A recombinant VP1 protein, test strip, and application of a foot-and-mouth disease virus SAT1 antibody

Abstract

This invention belongs to the field of biotechnology, specifically relating to a recombinant VP1 protein of foot-and-mouth disease virus (FMD) SAT1 type antibody, a test strip, and its applications. The amino acid sequence of the recombinant VP1 protein is shown in SEQ ID No. 1, exhibiting significant antigenicity and immunogenicity, making it a good target for diagnosing FMD SAT1 type. A colloidal gold test strip for detecting FMD SAT1 type antibody was also prepared. The colloidal gold test strip has a PVC base plate with a sample pad, a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad arranged sequentially. The gold-labeled pad is coated with gold-labeled recombinant VP1 protein. The test strip can specifically detect FMD SAT1 type antibody with a sensitivity of 1:128, and shows no cross-reactivity with bovine viral diarrhea virus, FMD type O virus, FMD type A virus, Seneca virus type A, brucellosis, and Escherichia coli infection, demonstrating good specificity and broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to a recombinant VP1 protein of foot-and-mouth disease virus SAT1 type antibody, a test strip, and its application. Background Technology

[0002] Foot-and-mouth disease (FMD) is an acute, febrile, and highly contagious disease of cloven-hoofed animals caused by the foot-and-mouth disease virus (FMDV). It has an extremely high infection rate, leading to high mortality rates in young animals and a severe decline in productivity in adult animals, significantly impacting the development of global livestock farming. FMDV includes seven different serotypes, such as O, A, C, SAT1, SAT2, SAT3, and Asia1. There is no cross-protection between different serotypes. Furthermore, the African buffalo, the natural reservoir host of FMDV, can carry the virus asymptomatically for more than five years, forming a stable natural focus of infection.

[0003] SAT1 foot-and-mouth disease virus is prevalent in southern Africa and has shown a risk of spreading beyond its traditional foci to other regions in recent years. SAT1 foot-and-mouth disease exhibits extremely rich genetic and antigenic diversity. This means that different lineages of SAT1 virus may have significant differences in antigenicity, making the diagnosis of SAT1 foot-and-mouth disease virus extremely complex and requiring diagnostic methods that can specifically differentiate SAT1 from other serotypes.

[0004] Therefore, in the absence of vaccine protection, in order to strictly prevent the introduction of SAT1 FMDV into my country, it is crucial to establish a field serological testing method for SAT1 FMDV as a technical reserve for import and export port quarantine and border area prevention and control screening. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a test strip capable of detecting SAT1 antibodies against foot-and-mouth disease virus. It is characterized by its ease of operation, lack of need for specialized equipment, and rapid result display, meeting the needs of rapid on-site testing. Specifically, it includes the following: In a first aspect, the present invention provides a recombinant VP1 protein for detecting SAT1 type antibodies against foot-and-mouth disease virus, the amino acid sequence of which is shown in SEQ ID No. 1.

[0006] In a second aspect, the present invention provides a gene sequence encoding the recombinant VP1 protein, the gene sequence being shown in SEQ ID No. 2.

[0007] Thirdly, the present invention provides the application of the recombinant VP1 protein in the preparation of reagents for detecting SAT1 type antibodies against foot-and-mouth disease virus.

[0008] Fourthly, the present invention provides a colloidal gold test strip for detecting SAT1 type antibodies against foot-and-mouth disease virus. The test strip includes a PVC base plate, a sample pad, a gold-labeled pad, a nitrocellulose membrane, an absorbent pad, a detection line, and a control line. The recombinant VP1 protein labeled with colloidal gold is immobilized on the gold-labeled pad.

[0009] Preferably, the sample pad, gold label pad, nitrocellulose membrane, and absorbent pad are stacked sequentially and placed on a PVC base plate, and the nitrocellulose membrane is provided with detection and quality control lines.

[0010] Preferably, the detection line is labeled with the recombinant VP1 protein as described in claim 1, and the quality control line is labeled with a polyclonal antibody against the recombinant VP1 protein.

[0011] Preferably, the amount of the recombinant VP1 protein of claim 1 labeled with colloidal gold is 5 μL / cm.

[0012] Preferably, the detection line is prepared by diluting the recombinant VP1 protein to 0.6 mg / mL and then drawing the line, with a coating volume of 1 μL / cm.

[0013] Preferably, the control line is prepared by diluting the recombinant VP1 protein polyclonal antibody to 2 mg / mL and then drawing the line, with a coating volume of 1 μL / cm.

[0014] Fifthly, the present invention provides a method for preparing the colloidal gold test strip, the method comprising the following steps: (1) Prepare recombinant VP1 protein and obtain a detection line by streaking it on a nitrocellulose membrane; (2) Prepare a polyclonal antibody against recombinant VP1 protein and obtain a quality control line by streaking it on a nitrocellulose membrane; (3) Prepare colloidal gold-labeled recombinant VP1 protein and fix the colloidal gold-labeled recombinant VP1 protein on a gold-labeled pad; (4) After wetting the sample pad and gold label pad with the blocking solution, place them in the oven to dry. Then assemble the PVC base plate, sample pad, gold label pad, nitrocellulose membrane and absorbent pad together to obtain the colloidal gold test strip for detecting SAT1 type antibody of foot-and-mouth disease virus.

[0015] In a sixth aspect, the present invention provides the application of the colloidal gold test strip in the detection of SAT1 type antibodies against foot-and-mouth disease virus for non-disease diagnosis purposes.

[0016] Preferably, the detection specifically includes the following steps: (1) Sample preparation: Collect blood samples from the animals to be tested, let them stand at room temperature or 4°C, centrifuge at 3000 rpm for 10-15 min, and dilute the serum with PBS at a ratio of 1:10. (2) Sample detection: Add 100 μL of serum sample to the sample pad of the test strip and observe the results after 10 min to 15 min at room temperature; (3) Result determination: Positive: When the gold-labeled recombinant VP1 protein can bind to the recombinant VP1 protein polyclonal antibody on the control line C, a red band will appear on the control line; if there is a foot-and-mouth disease SAT1 type VP1 protein antibody in the serum, it will bind to the gold-labeled recombinant VP1 protein on the gold pad and bind to the recombinant VP1 protein labeled on the test line T to form a double antigen sandwich, at which time a red band will appear on the T line and the result is judged as positive; Negative: When the gold-labeled recombinant VP1 protein can bind to the recombinant VP1 protein polyclonal antibody of the control line C, a red band appears on the control line. If there is no foot-and-mouth disease SAT1 type VP1 protein antibody in the serum, no red band appears on the T line, and the result is judged as negative. Invalid: If the gold-labeled recombinant VP1 protein does not bind to the recombinant VP1 protein polyclonal antibody on control line C, then there will be no red band on control line C, indicating that the test strip is invalid.

[0017] Compared with the prior art, the present invention has the following advantages: (1) The present invention obtains soluble recombinant VP1 protein by fusing the recombinant VP1 gene with the pMAL-c2X vector, expressing it in prokaryotes and purifying it. According to Western blot verification, the recombinant VP1 protein can react with positive serum of foot-and-mouth disease virus SAT1 subunit vaccine and can be used as a good target for diagnosing foot-and-mouth disease virus SAT1. (2) The present invention prepares a colloidal gold test strip for detecting SAT1 type antibodies of foot-and-mouth disease virus. The test strip has a sample pad, a gold-labeled pad, a nitrocellulose membrane and an absorbent pad arranged in sequence on the PVC base plate. The gold-labeled pad is coated with gold-labeled recombinant VP1 protein. The test strip of the present invention can specifically detect SAT1 type antibodies of foot-and-mouth disease virus with a sensitivity of 1:128. It has no cross-reactivity with bovine viral diarrhea virus, foot-and-mouth disease type O virus, foot-and-mouth disease type A virus, sheep stomatitis virus, brucellosis and coliosis, and has good specificity. (3) This invention uses polyclonal antibodies as a quality control line, which shortens the preparation time and reduces production costs; (4) The test strip of the present invention has the advantages of high sensitivity, good specificity, simple operation and rapid detection; (5) This invention is suitable for rapid on-site detection and helps in the early detection and control of foot-and-mouth disease virus type SAT1; (6) The preparation process of this invention is simple and easy to industrialize and promote. Attached Figure Description

[0018] Figure 1 The schematic diagram of the test strip structure provided by the present invention shows that 1 is a PVC base plate, 2 is a sample pad, 3 is a gold label pad, 4 is a nitrocellulose membrane, 5 is a detection line, 6 is a quality control line, and 7 is an absorbent pad.

[0019] Figure 2 SDS-PAGE analysis of recombinant VP1 protein was performed to assess its induced expression and purification. M represents the relative molecular mass of the protein. Lane 1 contains the supernatant from the ultrasonically disrupted pMAL-c2X-VP1 induced bacterial culture. Lane 2 contains the supernatant from the ultrasonically disrupted pMAL-c2X-VP1 induced bacterial culture. Lane 3 contains the washed-off protein. Lanes 4-9 contain the purified recombinant VP1 protein eluted from the previous lanes.

[0020] Figure 3 Western blot validation of reactivity of recombinant VP1 protein.

[0021] Figure 4 The test strip judgment criteria provided by this invention.

[0022] Figure 5 Determination of the optimal pH for colloidal gold labeling of recombinant VP1 protein.

[0023] Figure 6 Determination of the optimal protein amount for colloidal gold labeling of recombinant VP1 protein.

[0024] Figure 7 Specificity detection of colloidal gold test strips.

[0025] Figure 8 Sensitivity testing of colloidal gold test strips. Detailed Implementation

[0026] The technical solution of the present invention will be described in detail below through specific embodiments. It should be understood that these embodiments are only for illustrating the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various equivalent changes and modifications based on the teachings of the present invention without departing from the spirit and scope of the present invention.

[0027] It should be noted that the methods described in the following embodiments are all conventional methods, and the reagents described are all commercially available.

[0028] Example 1: Preparation of colloidal gold test strip for detecting SAT1 type antibody against foot-and-mouth disease virus This embodiment provides a colloidal gold test strip for detecting SAT1 type antibodies against foot-and-mouth disease virus, such as... Figure 1As shown: It includes five parts: a PVC base plate (1), a sample pad (2), a gold label pad (3), a nitrocellulose membrane (4), and an absorbent pad (7). The PVC base plate has a sample pad at one end, a gold label pad at the bottom, and a nitrocellulose membrane at the bottom. The absorbent pad is located at the other end of the PVC base plate and on top of the nitrocellulose membrane. The nitrocellulose membrane has a detection line (5) and a quality control line (6). The specific preparation includes the following steps: (1) Construction and identification of recombinant expression vectors A gene expressing the recombinant VP1 protein of foot-and-mouth disease virus SAT1 type was constructed through sequence optimization (SEQ ID No. 2, amino acid sequence as shown in SEQ ID No. 2), and was sent to Wuhan Jinkairui Biotechnology Co., Ltd. to synthesize and construct it into the pMAL-c2X vector. The recombinant plasmid was named pMAL-c2X-VP1. The constructed pMAL-c2X-VP1 plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and a single positive recombinant colony was picked and inoculated into LB liquid medium containing ampicillin.

[0029] (2) Prokaryotic expression, purification and identification of recombinant VP1 protein Select a single positive recombinant bacterium and inoculate it into LB liquid medium containing ampicillin and culture overnight. Then, add 200 mL of LB medium at a ratio of 1:100 and culture for 8-10 h. Finally, add IPTG (final concentration of 1 mmol / L) and incubate overnight at 16°C.

[0030] The bacterial culture was collected by centrifugation, the cells were resuspended in PBS, and after sonication, the supernatant was collected by centrifugation. The recombinant VP1 protein was then purified using a Dextrilin Beads 6FF column, specifically as follows: a. Prepare the Dextrin Beads 6FF column: Take an appropriate amount of Dextrin Beads 6FF column, load 2-3 ml of sample and equilibrate.

[0031] b. Cell disruption and protein extraction: Cells expressing recombinant VP1 protein were disrupted by sonication, centrifuged to remove cell debris and insoluble substances, and the supernatant was collected.

[0032] c. Sample loading: Slowly add the collected supernatant into the equilibrated Dextrin Beads 6FF column, control the flow rate, and collect the flow-through liquid.

[0033] d. Washing: Wash with binding buffer and collect the washing solution.

[0034] e. Elution: Elution is performed using an elution buffer containing maltose.

[0035] f. Collect the eluent and perform SDS-PAGE analysis to determine the purity of the eluted protein.

[0036] g. Protein storage: After the protein concentration is determined by the BCA method, the purified protein is aliquoted and stored at -80℃.

[0037] The results of SDS-PAGE analysis of recombinant VP1 protein induction expression and purification are as follows: Figure 2 As shown, after overnight induction of recombinant VP1 protein expression in E. coli at 16°C, SDS-PAGE results showed a clear expression band at approximately 66 kDa, consistent with the expected size, indicating successful protein expression. After sonication, the recombinant bacteria mainly expressed the protein in the form of soluble protein. The purified protein band obtained by maltose column chromatography had high purity and was consistent with the size of the expressed band, indicating successful purification of the target protein.

[0038] FMDV-SAT1 subunit vaccine immunogenic serum (provided by the National Foot-and-Mouth Disease Reference Laboratory) was used as the primary antibody (1:2000 dilution), and HRP-labeled goat anti-bovine antibody (1:5000 dilution) was used as the secondary antibody for Western blot identification. The Western blot identification steps are as follows: a. Electrophoresis: Load the processed recombinant VP1 protein sample into an SDS-PAGE gel. Perform electrophoresis at a constant current until the bromophenol blue indicator reaches the appropriate position.

[0039] b. Transfer: Place the SDS-PAGE adhesive and film in a transfer apparatus and perform the transfer using the wet transfer method under a constant current.

[0040] c. Membrane treatment: After transfer, wash with TBST and place the membrane in a 5% skim milk blocking solution, and gently shake to block for 2 h at room temperature.

[0041] d. Primary antibody incubation: After washing the membrane with TBST, add the specific primary antibody (1:200 dilution) and incubate overnight at 4°C.

[0042] e. Secondary antibody incubation: Wash the membrane with TBST, add HRP-labeled secondary antibody (1:5000 dilution), and incubate at room temperature for 1 h.

[0043] f. Color development: After mixing the luminescent solutions A and B, add them to the nitrocellulose membrane in the dark to ensure full adhesion. Then, place the membrane into a membrane scanning instrument for scanning and take a picture for storage.

[0044] The results of the reactivity identification of recombinant VP1 protein are as follows: Figure 3As shown, recombinant VP1 protein was identified by Western blot. After staining, a single and specific clear target band appeared, indicating that recombinant VP1 protein reacts well with positive serum.

[0045] (3) Preparation of polyclonal antibodies Recombinant VP1 protein was administered to New Zealand white rabbits via subcutaneous immunization at multiple sites on the back. After the third immunization, blood was collected from the heart, serum was separated by centrifugation, and polyclonal antibody titers were determined using an indirect ELISA method. IgG from the serum was purified using a GE Healthcare Protein G Sepharose™ antibody affinity chromatography column, and the concentration was measured by BCA before storage at -80°C for later use. The steps for purifying serum IgG using antibody affinity chromatography are as follows: a. Sample loading: Load the processed serum sample onto a Protein G Sepharose column that has been pre-equilibrated with equilibration buffer.

[0046] b. Washing: Wash the column with equilibration buffer (20 mM Na2HPO4, 0.15 M NaCl, pH 7.0) to remove non-specifically bound proteins.

[0047] c. Elution: Elute the specifically bound IgG using elution buffer (0.1 M glycine, pH 2.5).

[0048] d. Neutralization and Storage: The eluted IgG needs to be adjusted to neutral pH using neutralization buffer (1 M Tris) to restore antibody activity. The purified IgG can be stored in an appropriate buffer at low temperature.

[0049] (4) Preparation of recombinant VP1 protein-colloidal gold label 0.1 mol / L K2CO3 was slowly added to the colloidal gold solution until the pH reached 7. Recombinant VP1 protein with a final concentration of 0.24 mg / ml was added, and the mixture was stirred for 20 min. Then, bovine serum albumin (BSA) with a final concentration of 10% was added, and the mixture was stirred for 15 min. After centrifugation at 8000 rpm for 15 min, the supernatant was discarded. The reconstitution solution (10 mM Tris-HCl, 1% BSA, 10% sucrose, pH = 8.5) was reconstituted at one-tenth the volume of the original solution to obtain the recombinant VP1 protein-colloidal gold label. The recombinant VP1 protein-colloidal gold label was sprayed onto a gold-labeled pad and dried at 37 °C for 1 h to obtain the colloidal gold pad.

[0050] (5) Preparation of colloidal gold test strip for detecting SAT1 antibody against foot-and-mouth disease virus The sample pad and gold label pad are pre-soaked in blocking solution (PBS solution of 0.75% trehalose, 0.5% sucrose, 2% BSA, 0.5% Tween-20 and Triton X-100) and then dried in an oven. The PVC base plate, sample pad, gold label pad, nitrocellulose membrane and absorbent pad are then assembled together to obtain the colloidal gold test strip for detecting SAT1 antibody against foot-and-mouth disease virus.

[0051] (6) Criteria for judging test strips The criteria for judging the test strip are as follows: Figure 4 As shown, where: Positive: When the gold-labeled recombinant VP1 protein can bind to the recombinant VP1 protein polyclonal antibody on the control line C, a red band will appear on the control line; if there is VP1 protein antibody in the serum, it will bind to the gold-labeled recombinant VP1 protein on the gold pad and bind to the recombinant VP1 protein labeled on the test line T to form a double antigen sandwich, at which time a red band will appear on the T line and the result is judged as positive. Negative: When the gold-labeled recombinant VP1 protein can bind to the recombinant VP1 protein polyclonal antibody of the control line C, a red band appears on the control line. If there is no VP1 protein antibody in the serum, no red band appears on the T line, and the result is judged as negative. Invalid: If the gold-labeled recombinant VP1 protein does not bind to the recombinant VP1 protein polyclonal antibody on control line C, then there will be no red band on control line C, indicating that the test strip is invalid.

[0052] Example 2: Determination of optimal pH and optimal amount of labeled protein for colloidal gold-labeled recombinant VP1 protein (1) Optimal pH determination method for colloidal gold-labeled recombinant VP1 protein in this embodiment a. Take six 1.5 mL sterile centrifuge tubes, add 1 mL of colloidal gold solution to each, and adjust the pH to 5–9 with 0.1 mol / L K2CO3.

[0053] b. Add a fixed amount of protein to the gold solution at each pH gradient, let stand at room temperature for 2 h, and then centrifuge at 12000 rpm / min for 30 min at 4℃.

[0054] c. Collect the supernatant, coat it with an ELISA plate, use rabbit anti-VP1 protein serum as the primary antibody (1:100 dilution), and HRP-labeled goat anti-rabbit IgG as the secondary antibody (1:25,000 dilution). Detect the protein content in the supernatant using an indirect ELISA method.

[0055] d. Read OD using an ELISA reader 450nm Values, with pH value on the x-axis and OD value on the y-axis. 450nm Plot a scatter plot with OD values ​​on the ordinate. 450nmThe lowest value indicates that the amount of unlabeled protein in the gold solution supernatant is the least at this pH, which is the optimal pH.

[0056] The results are as follows Figure 5 As shown, the optimal pH for colloidal gold labeling of recombinant VP1 protein is 7.

[0057] (2) Method for determining the amount of colloidal gold-labeled recombinant VP1 protein in this embodiment a. Dilute VP1 protein with PBS to 1, 0.5, 0.2, 0.1, 0.05, 0.02, and 0.01 mg / mL.

[0058] b. Take 0.1 mL of the above solution and add it to the gold solution whose pH has been adjusted. Mix thoroughly and let stand at room temperature for 8 min to 10 min.

[0059] c. Add 0.1 mL of 10% NaCl solution, mix thoroughly, and observe the color change after 2 h.

[0060] d. The minimum amount of recombinant VP1 protein added to keep the solution red is the optimal amount of labeled protein.

[0061] The results are as follows Figure 6 As shown, the amount of labeled recombinant VP1 protein in colloidal gold was 0.24 mg / mL.

[0062] Experiment Example 3: Characteristic Detection of Colloidal Gold Test Strip Method 1. Specificity The test strips prepared above were used to detect positive sera for BVDV, FMDV-O, FMDV-A, ORFV, brucellosis, and Escherichia coli, with FMDV-SAT1 positive sera used as a control, thereby identifying their specificity (positive sera for BVDV, FMDV-O, FMDV-A, ORFV, brucellosis, and Escherichia coli were all identified and preserved by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences).

[0063] The results are as follows Figure 7 As shown, the test strip prepared according to the present invention was negative for BVDV, FMDV-O, FMDV-A, ORFV, brucellosis and Escherichia coli positive sera, indicating that the test strip has good specificity.

[0064] 2. Sensitivity FMDV SAT1 positive serum was serially diluted from 1:2 to 1:256 using sample dilution buffer. 100 μL of each solution was added to the sample pad and incubated at room temperature for 10 to 15 minutes to observe the results, thereby determining its sensitivity.

[0065] The results are as follows Figure 8 As shown, the positive or negative result was determined based on the appearance of the T line. The results showed that the lowest dilution for detecting positive serum of foot-and-mouth disease SAT1 type was 1:128, indicating that the test strip prepared in this study has good sensitivity.

[0066] 3. Stability The test strips prepared according to the present invention were stored at room temperature (18℃~25℃) and 4℃ respectively, and the stability of the test strips was tested at 0, 1, 3 and 6 months.

[0067] The results are shown in Table 1. The test strips can be stably stored for more than 6 months at both room temperature and 4 degrees Celsius.

[0068] Table 1. Results of stability test on colloidal gold test strips

[0069] In summary, this invention first prepared a recombinant VP1 protein of foot-and-mouth disease virus (FMD) SAT1 type antibody. The recombinant VP1 protein exhibits significant antigenicity and immunogenicity, making it a good target for diagnosing FMD SAT1 type. Secondly, this invention prepared a colloidal gold test strip for detecting FMD SAT1 type antibodies. The test strip has a PVC base plate with a sample pad, a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad arranged sequentially. The gold-labeled pad is coated with gold-labeled recombinant VP1 protein. The test strip of this invention can specifically detect FMD SAT1 type antibodies with a sensitivity of 1:128, and shows no cross-reactivity with bovine viral diarrhea virus, FMD type O virus, FMD type A virus, sheep stomatitis virus, brucellosis, or Escherichia coli infection, demonstrating good specificity and broad application prospects.

[0070] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0071] 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 recombinant VP1 protein of a foot-and-mouth disease virus SAT1 type antibody, characterized in that, The amino acid sequence of the recombinant VP1 protein is shown in SEQ ID No.

1.

2. The gene sequence encoding the recombinant VP1 protein of claim 1, characterized in that, The gene sequence is shown in SEQ ID No.

2.

3. The use of the recombinant VP1 protein as described in claim 1 in the preparation of a reagent for detecting SAT1 type antibodies against foot-and-mouth disease virus.

4. A colloidal gold test strip for detecting SAT1 type antibodies against foot-and-mouth disease virus, the test strip comprising a PVC base plate, a sample pad, a gold-labeled pad, a nitrocellulose membrane, an absorbent pad, a detection line, and a control line; characterized in that, The recombinant VP1 protein of claim 1 is immobilized on the gold-labeled pad with a colloidal gold label.

5. The colloidal gold test strip as described in claim 4, characterized in that, The sample pad, gold label pad, nitrocellulose membrane, and absorbent pad are sequentially overlapped and placed on a PVC base plate. The nitrocellulose membrane is equipped with detection and quality control lines.

6. The colloidal gold test strip as described in claim 5, characterized in that, The detection line is labeled with the recombinant VP1 protein as described in claim 1, and the quality control line is labeled with a polyclonal antibody against the recombinant VP1 protein.

7. The colloidal gold test strip as described in claim 4, characterized in that, The amount of recombinant VP1 protein used in claim 1 with colloidal gold labeling is 5 μL / cm.

8. The method for preparing colloidal gold test strips as described in any one of claims 4-7, characterized in that, The method includes the following steps: (1) Prepare recombinant VP1 protein and obtain a detection line by streaking it on a nitrocellulose membrane; (2) Prepare a polyclonal antibody against recombinant VP1 protein and obtain a quality control line by streaking it on a nitrocellulose membrane; (3) Prepare colloidal gold-labeled recombinant VP1 protein and fix the colloidal gold-labeled recombinant VP1 protein on a gold-labeled pad; (4) After wetting the sample pad and gold label pad with the blocking solution, place them in the oven to dry. Then assemble the PVC base plate, sample pad, gold label pad, nitrocellulose membrane and absorbent pad together to obtain the colloidal gold test strip for detecting SAT1 type antibody of foot-and-mouth disease virus.

9. The use of the colloidal gold test strip according to any one of claims 4-7 in the detection of SAT1 type antibodies against foot-and-mouth disease virus for non-disease diagnosis purposes.

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