Nanometer antibody specifically combined with Seneca virus A and application thereof

Abstract

The invention is applicable to the field of genetic engineering and biotechnology detection, and provides a nano antibody specifically bound with Seneca virus A and application of the nano antibody. The nucleotide sequence of the nano antibody is as shown in SEQ ID NO: 1, and the amino acid sequence of the nano antibody is as shown in SEQ ID NO: 2. The anti-SVA nano antibody provided by the invention is relatively high in titer and good in specificity; the detection method provided by the invention is used for detecting the Senecavirus A antibody, and has good specificity and sensitivity.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biotechnology detection, specifically to a nanobody that specifically binds to Seneca virus A and its applications. Background Technology

[0002] Senecavirus A (SVA) is the pathogen of swine idiopathic vesicular disease (SIVD). Infecting older pigs causes vesicles and ulcers on the mouth, snout, hooves, and coronary artery bands. The mortality rate in 1-3 day old piglets infected with SVA is as high as 70%-80%. The clinical symptoms of SVA are extremely similar to those of foot and mouth disease (FMD), swine vesicular disease (SVD), vesicular exanthema of swine (VES), and vesicular stomatitis (VS), making clinical diagnosis extremely difficult. This necessitates extensive diagnostic work and related trade restrictions, resulting in significant economic impacts. Nanobody (Nb) has advantages such as small molecular weight, structural stability, good solubility, high specificity, and the ability to recognize specific epitopes, making it a current hot topic in the clinical development of drugs for the detection and prevention of human and animal diseases. The advantages of nanobodies can significantly improve the sensitivity and specificity of the established competitive ELISA. Furthermore, competitive ELISA methods are simple to operate, have short detection times, and provide reliable results. Currently, kits for detecting SVA antibodies do not offer ideal specificity or sensitivity.

[0003] Therefore, in view of the above situation, there is an urgent need to provide a nanobody that specifically binds to Seneca virus A and its application, so as to overcome the shortcomings in current practical applications. Summary of the Invention

[0004] The purpose of this invention is to provide a nanobody that specifically binds to Seneca virus A and its application, thereby addressing the problems in the aforementioned background art.

[0005] The present invention is achieved by a nanobody that specifically binds to Seneca virus A, wherein the nucleotide sequence of the nanobody is shown in SEQ ID NO:1 and the amino acid sequence of the nanobody is shown in SEQ ID NO:2.

[0006] This invention also provides a method for detecting antibodies against Seneca virus A, comprising the following steps:

[0007] (1) Provide an ELISA plate coated with recombinant SVA-VP1 protein;

[0008] (2) Provide a detection antibody, wherein the detection antibody is an SVA-VP1 specific nanobody fused to express horseradish peroxidase in HEK293T cells; the SVA-VP1 specific nanobody is the nanobody according to claim 1;

[0009] (3) Mix the serum to be tested with the detection antibody and then incubate it in an ELISA plate;

[0010] (4) After washing, add colorimetric solution to develop color, measure OD value, calculate inhibition rate, and judge the result based on Cut-off value.

[0011] As a further aspect of the present invention, the coating amount of the SVA-VP1 protein is 400 ng / well.

[0012] As a further aspect of the present invention: the titer of the horseradish peroxidase-labeled SVA-VP1 specific nanobody is 1:10. 3 The dilution ratio is 1:2 4 .

[0013] As a further aspect of the present invention, it also includes the use of diluent, blocking solution, washing solution, positive serum, negative serum and termination solution.

[0014] As a further aspect of the present invention: the diluent is PBS; the washing buffer is PBST buffer; the blocking buffer is 2.5% milk powder dissolved in PBST; the positive serum is clinical positive serum detected by Western blotting; the negative serum is clinical negative serum detected by Western blotting and SPF porcine serum; the chromogenic solution is single-component TMB chromogenic solution; and the stop solution is 3M sulfuric acid aqueous solution.

[0015] The present invention also provides a kit for detecting Seneca virus A antibody, comprising the above-mentioned nanobody and an ELISA plate coated with SVA-VP1 protein.

[0016] The present invention also provides the use of the nanobody or the kit described above in the preparation of reagents or kits for detecting Seneca virus A antibodies.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The anti-SVA nanobody provided by this invention has high titer and good specificity.

[0019] The detection method of this invention is used to detect antibodies against Seneca virus A. It has good specificity and sensitivity, can diagnose whether pigs are infected with SVA, and provides reliable results. It can be distinguished from other vesicular diseases in pigs. The detection cost is low and the detection time is short, which can enable rapid, large-scale and accurate detection in farms. It has a good effect on the prevention and control of SVA infection. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 The target gene of a selected nanobody was transferred into HEK293T cells to express horseradish peroxidase (HRP) and synthesize the Nb84-HRP fusion protein. The expression was detected by indirect immunofluorescence (IFA). His monoclonal antibody was used as the primary antibody, and green fluorescence indicated the expression of SVA-VP1-HRP protein. The supernatant of untransfected HEK293T cells was used as a control.

[0022] Figure 2 It is an antibody protein coated with Nb84-HRP fusion protein. A colorimetric reaction is performed on the Nb84-HRP fusion protein to detect whether this antibody protein has HRP activity.

[0023] Figure 3 The Nb84-HRP fusion protein was coated with SVA-VP1 protein and ASFV-P30 protein respectively, and the binding ability and specificity of the Nb84-HRP fusion protein to SVA-VP1 protein were detected by direct ELISA.

[0024] Figure 4 The antibody titer of the Nb84-HRP fusion protein, which is coated with SVA-VP1 protein, was detected by serially diluting it 10-fold using a direct ELISA method.

[0025] Figure 5 The Nb84-HRP fusion protein was coated with SVA-VP1 protein, and the competitive ELISA method was used to detect the competitive effect of the Nb84-HRP fusion protein against Seneca virus A-positive serum.

[0026] Figure 6 This study evaluated the specificity of established competitive ELISA methods by comparing positive sera containing SVA, ASFV, PPRRSV, FMDV, and PCV pathogens.

[0027] Figure 7It involves sensitivity evaluation of established competing ELISA methods. Detailed Implementation

[0028] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The present invention will be further explained below with reference to specific embodiments.

[0030] Example 1: Screening of anti-SVA-VP1 protein nanobodies:

[0031] The purified SVA-VP1 protein was expressed and used to immunize Bactrian camels. After the antibody titer reached the target level, peripheral blood was collected, lymphocytes were isolated, and genomic RNA was extracted. The gene fragment encoding VHH was amplified using nested RT-PCR, ligated into pCANTAB5E to construct a recombinant phage vector, and electroporated into TG1 competent cells to construct a Bactrian camel heavy chain antibody variable region library. Phage display technology was used to pan for specific nanobodies against the SVA-VP1 protein. Finally, a specific nanobodies against the SVA-VP1 protein were obtained and named Nb84. The nucleotide and amino acid sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0032] Example 2: Expression of Nb84-HRP fusion protein:

[0033] The nanobody gene was cloned into the pEGFP-N1-RANbodies vector, and the ligation product was transformed into Trans5α competent cells. Cells were cultured at 37℃ for 14 h at 200 r / min. The plasmid was extracted using an endotoxin-free plasmid extraction kit. The pEGFP-Nb84-HRP recombinant plasmid was diluted with 100 μL of Opti-MEM, and 12 μL of PEI transfection reagent was also diluted with 100 μL of Opti-MEM. The resulting plasmids were then transfected into HEK293T cells for expression. Forty-eight hours after transfection, the cells were tested with anti-His antibody and incubated with FITC goat anti-mouse IgG antibody. The cells were then observed under a fluorescence microscope. The results are as follows: Figure 1 Nb84 successfully fused to express HRP.

[0034] Detection of whether the Nb84-HRP fusion protein has HRP activity:

[0035] Cell supernatant was directly coated onto a solid support, and the biological activity of expressed HRP was detected by colorimetric assay. The specific procedure was as follows: 100 μL of transfected and 100 μL of untransfected HEK293T cell supernatant were added to a 96-well ELISA plate and incubated overnight at 4°C. Untransfected cell supernatant served as a control. The plate was washed three times with 300 μL of PBS'T per well. 100 µL of TMB substrate was added per well, and the plate was incubated at room temperature in the dark for 15 min. 50 µL of 3 M sulfuric acid was added to each well to stop the reaction, and the OD of each well was measured. 450 nm Value. Result as follows: Figure 2 As shown, the Nb84-HRP protein possesses HRP activity.

[0036] The ability of Nb84-HRP to specifically bind to SVA-VP1 protein was determined by direct ELISA:

[0037] The purified SVA-VP1 and ASFV-P30 proteins were diluted with PBS, 100 ng / well, and added to a 96-well microplate. The plate was incubated overnight at 4°C. The plate was washed three times with PBST (300 μL / well). 300 μL of 2.5% milk powder blocking buffer was added, and the plate was blocked at 37°C for 1 h. The plate was washed three times with PBST (300 μL / well). 100 µL of Nb84-HRP fusion protein was added, and the plate was incubated at 37°C for 1 h. The plate was washed three times with PBST (300 μL / well). 100 µL of TMB substrate was added, and the plate was incubated at 37°C in the dark for 15 min. The reaction was stopped by adding 50 µL of 3 M sulfuric acid to each well, and the OD of each well was measured. 450 nm Value. Result as follows: Figure 3 As shown, the Nb84-HRP fusion protein can specifically bind to the SVA-VP1 protein.

[0038] The titer of the Nb84-HRP fusion protein was determined by direct ELISA:

[0039] The purified SVA-VP1 protein was diluted with PBS, 100 ng / well, and added to a 96-well microplate. The plate was incubated overnight at 4°C. The plate was washed three times with PBST (300 μL / well). 300 μL of 2.5% milk powder blocking buffer was added, and the plate was blocked at 37°C for 1 h. The plate was washed three times with PBST (300 μL / well). 100 µL of Nb84-HRP fusion protein (serially diluted 10-fold with blocking buffer) was added to each well, and the plate was incubated at 37°C for 1 h. The plate was washed three times with PBST (300 μL / well). 100 µL of TMB substrate was added to each well, and the plate was incubated at 37°C in the dark for 15 min. The reaction was stopped by adding 50 µL of 3 M sulfuric acid to each well, and the OD of each well was measured.450 nm Value. Result as follows: Figure 4 As shown, the valence of Nb84-HRP is 1×10⁻⁶. 3 .

[0040] Determination of the competitive effect of nanobody-HRP using competitive ELISA:

[0041] The purified SVA-VP1 protein was diluted with PBS, 100 ng / well, and added to a 96-well microplate. The plate was incubated overnight at 4°C. The plate was washed three times with PBST (300 μL / well). 300 μL of 2.5% milk powder blocking buffer was added, and the plate was blocked at 37°C for 1 h. The plate was washed three times with PBST (300 μL / well). A mixture of 1:10 diluted SVA-positive serum and 1:10 diluted Nb84-HRP fusion protein was added. After incubation at 37°C for 1 h, the plate was washed three times with PBST (300 μL / well). 100 µL of TMB substrate was added, and the plate was incubated at 37°C in the dark for 15 min. The reaction was stopped by adding 50 µL of 3 M sulfuric acid to each well, and the OD of each well was measured. 450 nm Value. Result as follows: Figure 5 As shown, SVA-positive serum has a good competitive ability against Nb84-HRP fusion protein.

[0042] Example 3: Establishment of a competitive ELISA method for detecting Seneca virus A antibodies

[0043] 1. Materials for the Seneca virus A antibody competitive ELISA detection method

[0044] The reagents and materials required for the competitive ELISA detection method for Seneca virus A antibody include: diluent, washing buffer, SVA-VP1 protein, 96-well ELISA plate, 2.5% milk powder, nanobody (Nb84-HRP), positive serum, negative serum, single-component TMB chromogenic solution, and stop solution.

[0045] (1) Diluent

[0046] The diluent is PBS, prepared as follows: Weigh 0.2g of KH2PO4, 2.9g of Na2HPO4·12H2O, 8g of NaCl, and 0.2g of KCl, dissolve them in distilled water, and bring the volume to 1L.

[0047] (2) Washing liquid

[0048] The washing solution is PBST, and the preparation method is as follows: Weigh 0.2g of KH2PO4, 2.9g of Na2HPO4·12H2O, 8g of NaCl, 0.2g of KCl and 0.5mL of Tween-20, dissolve them in distilled water and make up to 1L.

[0049] (3) SVA-VP1 was diluted with diluent and coated onto an ELISA plate.

[0050] A 4 μg / mL SVA-VP1 solution was prepared using diluent, and 100 μL was added to each well of the microplate. The plate was incubated overnight at 4°C. The microplate was then removed, washed three times with washing buffer (300 μL per well), and patted dry on clean, lint-free absorbent paper. 300 μL of a 2.5% (w / v) milk powder solution prepared with PBST as solvent was added to each well, and the plate was blocked at 37°C for 1 hour. The plate was then washed three times with washing buffer (300 μL per well), and patted dry on clean, lint-free absorbent paper. This yielded the microplate coated with SVA-VP1.

[0051] (4) Antibodies

[0052] Antibody solution for detection: The nanobody Nb84 fused with HRP expressed in Example 2 had an antibody titer of 1×10⁻⁶. 3 The dilution ratio is 1:2. 4 .

[0053] (5) Positive and negative serum

[0054] Positive serum: Positive serum refers to clinically positive serum detected by Western blotting.

[0055] Negative serum: Negative serum refers to clinically negative serum and SPF porcine serum detected by Western blotting.

[0056] (6) Single-component TMB colorimetric solution

[0057] The single-component TMB colorimetric solution was purchased from Solarbio, catalog number PR1200.

[0058] (7) Termination solution

[0059] The stop solution is a 3M sulfuric acid aqueous solution.

[0060] 2. Establishment of a competitive ELISA method for detecting Seneca virus A antibodies.

[0061] The method for detecting Seneca virus A serum antibodies using the kit of this invention is as follows: During detection, the nanobody and the serum to be tested are diluted to their respective dilution ratios using blocking buffer in the same dilution system to obtain a mixture of serum to be tested and detection antibody. 100 μL of the mixture is added to each well of an ELISA plate coated with recombinant protein SVA-VP1, and incubated at 37°C for 1 h. The liquid in the plate is discarded, and the plate is washed three times with washing buffer and patted dry on absorbent paper without fibers. 100 μL of single-component TMB chromogenic solution is added to each well, and the plate is incubated at 37°C in the dark for 15 min. The chromogenic reaction is terminated by adding stop solution (50 μL per well) in the dark, and the OD value at 450 nm is measured using an ELISA reader. Two positive control wells (denoted as PC) and two negative control wells (denoted as NC) are also included.

[0062] Determine the SVA-VP1 protein coating amount and the Nb84-HRP fusion protein dilution ratio:

[0063] The coating amounts of SVA-VP1 protein were set at 100 ng, 200 ng, 400 ng, and 800 ng, and the dilution ratio of the nanobody (Nb84) was 1:2. 2 1:2 3 1:2 4 1:2 5 1:2 6 1:2 7 Using direct ELISA combined with the checkerboard method, when OD 450nm When ≈1, the coating amount of SVA-VP1 protein was determined to be 400 ng / well, and the dilution ratio of the nanobody Nb84-HRP was determined to be 1:2. 4 The optimal dosage of SVA-VP1 protein and the optimal dilution ratio of Nb84-HRP are shown in Table 1.

[0064] Table 1 Optimal dosage of SVA-VP1 protein and optimal dilution ratio of Nb84-HRP

[0065]

[0066] Optimize serum dilution:

[0067] Positive and negative sera were diluted at ratios of 1:5, 1:10, 1:20, and 1:40, and co-incubated with nanobodies at the determined optimal dilution ratio. The positive serum value was denoted as P, and the negative serum value as N. The minimum P / N ratio was achieved when the serum dilution ratio was 1:10. (Refer to Table 2.)

[0068] Table 2. Optimization of serum dilution ratio in competitive enzyme-linked immunosorbent assay (ELISA)

[0069]

[0070] Optimization of color development time and reaction time:

[0071] Three SVA-positive sera and three SVA-negative sera were selected and diluted according to the previously determined optimal dilution. The incubation times were set to 30 min, 45 min, and 60 min, and the color development times were 10 min, 15 min, and 20 min, respectively. Other procedures were the same as described above. The optimal incubation time for serum was 1 h, and the TMB color development time was 15 min, as shown in Table 3.

[0072] Table 3. Optimization of color development time and incubation time using the checkerboard method.

[0073]

[0074] Determine the cut-off value for this cELISA method:

[0075] One hundred known negative serum samples were selected to determine the cut-off value for the competitive ELISA. The optimal reaction conditions were followed, and the inhibition rate (PI value) of the tested negative serum samples was calculated. Calculation method: Inhibition rate (PI%) = (1 - P / N) × 100%, where P is the OD of the tested serum. 450nm N represents the OD of the negative control serum. 450nm Cut-off value = mean PI + 3 × SD (variance). 100 swine serum samples with negative SVA antibody test results were tested. The mean PI was 2.2%, and the SD was 5.7%. A cut-off value of 19.4% was set; serum samples with PI ≥ 19.4% were classified as positive, while samples with PI < 19.4% were considered negative.

[0076] cELISA method specificity evaluation:

[0077] Ten positive sera for African swine fever (ASFV), porcine reproductive and respiratory syndrome (PRRS), foot-and-mouth disease (FMD), and porcine circovirus (PCV), along with ten positive sera for SVA, were selected from laboratory-preserved samples for testing. The results were statistically analyzed to evaluate the specificity of the established method. The results are as follows: Figure 6 As shown, except for SVA positive serum test, the positive serum test results for other pathogens were all negative, proving that the method has good specificity.

[0078] Sensitivity evaluation of cELISA method:

[0079] Three known positive and three negative porcine serum samples were selected and tested using an established competitive ELISA method. Positive and negative sera were diluted at ratios of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280. The highest detectable dilution of positive serum was determined using the established competitive ELISA method to evaluate the sensitivity of the method. Results are as follows: Figure 7 As shown, the highest dilution ratio at which positive serum can be detected is 1:40, indicating that the method has good sensitivity.

[0080] cELISA method repeatability evaluation:

[0081] Four positive serum samples and four negative serum samples were selected and diluted for testing according to the established method. The experiment was divided into three batches, with three replicates within each batch. Finally, the differences between batches and between different replicates within the same batch were statistically analyzed to evaluate the reproducibility of the method. The results showed that the intra-batch coefficient of variation was 1.44%-3.27%, and the inter-batch coefficient of variation was 1.36%-8.71%, both less than 10%, indicating good reproducibility of the method (see Table 4).

[0082] Table 4. Reproducibility of the competitive enzyme-linked immunosorbent assay (ELISA) determined by detecting intra- and inter-plate coefficients of variation (CV%).

[0083]

[0084] cELISA method consistency evaluation:

[0085] One hundred clinical swine serum samples were used for testing using a self-developed cELISA reagent, a commercially available ELISA kit, and Western blot. To assess the consistency of results between the self-developed cELISA reagent and the commercially available ELISA kit, as well as between the cELISA reagent and Western blot, the Kappa index was calculated using SPSS software. All serum samples were tested three times. A kappa value less than 0.4 indicated poor consistency, while a kappa value greater than 0.4 indicated good consistency. The closer the kappa value was to 1, the higher the consistency. The results showed an 84% concordance rate between this method and the commercially available kit, but a kappa value of 0.35 indicated poor consistency, suggesting the possibility of quality instability between different batches of the commercially available kit. Compared with the results of the Western blot method, the concordance rate was 99%, with a kappa value of 0.97, indicating excellent consistency (see Table 5).

[0086] Table 5 Comparison of concordance rates between cELISA results, commercial kit results, and Western blot results.

[0087]

[0088] Nanobody nucleotide sequence (SEQ ID NO:1) as follows: CTGCAGGAGTCTGGGGGAGACTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGACACACCTACAGTAGGTACTGCATGGGCTGGTACCGCCAGGCTCGTGGGAAGGAGCGCGAGGGGGTCGCAGTTATTGAGGTTGATGGTAGCGCAAGCTACGCAGACTCC GTGAAGGGCCGATTCACCATCTCCAAAGACAACGCCAAGAACACTCTATATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCTATCAACTGCCACGACCCTAGCGACTATGTTGGCGGCTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGC.

[0089] The amino acid sequence of the nanobody (SEQ ID NO:2) is as follows: LQESGGDSVQAGGSLRLSCAASGHTYSRYCMGWYRQARGKEREGVAVIEVDGSASYADSVKGRFTISKDNAKNTLYLQMNSLKPEDTAMYYCAAINCHDPSDYVGGYWGQGTQVTVSSAA.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanobody that specifically binds to Seneca virus A, characterized in that, The nucleotide sequence of the nanobody is shown in SEQ ID NO:1, and the amino acid sequence of the nanobody is shown in SEQ ID NO:

2.

2. A method for detecting antibodies against Seneca virus A, characterized in that, Includes the following steps: (1) Provide an ELISA plate coated with recombinant SVA-VP1 protein; (2) Provide a detection antibody, wherein the detection antibody is an SVA-VP1 specific nanobody fused to express horseradish peroxidase in HEK293T cells; the SVA-VP1 specific nanobody is the nanobody according to claim 1; (3) Mix the serum to be tested with the detection antibody and then incubate it in an ELISA plate; (4) After washing, add colorimetric solution to develop color, measure OD value, calculate inhibition rate, and judge the result based on Cut-off value.

3. The method according to claim 2, characterized in that, The coating amount of the SVA-VP1 protein was 400 ng / well.

4. The method according to claim 2, characterized in that, The titer of the horseradish peroxidase-labeled SVA-VP1 specific nanobody was 1:

10. 3 The dilution ratio is 1:2 4 .

5. The method according to claim 2, characterized in that, It also includes the use of diluent, blocking solution, washing solution, positive serum, negative serum and stop solution.

6. The method according to claim 5, characterized in that, The diluent is PBS; the washing buffer is PBST buffer; the blocking buffer is 2.5% milk powder dissolved in PBST; the positive serum is clinical positive serum detected by Western blotting; the negative serum is clinical negative serum detected by Western blotting and SPF porcine serum; the chromogenic solution is single-component TMB chromogenic solution; and the stop solution is 3M sulfuric acid aqueous solution.

7. A kit for detecting Seneca virus A antibodies, characterized in that, The nanobody as described in claim 1 and the ELISA plate coated with SVA-VP1 protein.

8. The use of a nanobody as described in claim 1 or a kit as described in claim 7 in the preparation of a reagent or kit for detecting Seneca virus A antibodies.

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