen provided by the present application and an ELISA test kit for detecting porcine respiratory and reproductive virus antibodies based on the recombinant antigen. This article uses specific examples to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A recombinant antigen, characterized in that: The recombinant antigen is selected from the structure shown in formula (I): AAS1-L-AAS2 (I) Wherein, AAS1 and AAS2 each independently represent the amino acid sequence shown in SEQ ID NO.8 or the amino acid sequence shown in SEQ ID NO.9, and AAS1 and AAS2 represent different amino acid sequences; L represents the amino acid sequence from N-terminus to C-terminus n is selected from integers from 1 to 5.

2. The recombinant antigen according to claim 1, characterized in that AAS1 represents the amino acid sequence shown in SEQ ID NO.8, and AAS2 represents the amino acid sequence shown in SEQ ID NO.9; Optionally, n is selected from an integer from 2 to 4; Optionally, the recombinant antigen is selected from the amino acid sequence shown in SEQ ID NO.

7.

3. A gene encoding a recombinant antigen according to any one of claims 1 to 2, characterized in that: The coding gene is selected from the structure shown in formula (II): E1-NAS1-L'-NAS2-E2 (II) Wherein, NAS1 and NAS2 each independently represent the nucleotide sequence shown in SEQ ID NO.2 or the nucleotide sequence shown in SEQ ID NO.3, and NAS1 and NAS2 represent different nucleotide sequences; The nucleotide sequence shown in SEQ ID NO.2 encodes the amino acid sequence shown in SEQ ID NO.8; The nucleotide sequence shown in SEQ ID NO.3 encodes the amino acid sequence shown in SEQ ID NO.9; L' represents the nucleotide sequence from 5' to 3' n' is selected from integers from 1 to 5; E1 and E2 are each independently selected from restriction enzyme cleavage sites.

4. The coding gene according to claim 3, characterized in that NAS1 represents the nucleotide sequence shown in SEQ ID NO.2, and NAS2 represents the nucleotide sequence shown in SEQ ID NO.3; Optionally, n' is selected from an integer from 2 to 4; Optionally, E1 and E2 are each independently selected from an Nde I restriction site and an Xho I restriction site; Optionally, E1 is selected from the Nde I restriction site; Optionally, E2 is selected from the Xho I restriction site; Optionally, the NdeI restriction site is selected from the nucleotide sequence shown in SEQ ID NO.5; Optionally, the Xho I restriction site is selected from the nucleotide sequence shown in SEQ ID NO.6; Optionally, the encoding gene is selected from the nucleotide sequence shown in SEQ ID NO.

1.

5. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the coding gene according to any one of claims 3 to 4.

6. Use of any of the following in the preparation of a product for detecting antibodies to porcine respiratory and reproductive virus: (I), the recombinant antigen according to any one of claims 1 to 2; and / or (II), the coding gene according to any one of claims 3 to 4; and / or (III) The recombinant expression vector according to claim 5.

7. The use according to claim 6, characterized in that The product comprises at least one of an ELISA detection kit and a magnetic particle chemiluminescence detection kit.

8. An ELISA test kit for detecting porcine respiratory and reproductive virus antibodies based on the recombinant antigen described in any one of claims 1 to 2.

9. The ELISA test kit according to claim 8, characterized in that The ELISA detection kit comprises a coating antigen and a competitive antigen; Wherein, the coating antigen is selected from the recombinant antigen according to any one of claims 1 to 2.

10. The ELISA detection kit according to claim 9, characterized in that: The competing antigen is selected from goat anti-pig IgG labeled with horseradish peroxidase.

11. The ELISA detection kit according to any one of claims 9 to 10, characterized in that: The ELISA detection kit also includes a sample diluent; Wherein, the sample diluent is selected from a buffer solution or a buffer solution containing an immunoblocker; Optionally, the immunoblocking agent comprises at least one of component 1, component 2 and component 3; The component 1 comprises at least one of bovine serum albumin, casein, gelatin, skim milk, and whey protein; The component 2 includes at least one of pig serum, fetal bovine serum, and mouse serum; The component 3 comprises at least one of mouse IgG antibody, human IgG antibody, goat IgG antibody, sheep IgG antibody, and rabbit IgG antibody; Optionally, the immunoblocking agent comprises component 1 and component 3; Optionally, the immunoblocking agent comprises component 1, component 2 and component 3; Optionally, the concentration of component 1 is (1-10)% (W / V); Optionally, the concentration of component 2 is (0.1-5)% (V / V); Optionally, the concentration of component 3 is (5-100) μg / mL.

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