M-gp5 recombinant protein-based porcine reproductive and respiratory syndrome virus elisa detection kit

Abstract

This invention relates to an ELISA detection kit for porcine reproductive and respiratory syndrome virus (PRRSV) based on M-GP5 recombinant protein, and belongs to the field of ELISA detection technology. The kit includes recombinant (M-GP5) antigen protein, an ELISA plate, blocking buffer, primary antibody dilution buffer, enzyme-labeled secondary antibody, secondary antibody dilution buffer, chromogenic solution, stop solution, negative serum, and positive serum. It features high sensitivity, high specificity, and good stability, and can accurately detect PRSV antibodies.

Description

Technical Field

[0001] This invention relates to the field of ELISA detection technology, and to a porcine reproductive and respiratory syndrome virus ELISA detection kit based on M-GP5 recombinant protein. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease in pigs caused by Porcine reproductive and respiratory syndrome virus (PRRSV), also known as "blue ear disease." Its main characteristics include reproductive disorders in pregnant sows (abortion, stillbirth, mummified fetuses) and respiratory diseases in pigs of all ages, especially piglets. PRRSV is a single-stranded positive-sense small RNA virus belonging to the order Tibervirales, family Arterividdae, and genus Arterivirus. It is mainly divided into two types: the American type and the European type. The PRRSV genome is approximately 15kb in length and contains 10 open reading frames (ORFs). Among them, ORF1a / 1b encodes non-structural proteins, while ORF2a / 2b / 3-7 encode structural proteins. The main structural proteins encoded are GP2a, E, GP3, GP4, GP5, GP5a, M, and N, and at least 13 non-structural proteins, including NSP1α, NSP1β, NSP2-NSP8, and NSP9-NSP12.

[0003] GP5 protein, as the most important structural protein of PRRSV, contains neutralizing epitopes that can induce high levels of neutralizing antibodies. However, GP5's neutralizing epitopes are mainly concentrated in the N-terminal extracellular region, and its glycosylation sites can shield some of these epitopes, leading to immune escape. Therefore, the application of GP5 as a diagnostic protein is currently rare. For the prevention and screening of porcine reproductive and respiratory syndrome (PRRS) in pig farms, most tests use PRRSV N protein detection kits to assess the effectiveness of swine herd immunity. However, with the rapid increase in pig populations and the strong variability of the virus, N protein detection kits are no longer sufficient for large-scale testing. It is evident that existing technologies require further improvement. Summary of the Invention

[0004] To address the above problems, this invention provides an ELISA detection kit for porcine reproductive and respiratory syndrome virus (PRRSV) based on M-GP5 recombinant protein. This ELISA detection kit is characterized by high sensitivity, high specificity, and good stability, and can accurately detect PRSV antibodies.

[0005] This invention provides an ELISA detection kit for porcine reproductive and respiratory syndrome virus based on M-GP5 recombinant protein. The ELISA detection kit includes: M-GP5 recombinant protein, ELISA plate, blocking buffer, primary antibody dilution buffer, ELISA-labeled secondary antibody, secondary antibody dilution buffer, chromogenic solution, stop solution, negative serum, and positive serum. The M-GP5 recombinant protein includes a vector and a GP5 protein. The vector includes an M protein sequence, which is the amino acid sequence from position 1 to position 78 of the M protein amino acid sequence.

[0006] In one embodiment, the M protein sequence is linked to the GP5 protein using a flexible peptide linker; the M-GP5 recombinant protein has a protective base, an Nhe I restriction endonuclease cleavage site, and a Kozak sequence before the start codon, and a terminator, an MluI restriction endonuclease cleavage site, and a protective base at the end, with an Igk signal peptide and a his tag added to the N-terminus.

[0007] In one embodiment, the sequence of the M-GP5 recombinant protein is shown in SEQ ID NO.1.

[0008] The amino acid sequence of the M-GP5 recombinant protein is as shown in SEQ ID NO.1: MHHHHHHMDQPLTVPLELVQVYLLALVAGLFFCLYQALVFGAYFCCCRSHKICRPVTDWVLNRAKLTQEVNEVVRGDVRSLYRTGGRGKGGGGSGGGGSGGGGSMARLVLLCALLLFLAVTASALCLVGAQALHTAIRQYEQPVNTQDLGFFGLFIPFIAAMMWYIYMQNFINQAVLPPQPIYA YSKIKGQKVVFGLVYNVTPQTLQVRRKFQPVEYAFYKLDQVIHNPTGHVRFQEEKAALQAPVTAATVTPTTTTTETTTTTTVATATTTTETTTTTTSQVEQPLKGRYVLAQPPSLEPVSLPVSLPQPGLPGLNGLPGPDPGIPGLPGLPGPPGLPGIPGLPGIPGLPGLPGLPGLPGLPGLPGLS.

[0009] By comparing the M amino acid sequences of different strains, highly conserved amino acids 1-78 and GP5 amino acid sequences were selected, codon optimization was performed, and the M-GP5 recombinant protein of this invention was synthesized. The specific sequence was synthesized by Qingke Biotechnology Co., Ltd. (Beijing).

[0010] In one embodiment, the M-GP5 recombinant protein is obtained by expression in mammalian cells.

[0011] In one embodiment, the enzyme-labeled secondary antibody includes HRP-labeled goat anti-pig IgG secondary antibody.

[0012] In one embodiment, the blocking solution comprises a milk powder solution with a mass fraction of 10-15%, the primary antibody diluent comprises a milk powder solution with a mass fraction of 10-15%, and the secondary antibody diluent comprises a milk powder solution with a mass fraction of 10-15%.

[0013] The present invention also provides a method for preparing the M-GP5 recombinant protein in the ELISA detection kit, comprising the following steps: inserting the M-GP5 recombinant protein into a lentiviral vector, transforming competent cells to obtain a recombinant lentiviral vector; transfecting the recombinant lentiviral vector into mammalian cells, culturing them to obtain a cell line expressing the M-GP5 recombinant protein; collecting the cell supernatant, purifying, eluting, and dialysis to obtain the M-GP5 recombinant protein.

[0014] The use of mammalian cells in a eukaryotic expression system to express the (M-GP5) antigen protein reduces the problem of decreased immunogenicity caused by inclusion bodies in the prokaryotic system, and improves the sensitivity and stability of the (M-GP5) antigen protein.

[0015] In one embodiment, the mammalian cells include HEK-293T cells.

[0016] In one embodiment, the culture time for the M-GP5 recombinant protein preparation method is 65-80 hours.

[0017] In one embodiment, the method for preparing the M-GP5 recombinant protein includes: inserting the synthesized M-GP5 recombinant protein amino acid sequence into a lentiviral vector, transforming DH5α competent cells to obtain a recombinant lentiviral vector; transfecting the recombinant lentiviral vector into HEK-293T cells using Lipo8000™ reagent and culturing for 72 h to construct a cell line stably expressing the recombinant (M-GP5) antigen protein; collecting the cell supernatant and purifying it using a HisTrap™ HP column, eluting the target protein with 50 mM imidazole elution buffer, and dialysis the elution buffer with PBS buffer to obtain high-purity (M-GP5) antigen protein.

[0018] This invention also provides a method for detecting GP5 antibodies against porcine reproductive and respiratory syndrome (PRRS) for non-diagnostic purposes, using the aforementioned ELISA detection kit, comprising the following steps: Antigen coating: Add M-GP5 recombinant protein solution to the ELISA plate, coat, discard the solution in the wells, wash, and pat dry; Blocking: Add blocking solution to the wells of the microplate, block, discard the solution in the wells, wash, and pat dry; Reaction: The test sample, negative serum, and positive serum were diluted separately using primary antibody diluent. The resulting test sample diluent, negative serum diluent, and positive serum diluent were added to the wells of the ELISA plate, incubated, the solution in the wells was discarded, washed, and patted dry. The ELISA-labeled secondary antibody was diluted using secondary antibody diluent. The resulting ELISA-labeled secondary antibody diluent was added to the wells of the ELISA plate, incubated, the solution in the wells was discarded, washed, and patted dry. Color development: Add color development solution, develop color in the dark, add to the plant, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value to determine the sample to be tested.

[0019] In one embodiment, the detection method includes the following steps: Antigen coating: Add a recombinant (M-GP5) antigen protein solution with a concentration of 0.5~1μg / ml to the ELISA plate, coat at 37℃ for 2h, discard the solution in the wells, wash, and pat dry; Blocking: Add 10% skim milk powder solution to the wells of the ELISA plate and block at 37℃ for 2 hours. After blocking, discard the solution in the wells, wash, and pat dry. Reaction: Dilute the test sample, negative serum, and positive serum separately with primary antibody diluent to obtain three serum diluents. Add the three serum diluents to the wells of the ELISA plate, incubate, discard the solution in the wells, wash, and pat dry. Dilute the enzyme-labeled secondary antibody with secondary antibody diluent to obtain enzyme-labeled secondary antibody diluent. Add the enzyme-labeled secondary antibody diluent to the wells of the ELISA plate, incubate, discard the solution in the wells, wash, and pat dry. Color development: Add colorimetric solution, develop color in the dark, add stop solution, and measure the absorbance value at a wavelength of 450 nm using an ELISA reader. When the sample OD... 450nm A value ≤0.1045 is considered negative, while a value above this value is considered positive. In one embodiment, the test sample is diluted 200 times with the primary antibody diluent; the incubation temperature is 37°C, and the incubation time is 30 minutes.

[0020] In one embodiment, when diluting the enzyme-labeled secondary antibody with the secondary antibody dilution buffer, the dilution factor of the enzyme-labeled secondary antibody was 20,000 times; the incubation temperature was 37°C and the incubation time was 1 hour.

[0021] In one embodiment, the color development step involves developing the color in the dark for 15 minutes at a temperature of 37°C.

[0022] In one embodiment, the detection method uses phosphate-Tween buffer (PBST) containing 1‰ (mass fraction) Tween-20 for washing.

[0023] In one embodiment, the rule for determination includes: when the sample OD 450nm If the OD value is ≤0.1045, it is considered negative; when the sample OD value is ≤0.1045, it is considered negative. 450nm A value greater than 0.1045 is considered positive.

[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention relates to an ELISA detection kit for porcine reproductive and respiratory syndrome virus (PRRSV) based on the M-GP5 recombinant protein. The M-GP5 recombinant protein in this kit is produced using a lentiviral vector and a eukaryotic expression system. Based on the stability of the M protein, the M-GP5 recombinant protein is linked to the highly variable GP5 protein, forming a stable M-GP5 fusion protein and synergistically enhancing the sensitivity of PRRSV detection and diagnosis. The kit based on the M-GP5 recombinant protein exhibits advantages such as high sensitivity, strong stability, and good reproducibility. Detailed Implementation

[0025] To facilitate understanding of the present invention, a more complete description will be given below with reference to relevant embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0027] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0028] Source: In the following experiments, negative serum, positive serum, and porcine clinical serum samples were all provided by Guangzhou Berniz Biotechnology Co., Ltd. Unmodified GP5 antigen protein was stored at Guangzhou Berniz Biotechnology Co., Ltd. The enzyme-labeled secondary antibody was HRP-labeled goat anti-porcine IgG secondary antibody, manufactured by Solarbio, with an IgG content of 4.5 mg / ml and an HRP content of 2 mg / ml.

[0029] Example 1 I. Preparation method of M-GP5 recombinant protein By comparing the amino acid sequences of M from different strains, highly conserved amino acids 1-78 were selected and linked to the GP5 amino acid sequence. The M protein sequence and the GP5 protein were connected using a flexible peptide linker. The start codon of the M-GP5 recombinant protein is preceded by a protective base, an Nhe I restriction endonuclease cleavage site, and a Kozak sequence. The end of the protein is preceded by a terminator, an MluI restriction endonuclease cleavage site, and a protective base. An Igk signal peptide and a his tag are added to the N-terminus. The final sequence of the M-GP5 recombinant protein is shown in SEQ ID NO.1.

[0030] The amino acid sequence of the M-GP5 recombinant protein is as shown in SEQ ID NO.1: MHHHHHHMDQPLTVPLELVQVYLLALVAGLFFCLYQALVFGAYFCCCRSHKICRPVTDWVLNRAKLTQEVNEVVRGDVRSLYRTGGRGKGGGGSGGGGSGGGGSMARLVLLCALLLFLAVTASALCLVGAQALHTAIRQYEQPVNTQDLGFFGLFIPFIAAMMWYIYMQNFINQAVLPPQPIYA YSKIKGQKVVFGLVYNVTPQTLQVRRKFQPVEYAFYKLDQVIHNPTGHVRFQEEKAALQAPVTAATVTPTTTTTETTTTTTVATATTTTETTTTTTSQVEQPLKGRYVLAQPPSLEPVSLPVSLPQPGLPGLNGLPGPDPGIPGLPGLPGPPGLPGIPGLPGIPGLPGLPGLPGLPGLPGLPGLS.

[0031] The synthesized M-GP5 recombinant protein amino acid sequence was inserted into a lentiviral vector and transformed into DH5α competent cells to obtain a recombinant lentiviral vector. The recombinant lentiviral vector was transfected into HEK-293T cells using Lipo8000™ reagent and cultured for 72 h to construct a cell line stably expressing the recombinant (M-GP5) antigen protein. The cell supernatant was collected and purified using a HisTrap™ HP column. The target protein was eluted with 50 mM imidazole elution buffer, and the elution buffer was dialyzed against PBS buffer to obtain high-purity (M-GP5) antigen protein.

[0032] II. Optimization of the optimal coating amount of antigen protein and the optimal dilution factor of serum The steps of the test method are as follows: Step A001. Antigen Coating: Prepare recombinant (M-GP5) antigen protein solutions using 50mM carbonate buffer at pH 9.6, with concentrations of 8μg / ml, 4μg / ml, 2μg / ml, 1μg / ml, 0.5μg / ml, and 0.25μg / ml. Add 100μL / well to a 96-well microplate, with each concentration in a separate column, and coat at 4℃ for 16h. After coating, wash four times with PBST containing 1‰ Tween-20, allowing the plate to stand for 1min after each addition of washing buffer before discarding the washing buffer. Finally, blot dry. Step A002. Blocking: Add a 10% (w / w) skim milk powder solution to the wells of the microplate and block at 37°C for 2 hours. After blocking, discard the solution in the wells, wash, and pat dry. Step A003. Serially dilute negative and positive sera with 10% skim milk powder solution to obtain serum dilutions of 100, 200, 400, and 800 times. Add each serum dilution to an ELISA plate coated with recombinant (M-GP5) antigen protein, with two replicates for each dilution, 100 μL / well, and incubate at 37°C for 30 min. Wash four times with PBST containing 1‰ Tween-20, allowing the plate to stand for 1 min after each addition of washing buffer before discarding the washing buffer in the well. Drain the plate completely on the last wash. Step A004. Dilute the HRP-labeled goat anti-pig IgG secondary antibody with a 10% (w / w) skim milk powder solution to a dilution factor of 10000 times to obtain the enzyme-labeled secondary antibody dilution solution. Add 100 μl of the enzyme-labeled secondary antibody dilution solution to each well of the ELISA plate and incubate at 37°C for 60 min. Wash four times with PBST containing 1‰ Tween-20. After each addition of washing buffer, let stand for 1 min and discard the washing buffer in the well. Drain the plate completely on the last wash. Step A005. Add 100 μl of TMB chromogenic solution to each well, incubate in the dark for 15 min, add 50 μl of 2M H2SO4 stop solution to each well, and measure the absorbance at 450 nm using a microplate reader. Take the average value of each dilution and calculate the P / N value.

[0033] The results are shown in Table 1.

[0034] Table 1. P / N values ​​for each dilution

[0035] Note: The symbol "+" represents the OD450 of the positive serum group; the symbol "-" represents the OD450 of the negative serum group; "P / N" represents the ratio of positive serum OD450 to negative serum OD450.

[0036] As shown in Table 1, the P / N ratio was highest (34.330) in antigen coating when the concentration of the recombinant (M-GP5) antigen protein solution was 0.5 μg / ml and the serum dilution factor was 200-fold. Even when the concentration of the recombinant (M-GP5) antigen protein solution was 1 μg / ml, the P / N ratio was still 34.229, indicating that the optimal concentration range for coated antigen protein is 0.5–1 μg / ml.

[0037] Example 2 Optimization of primary antibody serum incubation time The test method in Example 2 differs from that in Example 1 in that: In step A001, a recombinant (M-GP5) antigen protein solution with a concentration of 1 μg / ml was used for coating; the coating conditions were 4℃ for 16 h. In step A003, the negative serum and positive serum were diluted 200 times; different conditions were selected for comparison during incubation, namely: (1) incubation at 37℃ for 30 min; (2) incubation at 37℃ for 60 min; (3) incubation at 37℃ for 120 min.

[0038] The results are shown in Table 2: Table 2. P / N values ​​for optimizing antiserum incubation time.

[0039] Note: The symbol "+" represents the OD450 of the positive serum group; the symbol "-" represents the OD450 of the negative serum group; "P / N" represents the ratio of positive serum OD450 to negative serum OD450.

[0040] As can be seen from the results in Table 2, the P / N value was the highest and the incubation effect was the best when the serum was incubated in a 37°C incubator for 30 minutes.

[0041] Example 3 Optimization of enzyme-labeled secondary antibody dilution factor and incubation time The difference between the test method in Example 3 and the test method in Example 1 is as follows: In step A001, a recombinant (M-GP5) antigen protein solution with a concentration of 1 μg / ml was used for coating; the coating conditions were 4℃ for 16 h. In step A003, the negative serum and positive serum were diluted 200 times; the incubation conditions were: 37℃ for 30 minutes. In step A004, the goat anti-pig IgG secondary antibody was compared at different dilutions: 5000, 10000, 20000, and 40000. Different incubation conditions were selected for comparison: (1) 37℃ for 30 min; (2) 37℃ for 60 min; (3) 37℃ for 90 min; (4) 37℃ for 120 min. The results are shown in Table 3: Table 3. Optimized P / N values ​​for each enzyme-labeled secondary antibody dilution factor and incubation time.

[0042] Note: The symbol "+" represents the OD450 of the positive serum group; the symbol "-" represents the OD450 of the negative serum group; "P / N" represents the ratio of positive serum OD450 to negative serum OD450. As can be seen from the results in Table 3, the P / N value was the highest when the enzyme-labeled secondary antibody was diluted 20,000 times and the incubation time was 60 min, indicating that the incubation and detection effect was the best under these conditions.

[0043] Example 4 Optimization of color development time The difference between the test method in Example 4 and the test method in Example 1 is as follows: In step A001, a recombinant (M-GP5) antigen protein solution with a concentration of 1 μg / ml was used for coating; the coating conditions were 4℃ for 16 h. In step A003, the negative serum and positive serum are diluted 200 times; the incubation conditions are 37°C for 30 minutes.

[0044] In step A004, the goat anti-pig IgG secondary antibody was diluted 20,000 times and incubated at 37°C for 60 minutes.

[0045] In step A005, different color development times were selected for comparison, namely 10 min, 15 min, and 20 min.

[0046] The results are shown in Table 4: Table 4. P / N values ​​for each color development time optimization.

[0047] Note: The symbol "+" represents the OD450 of the positive serum group; the symbol "-" represents the OD450 of the negative serum group; "P / N" represents the ratio of positive serum OD450 to negative serum OD450. As can be seen from the results in Table 4, the P / N value is the highest when the color development time is 15 minutes, indicating that the incubation detection effect is best at a color development time of 15 minutes.

[0048] Example 5 Optimization of wrapping conditions The test method in Example 5 differs from that in Example 1 in that: In step A001, a recombinant (M-GP5) antigen protein solution with a concentration of 1 μg / ml was used for coating; the coating conditions were as follows: (1) 37℃ for 2 h; (2) 37℃ for 4 h; (3) 4℃ for 16 h.

[0049] In step A003, the negative serum and positive serum are diluted 200 times; the incubation conditions are 37°C for 30 minutes.

[0050] In step A004, the goat anti-pig IgG secondary antibody was diluted 20,000 times and incubated at 37°C for 60 minutes.

[0051] The results are shown in Table 5: Table 5. P / N values ​​for each package condition optimization

[0052] Note: The symbol "+" represents the OD450 of the positive serum group; the symbol "-" represents the OD450 of the negative serum group; "P / N" represents the ratio of positive serum OD450 to negative serum OD450. As can be seen from the results in Table 5, the P / N value was the largest when the coating condition was 37℃ for 2 hours, indicating that the detection effect was the best under this condition.

[0053] Example 6 Optimization of sealing fluid The test method in Example 6 differs from that in Example 1 in that: In step A001, a recombinant (M-GP5) antigen protein solution with a concentration of 1 μg / ml was used for coating; the coating conditions were: 37℃ for 2 h.

[0054] In step A002, the blocking solutions used were (1) 5% BSA; (2) 1% BSA; (3) 10% skim milk powder; and (4) 5% skim milk powder. In step A003, the negative serum and positive serum are diluted 200 times; the incubation conditions are 37°C for 30 minutes.

[0055] In step A004, the goat anti-pig IgG secondary antibody was diluted 20,000 times and incubated at 37°C for 60 minutes.

[0056] The results are shown in Table 6: Table 6. Optimized P / N values ​​for each sealing fluid.

[0057] Note: The symbol "+" represents the OD450 of the positive serum group; the symbol "-" represents the OD450 of the negative serum group; "P / N" represents the ratio of positive serum OD450 to negative serum OD450.

[0058] As can be seen from the results in Table 6, the P / N value was the highest when 10% skim milk powder was used, indicating that the detection effect was the best under this condition.

[0059] Example 7 Determination of the critical value of the reaction Thirty-five latent samples of porcine reproductive and respiratory syndrome (PRRS) were obtained and tested using ELISA under optimized ELISA conditions.

[0060] The steps are as follows: (1) Antigen coating: Dilute the antigen protein to 1 μg / ml with 50 mM pH 9.6 carbonate buffer, and add 100 μL / well to a 96-well microplate. Coat at 37°C for 2 h. After coating, wash 4 times with PBST containing 1‰ (mass fraction) Tween-20, and let stand for 1 min after each wash. Finally, pat dry. (2) Blocking: Prepare a 10% skim milk powder solution with PBST containing 1‰ Tween-20, add 300 μl to each well of the 96-well microplate, and block at 37℃ for 2 h; wash 4 times with PBST, let stand for 1 min each time, and pat dry on the last wash; (3) Primary antibody serum incubation: Dilute the serum to be tested with PBST containing 10% skim milk powder at a dilution of 1:200. Add 100 μl of sample diluent to the coated microplate and set up one blank well. Do not add any diluent to the blank well. Incubate at 37°C for 30 min. Wash with PBST 4 times, let stand for 1 min after each wash, and pat dry after the last wash. (4) Enzyme-labeled secondary antibody incubation: HRP goat anti-pig IgG secondary antibody was diluted with PBST containing 10% skim milk powder at a dilution of 1:20000 to obtain enzyme-labeled secondary antibody dilution solution. Then, the enzyme-labeled secondary antibody dilution solution was added to a 96-well microplate at a volume of 100 μl / well and incubated at 37℃ for 60 min. Wash with PBST 4 times, and let stand for 1 min after each wash. Finally, pat dry. (5) Color development: Add 100 μl of TMB color development solution to each well and develop the color at 37°C in the dark for 15 min. (6) Termination: Add 50 μl of 2M H2SO4 termination solution to each well; (7) Reading: Within 10 minutes of adding the stop solution, turn on the microplate reader and read the absorbance at a wavelength of 450 nm.

[0061] Calculate the mean (X) and standard deviation (SD) of 35 negative samples. According to statistical principles, when the sample yields an OD value of 1,000, the mean (X) and standard deviation (SD) are calculated. 450nm A sample is considered negative if its value is less than or equal to the mean of negative samples (x) + 3 × standard deviation (SD); otherwise, it is considered positive.

[0062] The results are shown in Table 7: Table 7. Determination of Reaction Critical Values

[0063] The results in Table 7 show that the average OD450 value (X) of the 35 negative serum samples was 0.0622, and the standard deviation was 0.0141. According to the formula above, when the OD450 value is ≤0.1045, the sample can be judged as negative, and otherwise it can be judged as positive.

[0064] Example 8 Specificity test Following the method described in Example 7, ELISA was performed on positive serum samples for porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV-2), and porcine pseudorabies virus (PRV). A PRRSV-negative serum sample was used as a control. The results are shown in Table 8. Table 8. Specificity Experiment Results

[0065] As can be seen from the results in Table 8, the values ​​of PEDV-positive serum, PCV-2-positive serum, and PRV-positive serum were all below 0.1045, and were therefore considered negative. The PRRSV-positive serum result was positive, indicating that the detection method of the present invention has good specificity.

[0066] Example 9 Sensitivity test The recombinant (M-GP5) antigen protein was used as the coating antigen, and an ELISA assay was performed according to the method in Example 7.

[0067] PRRSV-positive serum was serially diluted at 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, and 1:51200, with each dilution performed in duplicate. The test results are shown in Table 9. Table 9 Sensitivity Test Results

[0068] As can be seen from the results in Table 9, when the positive serum dilution is 1:25600, the result is negative, indicating that when the recombinant (M-GP5) antigen protein is used as the coating antigen, positive serum diluted 12800 times can be detected.

[0069] Example 10 Repeatability test Multiple batches of ELISA plates were prepared using recombinant (M-GP5) antigen protein as the coating antigen, and ELISA assays were performed according to the method in Example 7. Three positive sera and three negative sera were selected for intra-batch and inter-batch replicate assays, respectively. The detection steps were performed according to the method in Example 7, with three replicates for each sample. The repeatability of the results was evaluated, and the results are shown in Table 10. Table 10 Results of Repeatability Tests

[0070] As can be seen from the results in Table 10, the coefficient of variation for intra-batch replication was 0.15%~5.11%, and the coefficient of variation for inter-batch replication was 1.13%~5.07%, indicating that the reproducibility of using recombinant (M-GP5) antigen protein as the coating antigen was good.

[0071] Based on the above experimental results, it can be seen that by modifying and designing the M protein and GP5 protein to form a recombinant (M-GP5) antigen protein, the recombinant protein expressed and secreted by the eukaryotic expression system has good sensitivity, strong stability and good reproducibility when used as the coating antigen of the ELISA detection kit.

[0072] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0073] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A porcine reproductive and respiratory syndrome virus (PRRSV) ELISA detection kit based on M-GP5 recombinant protein, characterized in that, This ELISA kit includes: M-GP5 recombinant protein, microplate, blocking buffer, primary antibody dilution buffer, enzyme-labeled secondary antibody, secondary antibody dilution buffer, chromogenic solution, stop solution, negative serum, and positive serum; The M-GP5 recombinant protein includes a vector and a GP5 protein. The vector includes an M protein sequence, which is the amino acid sequence from position 1 to position 78 of the M protein amino acid sequence.

2. The ELISA detection kit according to claim 1, characterized in that, The M protein sequence is linked to the GP5 protein using a flexible peptide linker; the M-GP5 recombinant protein has a protective base, an Nhe I restriction endonuclease cleavage site and a Kozak sequence before the start codon, and a terminator, an MluI restriction endonuclease cleavage site and a protective base at the end, with an Igk signal peptide and a his tag added to the N-terminus.

3. The ELISA detection kit according to claim 2, characterized in that, The sequence of the M-GP5 recombinant protein is shown in SEQ ID NO.

1.

4. The ELISA detection kit according to any one of claims 1-3, characterized in that, The M-GP5 recombinant protein was obtained by expression in mammalian cells.

5. The ELISA detection kit according to claim 1, characterized in that, The enzyme-labeled secondary antibody includes HRP-labeled goat anti-pig IgG secondary antibody.

6. A method for preparing the M-GP5 recombinant protein in the ELISA detection kit according to any one of claims 1-5, characterized in that, The procedure includes the following steps: inserting the M-GP5 recombinant protein into a lentiviral vector, transforming competent cells to obtain a recombinant lentiviral vector; transfecting the recombinant lentiviral vector into mammalian cells, culturing them to obtain a cell line expressing the M-GP5 recombinant protein; collecting the cell supernatant, purifying it, eluting it, and dialysis to obtain the M-GP5 recombinant protein.

7. The preparation method according to claim 6, characterized in that, The mammalian cells include HEK-293T cells.

8. The preparation method according to claim 6, characterized in that, In the preparation method of the M-GP5 recombinant protein, the culture time is 65-80 h.

9. A method for detecting GP5 antibodies against porcine reproductive and respiratory syndrome (PRRS) for non-diagnostic purposes, characterized in that, The method employs the ELISA detection kit described in any one of claims 1-5, and includes the following steps: Antigen coating: Add M-GP5 recombinant protein solution to the ELISA plate, coat, discard the solution in the wells, wash, and pat dry; Blocking: Add blocking solution to the wells of the microplate, block, discard the solution in the wells, wash, and pat dry; Reaction: The test sample, negative serum, and positive serum were diluted separately using primary antibody diluent. The resulting test sample diluent, negative serum diluent, and positive serum diluent were added to the wells of the ELISA plate, incubated, the solution in the wells was discarded, washed, and patted dry. The ELISA-labeled secondary antibody was diluted using secondary antibody diluent. The resulting ELISA-labeled secondary antibody diluent was added to the wells of the ELISA plate, incubated, the solution in the wells was discarded, washed, and patted dry. Color development: Add color development solution, develop color in the dark, add to the plant, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value to determine the sample to be tested.

10. The detection method according to claim 9, characterized in that, The rules for determination include: when the sample OD 450nm If the OD value is ≤0.1045, it is considered negative; when the sample OD value is ≤0.1045, it is considered negative. 450nm A value greater than 0.1045 is considered positive.

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