A colloidal gold test strip for detecting sheep pox virus antibodies, its preparation method and application

Abstract

This invention relates to a colloidal gold test strip for detecting sheep pox virus antibodies, its preparation method, and its application. First, this invention prepares recombinant 122 protein, which exhibits significant antigenicity and immunogenicity, making it a good target for diagnosing sheep pox virus. Second, this invention prepares a colloidal gold test strip for detecting sheep pox virus 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 122 protein. The test strip of this invention can simultaneously detect antibodies against sheep pox virus, bovine nodular dermatosis virus, and goat pox virus, with sensitivities of 1:128, 1:128, and 1:64, respectively. It shows no cross-reactivity with bovine viral diarrhea virus, foot-and-mouth disease virus type O, foot-and-mouth disease virus type A, sheep stomatitis virus, brucellosis, and Escherichia coli, exhibiting good specificity and a diagnostic concordance rate of 93%.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to a colloidal gold test strip for detecting sheep pox virus antibodies, its preparation method, and its application. Background Technology

[0002] Capripoxvirus (CaPV) is a member of the Chorlocvirinae subfamily of the Poxviridae family. It comprises bovine nodular skin disease virus (LSDV), goatpox virus (GTPV), and sheep pox virus (SPPV), which can infect cattle, goats, and sheep, causing bovine nodular skin disease, goatpox, and sheep pox, respectively. Infected animals exhibit widespread nodules and edema on the skin and organ surfaces, a sharp decrease in milk production, and a significant decline in coat quality. Due to its potential for rapid cross-species transmission, it can spread rapidly in endemic areas, causing substantial economic losses and severely impacting cattle and sheep farming. The World Organization for Animal Health (WOAH) lists it as a reportable animal disease.

[0003] CaPV has a severe impact on livestock development and causes significant losses to the global economy and trade. Currently, there are no effective treatments for CaPV; prevention is the primary focus. Monitoring the disease using sensitive and specific diagnostic methods is the best way to control its incidence and eradicate it in any region. The CaPV genome is a double-stranded, linear, covalently closed DNA, approximately 150 kb in length, encoding about 150 proteins. The p32 protein is a major structural protein of the sheep poxvirus genus, involved in viral particle assembly, highly immunogenic, and contains major antigenic epitopes. In the early stages of infection, it can stimulate the host to produce antibodies, thereby preventing viral spread. Several serological detection methods for sheep pox virus have been established based on this protein.

[0004] Methods for detecting sheep pox virus antibodies include virus neutralization assay (VNT), enzyme-linked immunosorbent assay (ELISA), agar diffusion assay (AGID), and indirect immunofluorescence assay (IFA). While VNT is currently the gold standard for serological detection of CaPV, it is time-consuming and lacks sufficient sensitivity. ELISA, AGID, and IFA are respectively characterized by cumbersome procedures, low sensitivity, time consumption, and high cost. Antigen diagnostic methods include PCR, qPCR, and virus isolation and identification. These methods require expensive equipment and professional personnel, and are time-consuming, hindering their widespread adoption at the grassroots level. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a test strip capable of simultaneously detecting antibodies against three viruses: sheep poxvirus, bovine nodular dermatosis virus, and goat poxvirus. 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:

[0006] In a first aspect, the present invention provides a recombinant 122 protein for detecting sheep pox virus antibodies, the amino acid sequence of which is shown in SEQ ID No. 1.

[0007] Secondly, the present invention provides the application of the recombinant 122 protein described in the first aspect above in the preparation of reagents for detecting sheep pox virus antibodies.

[0008] Preferably, the sheep pox virus includes sheep pox virus, bovine nodular dermatosis virus, and goat pox virus.

[0009] Thirdly, the present invention provides a colloidal gold test strip for detecting sheep pox virus antibodies, 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; the gold-labeled pad is immobilized with the recombinant 122 protein described in the first aspect above, which is labeled with colloidal gold.

[0010] Preferably, the sample pad is placed at one end of the PVC base plate, a gold label pad is placed under the sample pad, a nitrocellulose membrane is placed under the gold label pad, the absorbent pad is placed at the other end of the PVC base plate and on top of the nitrocellulose membrane, and a detection and quality control line is provided on the nitrocellulose membrane between the gold label pad and the absorbent pad.

[0011] Preferably, the detection line is labeled with the recombinant 122 protein described in the first aspect above, and the control line is labeled with a polyclonal antibody against the recombinant 122 protein.

[0012] Preferably, the amount of the colloidal gold-labeled recombinant 122 protein described in the first aspect above is 5 μL / cm.

[0013] Preferably, the detection line is prepared by diluting the recombinant 122 protein to 0.6 mg / mL and then drawing the line, with a coating volume of 1 μL / cm.

[0014] Preferably, the control line is prepared by diluting the recombinant 122 protein polyclonal antibody to 2 mg / mL and then drawing the line, with a coating volume of 1 μL / cm.

[0015] Fourthly, the present invention provides a method for preparing the colloidal gold test strip described in the third aspect above, the method comprising the following steps:

[0016] (1) Prepare recombinant 122 protein and obtain detection lines by streaking it on a nitrocellulose membrane;

[0017] (2) Prepare a polyclonal antibody against recombinant 122 protein and obtain a quality control line by streaking it on a nitrocellulose membrane;

[0018] (3) Prepare colloidal gold-labeled recombinant 122 protein and fix the colloidal gold-labeled recombinant 122 protein onto a gold-labeled pad to prepare a gold-labeled pad;

[0019] (4) After wetting the sample pad and gold label pad with the sealing solution, place them in an 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 sheep pox virus antibodies.

[0020] Preferably, the preparation method of the recombinant 122 protein is as follows: the 122 gene shown in SEQ ID No. 2 is constructed into the pET-SUMO vector to obtain the recombinant plasmid pET-SUMO-122; the constructed pET-SUMO-122 plasmid is transformed into Escherichia coli BL21(DE3) competent cells, a single positive recombinant colony is picked and inoculated into LB liquid medium containing kanamycin and cultured overnight, 200 mL of LB medium is added at a ratio of 1:100 and cultured for 8 h to 10 h, then IPTG (final concentration of 1 mmol / L) is added and cultured overnight at 16℃; the bacterial culture is collected by centrifugation, the bacterial cells are resuspended in PBS, the cells are sonicated and centrifuged to collect the supernatant, and the recombinant 122 protein is purified by Ni-NTA to obtain the recombinant 122 protein;

[0021] Preferably, the preparation method of the polyclonal antibody is as follows: the prepared recombinant 122 protein is used to immunize New Zealand white rabbits subcutaneously at multiple sites on the back. After the third immunization, blood is collected from the heart, the serum is separated by centrifugation, and the IgG in the serum is purified using a GE Healthcare ProteinGS Sepharose™ antibody affinity chromatography column to obtain the polyclonal antibody of the recombinant 122 protein. After BCA concentration measurement, it is stored at -80°C for later use.

[0022] Preferably, the preparation method of the gold-labeled pad is as follows: 0.1 mol / L K2CO3 is slowly added to the colloidal gold solution until the pH is 7, recombinant 122 protein with a final concentration of 0.24 mg / ml is added, and after stirring for 20 min, bovine serum albumin (BSA) with a final concentration of 10% is added. After stirring for 15 min, the mixture is centrifuged, the supernatant is discarded, and the reconstituted solution is reconstituted at one-tenth the volume of the original solution to obtain the recombinant 122 protein-colloidal gold label. The recombinant 122 protein-colloidal gold label is sprayed onto the gold-labeled pad and dried at 37°C for 1 h to obtain the colloidal gold pad.

[0023] Preferably, the concentration of recombinant 122 protein in the recombinant 122 protein-colloidal gold label is 0.24 mg / ml, and the amount used is 5 μL / cm.

[0024] Preferably, the detection line is prepared by diluting the recombinant 122 protein with PBS to 0.6 mg / mL and then drawing the line, with a coating volume of 1 μL / cm.

[0025] Preferably, the control line is prepared by diluting the polyclonal antibody of recombinant 122 protein to 2 mg / mL with PBS and then drawing the line, with a coating volume of 1 μL / cm.

[0026] Preferably, the centrifugation speed during the preparation of the gold label pad is 8000 rpm / min, and the centrifugation time is 15 min.

[0027] Preferably, the reconstitution solution in the preparation process of the gold label pad is 10mM Tris-HCl, 1% BSA, 10% sucrose, and pH=8.5.

[0028] Preferably, the blocking solution is a PBS solution containing 0.75% trehalose, 0.5% sucrose, 2% BSA, 0.5% Tween-20 and Triton X-100.

[0029] Fourthly, the present invention provides the application of the colloidal gold test strip described in the third aspect above in the detection of antibodies against sheep poxvirus, bovine nodular dermatovirus, and goat poxvirus for non-disease diagnosis purposes.

[0030] Preferably, the application includes the following steps:

[0031] (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.

[0032] (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;

[0033] (3) Result determination:

[0034] Positive: When the gold-labeled recombinant 122 protein can bind to the recombinant 122 protein polyclonal antibody on the control line C, a red band will appear on the control line; if there is 122 protein antibody in the serum, it will bind to the gold-labeled recombinant 122 protein on the gold pad and bind to the recombinant 122 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;

[0035] Negative: When the gold-labeled recombinant 122 protein can bind to the recombinant 122 protein polyclonal antibody of the control line C, a red band appears on the control line. If there is no 122 protein antibody in the serum, no red band appears on the T line, and the result is judged as negative.

[0036] Invalid: If the gold-labeled recombinant 122 protein does not bind to the recombinant 122 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.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] (1) The present invention obtains recombinant 122 protein by fusing the 122 gene with the pET-SUMO vector, expressing it in prokaryotes and purifying it. Western blot verification shows that the recombinant 122 protein can react with sheep pox virus, bovine nodular dermatovirus and goat pox virus, and can be used as a good target for diagnosing sheep pox virus.

[0039] (2) This invention prepares a colloidal gold test strip for detecting sheep pox virus antibodies. The test strip has a sample pad, a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad arranged sequentially on a PVC base plate. The gold-labeled pad is coated with gold-labeled recombinant 122 protein. The test strip of this invention can simultaneously detect antibodies against sheep pox virus, bovine nodular dermatosis virus, and goat pox virus, with sensitivities of 1:128, 1:128, and 1:64, respectively. It shows 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 Escherichia coli, exhibiting good specificity and a diagnostic accuracy rate of 93%.

[0040] (3) This invention uses polyclonal antibodies as quality control lines, which shortens the preparation time and reduces production costs;

[0041] (4) The test strip of the present invention has the advantages of high sensitivity, good specificity, simple operation and rapid detection;

[0042] (5) This invention is suitable for rapid on-site detection and helps in the early detection and control of sheep pox virus;

[0043] (6) The preparation process of this invention is simple and easy to industrialize and promote. Attached Figure Description

[0044] Figure 1 Results of ORF122 gene analysis;

[0045] Figure 2 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.

[0046] Figure 3 SDS-PAGE analysis of recombinant protein showed its induction expression and purification, where M represents the relative molecular mass of the protein; lane 1 was the uninduced pET-SUMO-122 bacterial culture; lane 2 was the induced pET-SUMO-122 bacterial culture; lane 3 was the supernatant of the induced pET-SUMO-122 bacterial culture after sonication; lane 4 was the precipitate of the induced pET-SUMO-122 bacterial culture after sonication; and lane 5 was the purified recombinant 122 protein.

[0047] Figure 4 Recombinant protein reactivity was determined, where M is the relative molecular mass of the protein; lane 1 is SPPV positive serum; lane 2 is SPPV negative serum; lane 3 is LSDV positive serum; lane 4 is LSDV negative serum; lane 5 is GTPV positive serum; and lane 6 is GTPV negative serum.

[0048] Figure 5 The test strip judgment criteria provided by this invention.

[0049] Figure 6 Determination of the optimal pH for colloidal gold labeling of recombinant 122 protein.

[0050] Figure 7 Determination of the optimal protein amount for colloidal gold labeling of recombinant 122 protein.

[0051] Figure 8 Specificity detection of colloidal gold test strips.

[0052] Figure 9 Sensitivity testing of colloidal gold test strips, where A represents the sensitivity of the test strip to SPPV, B represents the sensitivity of the test strip to LSDV, C represents the sensitivity of the test strip to GTPV, and D represents the sensitivity of the ELISA antibody detection kit to SPPV, LSDV, and GTPV, respectively.

[0053] Figure 10 Repeatability testing of colloidal gold test strips, where A is LSDV positive serum and B is LSDV negative serum. Detailed Implementation

[0054] 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.

[0055] This invention selected 17 CaPV strains, including 4 GTPV strains, 9 LSDV strains, and 4 SPPV strains, for homology analysis. Gene sequence comparison analysis showed that the homology among the 17 GTPV, LSDV, and SPPV strains was no less than 93.4%, confirming the high conservation of the ORF122 gene (e.g., Figure 1 (As shown).

[0056] Example 1: Preparation of colloidal gold test strip for detecting sheep pox virus antibodies

[0057] This embodiment provides a colloidal gold test strip for detecting sheep pox virus antibodies, such as... Figure 2 As 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:

[0058] (1) Construction and identification of recombinant expression vectors

[0059] The 122 gene (accession number: OP508345.1) was retrieved from the GenBank database and truncated. The truncated 122 gene (SEQ ID No. 2) was sent to Wuhan Jinkairui Biotechnology Co., Ltd. to synthesize and construct it into the pET-SUMO vector. The recombinant plasmid was named pET-SUMO-122. The constructed pET-SUMO-122 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 kanamycin.

[0060] (2) Prokaryotic expression, purification and identification of recombinant 122 protein

[0061] Select a single positive recombinant bacterium and inoculate it into LB liquid medium containing kanamycin and culture it overnight. Then, add 200 mL of LB medium at a ratio of 1:100 and culture for 8-10 hours. Finally, add IPTG (final concentration of 1 mmol / L) and incubate overnight at 16°C.

[0062] The bacterial culture was collected by centrifugation, the bacterial cells were resuspended in PBS, and after sonication, the supernatant was collected by centrifugation. The recombinant 122 protein was purified using Ni-NTA, specifically as follows:

[0063] a. Prepare Ni-NTA resin: Take an appropriate amount of Ni-NTA resin, load 2-3 ml of the sample and equilibrate.

[0064] b. Cell disruption and protein extraction: Cells expressing recombinant 122 protein were disrupted by sonication, centrifuged to remove cell debris and insoluble substances, and the supernatant was collected.

[0065] c. Sample loading: Slowly add the collected supernatant into the equilibrated Ni-NTA resin column, controlling the flow rate, and collect the flow liquid.

[0066] d. Washing: Wash the resin column with a washing buffer containing 8M urea and collect the washing solution.

[0067] e. Elution: Elute the resin column using 250mM imidazole eluent containing 8M urea.

[0068] f. Collect the eluent and perform SDS-PAGE analysis to determine the purity of the eluted protein.

[0069] g. Protein storage: After the protein concentration is determined by the BCA method, the protein is aliquoted and stored at -80℃.

[0070] The results of SDS-PAGE analysis of recombinant protein induction expression and purification are as follows: Figure 3 As shown, after overnight induction of recombinant protein 122 in E. coli at 16°C, SDS-PAGE results showed a clear expression band at approximately 35 kDa, consistent with the expected size, indicating successful protein expression. Protein products were present in both the supernatant and precipitate after sonication of the recombinant bacteria, but mainly expressed in the form of soluble protein. The protein band purified by nickel column chromatography was single and clear, and consistent with the size of the expressed band, indicating successful purification of the target protein.

[0071] Standard positive and negative sera of SPPV, LSDV, and GTPV were used as primary antibodies (1:200 dilution), and HRP-labeled rabbit anti-mongol / goat IgG and goat anti-bovine IgG (1:5000 dilution) were used as secondary antibodies for Western blot identification. The Western blot identification steps are as follows:

[0072] a. Electrophoresis: Load the processed recombinant 122 protein sample into an SDS-PAGE gel. Perform electrophoresis at a constant current until the bromophenol blue indicator reaches the appropriate position.

[0073] 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.

[0074] c. Membrane treatment: After transfer, wash with TBST and place the membrane in a 5% skim milk powder blocking solution, and gently shake to block for 2 hours at room temperature.

[0075] d. Primary antibody incubation: After washing the membrane with TBST, add the specific primary antibody (1:200 dilution) and incubate overnight at 4°C.

[0076] 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.

[0077] f. Color development: After mixing the luminescent solutions A and B, add them to the nitrocellulose membrane in the dark to ensure full bonding. Then, place the membrane into a membrane scanning instrument for scanning and take a picture for storage.

[0078] The results of recombinant protein reactivity identification are as follows: Figure 4 As shown, the 122 recombinant protein was identified by Western blot. After staining, a single and specific clear target band appeared, indicating that the 122 recombinant protein reacted well with all CaPV positive sera.

[0079] (3) Preparation of polyclonal antibodies

[0080] New Zealand white rabbits were immunized subcutaneously at multiple sites on the back with recombinant protein 122. After the third immunization, blood was collected from the heart, serum was separated by centrifugation, and polyclonal antibody titers were determined by indirect ELISA. IgG from the serum was purified using a GE Healthcare Protein G Sepharose™ antibody affinity chromatography column. After BCA concentration determination, the serum was stored at -80°C for later use. The steps for purifying serum IgG using antibody affinity chromatography are as follows:

[0081] a. Sample loading: Load the processed serum sample onto a Protein G Sepharose column that has been pre-equilibrated with equilibration buffer.

[0082] b. Washing: Wash the column with equilibration buffer (20mM Na2HPO4, 0.15M NaCl, pH 7.0) to remove non-specifically bound proteins.

[0083] c. Elution: Elute the specifically bound IgG using elution buffer (0.1M glycine, pH 2.5).

[0084] d. Neutralization and Storage: The eluted IgG needs to be adjusted to neutral pH using neutralization buffer (1M Tris) to restore antibody activity. The purified IgG can be stored in an appropriate buffer at low temperature.

[0085] (4) Preparation of recombinant 122 protein-colloidal gold marker

[0086] 0.1 mol / L K2CO3 was slowly added to the colloidal gold solution until the pH reached 7. Recombinant 122 protein was then added to a final concentration of 0.24 mg / ml. After stirring for 20 min, bovine serum albumin (BSA) to a final concentration of 10% was added. After stirring for 15 min, the mixture was centrifuged at 8000 rpm for 15 min. The supernatant was discarded, and 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 122 protein-colloidal gold label. The recombinant 122 protein-colloidal gold label was then sprayed onto a gold-labeled pad and dried at 37 °C for 1 h to obtain the colloidal gold pad.

[0087] (5) Preparation of colloidal gold test strips for detecting sheep pox virus antibodies

[0088] 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 sheep pox virus antibodies.

[0089] (6) Judgment criteria for test strips

[0090] The criteria for judging the test strip are as follows: Figure 5 As shown, where:

[0091] Positive: When the gold-labeled recombinant 122 protein can bind to the recombinant 122 protein polyclonal antibody on the control line C, a red band will appear on the control line; if there is 122 protein antibody in the serum, it will bind to the gold-labeled recombinant 122 protein on the gold pad and bind to the recombinant 122 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;

[0092] Negative: When the gold-labeled recombinant 122 protein can bind to the recombinant 122 protein polyclonal antibody of the control line C, a red band appears on the control line. If there is no 122 protein antibody in the serum, no red band appears on the T line, and the result is judged as negative.

[0093] Invalid: If the gold-labeled recombinant 122 protein does not bind to the recombinant 122 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.

[0094] Example 2: Determination of optimal pH and optimal amount of labeled protein for colloidal gold labeling of recombinant 122 protein.

[0095] (1) The optimal pH determination method for colloidal gold-labeled recombinant 122 protein in this embodiment

[0096] a. Take six 1.5 mL sterile centrifuge tubes, add 1 mL of colloidal gold solution to each, and adjust the pH to 4–9 with 0.1 mol / L K2CO3.

[0097] b. Add a fixed amount of protein to the gold solution at each pH gradient, let stand at room temperature for 2 hours, and then centrifuge at 12,000 rpm / min for 30 minutes at 4°C.

[0098] c. Collect the supernatant, coat it with an ELISA plate, use rabbit anti-122 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.

[0099] 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. 450nm The 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.

[0100] The results are as follows Figure 6 As shown, the optimal pH for colloidal gold labeling of recombinant 122 protein is 7.

[0101] (2) Method for determining the amount of colloidal gold-labeled recombinant 122 protein in this embodiment

[0102] a. Dilute protein 122 with PBS to 0.8, 0.4, 0.2, 0.1, 0.05, 0.02, 0.01 and 0.005 mg / mL.

[0103] b. Take 0.1 mL of the above solution and add it to the gold solution whose pH has been adjusted. Mix well and let stand at room temperature for 8 min to 10 min.

[0104] c. Add 0.1 mL of 10% NaCl solution, mix thoroughly, and observe the color change after 2 hours.

[0105] d. The minimum amount of recombinant 122 protein added to keep the solution red is the optimal amount of labeled protein.

[0106] The results are as follows Figure 7 As shown, the amount of labeled recombinant 122 protein by colloidal gold labeling was 0.24 mg / mL.

[0107] Example 3: Characteristic detection of the colloidal gold test strip method

[0108] 1. Specificity

[0109] The test strips prepared above were used to detect positive sera for BVDV, FMDV-O, FMDV-A, ORFV, brucellosis, and Escherichia coli infection, with LSDV positive sera used as a control, thereby identifying their specificity.

[0110] The results are as follows Figure 8 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.

[0111] 2. Sensitivity

[0112] SPPV, LSDV, and GTPV positive sera were diluted from 1:2 to 1:512 using sample dilution buffer. 100 μL of each sera were added to the sample pad and incubated at room temperature for 10 to 15 minutes to observe the results. The sensitivity was then assessed and compared with the results of the commercially available Biovetest sheep pox antibody detection kit.

[0113] The results are as follows Figure 9 As shown, the positive or negative result was determined based on the appearance of the T-line. The results showed that the lowest dilutions for SPPV, LSDV, and GTPV using this method were 1:64, 1:128, and 1:128, respectively, while the lowest dilutions for SPPV, LSDV, and GTPV using the Biovetest sheep pox antibody detection kit were 1:128, 1:256, and 1:256, respectively. Compared with ELISA results, the test strips prepared in this study exhibited good sensitivity.

[0114] 3. Repeatability

[0115] The test strips prepared using this invention were subjected to repeatability tests on three different batches, and the repeatability of the test strips was determined based on the appearance of the test strips.

[0116] The results are as follows Figure 10 As shown, the results between and within batches are relatively consistent, indicating that the test strips have good repeatability.

[0117] 4. Stability

[0118] The test strips prepared according to the present invention were stored at room temperature (18℃~25℃) and 4℃, respectively, and stability tests were conducted on the test strips at 0, 1, 3, 6, 9, 12 and 15 months.

[0119] The results are shown in Table 1. The test strips can be stably stored at room temperature for 6 months and at 4°C for 9 months.

[0120] Table 1. Results of stability test on colloidal gold test strips

[0121]

[0122] 5. Clinical sample testing and compliance comparison, the methods are as follows:

[0123] The colloidal gold test strip for sheep pox virus antibody prepared in this study and the Biovetest sheep pox virus indirect ELISA antibody detection kit were used to simultaneously detect 130 clinical serum samples. The test results were statistically analyzed and the concordance rate of the two methods was compared.

[0124] The results are shown in Table 2. Of the 130 clinical serum samples, the Biovetest kit detected 45 positive serum samples and 85 negative serum samples; the test strip prepared in this invention detected 41 positive serum samples and 89 negative serum samples. The test strip and the Biovetest antibody detection kit showed consistent results in 126 tests, with a Kappa value of 0.93, indicating good accuracy of the colloidal gold test strip.

[0125] Table 2 Comparison of detection methods

[0126]

[0127] In summary, this invention first prepared recombinant 122 protein, which exhibits significant antigenicity and immunogenicity, making it a good target for diagnosing sheep pox virus. Secondly, this invention prepared a colloidal gold test strip for detecting sheep pox virus 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 122 protein. The test strip of this invention can simultaneously detect antibodies against sheep pox virus, bovine nodular dermatosis virus, and goat pox virus, with sensitivities of 1:64, 1:128, and 1:128, respectively. It shows 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 Escherichia coli, exhibiting good specificity and a diagnostic concordance rate of 93%.

[0128] 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.

Claims

1. A recombinant 122 protein for detecting sheep pox virus antibodies, characterized in that, The amino acid sequence of the recombinant 122 protein is shown in SEQ ID No.

1.

2. The use of the recombinant 122 protein as described in claim 1 in the preparation of reagents for detecting sheep pox virus antibodies.

3. A colloidal gold test strip for detecting sheep pox virus antibodies, 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 122 protein of claim 1 is immobilized on the gold-labeled pad with a colloidal gold label.

4. The colloidal gold test strip as described in claim 3, characterized in that, The sample pad is placed at one end of the PVC base plate, with a gold label pad underneath and a nitrocellulose membrane underneath the gold label pad. The absorbent pad is placed at the other end of the PVC base plate and on top of the nitrocellulose membrane. Detection and quality control lines are provided on the nitrocellulose membrane between the gold label pad and the absorbent pad.

5. The colloidal gold test strip as described in claim 4, characterized in that, The detection line is labeled with the recombinant 122 protein as described in claim 1, and the control line is labeled with a polyclonal antibody against the recombinant 122 protein.

6. The colloidal gold test strip as described in claim 5, characterized in that, The concentration of the recombinant 122 protein of claim 1 labeled with colloidal gold is 0.24 mg / ml, and the amount used is 5 μL / cm.

7. The colloidal gold test strip as described in claim 6, characterized in that, The detection line was prepared by diluting the recombinant 122 protein to 0.6 mg / mL and then drawing the line, with a coating volume of 1 μL / cm.

8. The colloidal gold test strip as described in claim 7, characterized in that, The control line was prepared by diluting the recombinant 122 protein polyclonal antibody to 2 mg / mL and then drawing the line, with a coating volume of 1 μL / cm.

9. The method for preparing colloidal gold test strips as described in any one of claims 3-8, characterized in that, The method includes the following steps: (1) Prepare recombinant 122 protein and obtain detection lines by streaking it on a nitrocellulose membrane; (2) Prepare a polyclonal antibody against recombinant 122 protein and obtain a quality control line by streaking it on a nitrocellulose membrane; (3) Prepare colloidal gold-labeled recombinant 122 protein and fix the colloidal gold-labeled recombinant 122 protein on a gold-labeled pad; (4) After wetting the sample pad and gold label pad with the sealing solution, place them in an 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 sheep pox virus antibodies.

10. The use of the colloidal gold test strip according to any one of claims 3-8 in the detection of antibodies against sheep poxvirus, bovine nodular dermatovirus and goat poxvirus for non-disease diagnosis purposes.

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