Colloidal gold test strip for detecting sheep pox virus antibody as well as preparation method and application of colloidal gold test strip

Abstract

The invention relates to a colloidal gold test strip for detecting a sheep pox virus antibody as well as a preparation method and application of the colloidal gold test strip. According to the invention, firstly, a recombinant 122 protein is prepared, and the recombinant 122 protein has remarkable antigenicity and immunogenicity and can be used as a good target for diagnosing the sheep pox virus; secondly, the colloidal gold test strip for detecting the sheep pox virus antibody is prepared, a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on a PVC base plate of the test strip, and the gold-labeled pad is coated with gold-labeled recombinant 122 protein; the test strip can simultaneously detect antibodies of the sheep pox virus, the bovine nodular skin disease virus and the goat pox virus, the sensitivity can reach 1: 128, 1: 128 and 1: 64 respectively, the test strip has no cross reaction with bovine viral diarrhea virus, foot-and-mouth disease O-type virus, foot-and-mouth disease A-type virus, orf virus, brucellosis and colibacillosis, the specificity is good, and the diagnosis coincidence rate reaches 93%.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a colloidal gold test strip for detecting sheep pox virus antibodies, a preparation method and an application thereof. Background Art

[0002] Capripoxvirus (CaPV) is a member of the Chordata subfamily of the Poxviridae family. It consists of Lumpy skin disease virus (LSDV), Goatpox virus (GTPV) and Sheeppox virus (SPPV), which can infect cattle, goats and sheep, respectively, causing bovine lumpy skin disease, goat pox and sheep pox. Sick animals have extensive nodules and edema on the surface of their skin and organs, their milk production decreases sharply, and the quality of their fur is greatly reduced. Due to its potential for rapid cross-border transmission, it can spread rapidly in epidemic areas and cause huge economic losses, causing serious impacts on the cattle and sheep farming industries. The World Organization for Animal Health (WOAH) has listed it as a reportable animal disease.

[0003] CaPV has a serious impact on the development of animal husbandry and has caused serious losses to the world economy and trade. At present, there is no effective treatment for CaPV, and prevention is the main approach for the disease. Monitoring the disease through sensitive and specific diagnostic methods is the best way to control the incidence of the disease in any region and eradicate the disease. The CaPV genome is a double-stranded linear DNA with covalent closure at both ends, about 150kb in length, encoding about 150 proteins. The P32 protein is a major structural protein of the genus Capripoxvirus, involved in the assembly of viral particles, with strong immunogenicity and containing major antigenic epitopes. It can stimulate the host to produce antibodies in the early stage of infection to prevent the spread of the virus. Currently, a variety of serological detection methods for Capripoxvirus have been established based on this protein.

[0004] The methods for detecting antibodies to the sheep pox virus include virus neutralization test (VNT), enzyme-linked immunosorbent assay (ELISA), agar diffusion test (AGID) and indirect immunofluorescence (IFA). Among them, although VNT is the gold standard for the current serological method for detecting CaPV, it is time-consuming and insensitive, and ELISA, AGID and IFA have the disadvantages of cumbersome steps, low sensitivity, time-consuming and high cost; antigen diagnosis methods include PCR, qPCR and virus isolation and identification, which require expensive instruments and professionals, are time-consuming and are not conducive to promotion at the grassroots level. Summary of the invention

[0005] In view of the above technical problems, the present invention prepares a test strip that can simultaneously detect three virus antibodies of sheep pox virus, bovine nodular skin disease virus and goat pox virus, which has the characteristics of simple operation, no need for special instruments and equipment, and rapid results, and meets the needs of on-site rapid detection. Specifically, it includes the following contents:

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

[0007] In a second aspect, the present invention provides the use of the recombinant 122 protein described in the first aspect in the preparation of a reagent for detecting antibodies against capripoxvirus.

[0008] Preferably, the sheeppox virus includes sheeppox virus, bovine lumpy skin disease virus and goatpox virus.

[0009] In a third aspect, 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 label pad, a nitrocellulose membrane, a water-absorbing pad, a test line and a quality control line; the recombinant 122 protein described in the first aspect above and labeled with colloidal gold is fixed on the gold label pad.

[0010] Preferably, the sample pad is arranged at one end of the PVC bottom plate, a gold label pad is arranged under the sample pad, a nitrocellulose membrane is arranged under the gold label pad, the absorbent pad is arranged at the other end of the PVC bottom plate and on the upper layer of the nitrocellulose membrane, and a detection and quality control line is arranged on the nitrocellulose membrane between the gold label pad and the absorbent pad.

[0011] Preferably, the detection line is marked with the recombinant 122 protein described in the first aspect above, and the quality control line is marked 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 is 5 μL / cm.

[0013] Preferably, the test 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 quality 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] In a fourth aspect, the present invention provides a method for preparing the colloidal gold test strip described in the third aspect, the method comprising the following steps:

[0016] (1) preparing recombinant 122 protein and streaking on a nitrocellulose membrane to obtain a detection line;

[0017] (2) preparing polyclonal antibodies against the recombinant 122 protein and streaking on a nitrocellulose membrane to obtain a quality control line;

[0018] (3) preparing colloidal gold-labeled recombinant 122 protein, and fixing the colloidal gold-labeled recombinant 122 protein on a gold label pad to prepare a gold label pad;

[0019] (4) After the sample pad and the gold label pad are soaked with the blocking solution, they are placed in an oven for drying, and then the PVC base plate, the sample pad, the gold label pad, the nitrocellulose membrane and the absorbent pad are assembled together to obtain a colloidal gold test strip for detecting sheep pox virus antibodies.

[0020] Preferably, the preparation method of the recombinant 122 protein is as follows: constructing the 122 gene shown in SEQ ID No. 2 into the pET-SUMO vector to obtain the recombinant plasmid pET-SUMO-122; transforming the constructed pET-SUMO-122 plasmid into Escherichia coli BL21 (DE3) competent cells, picking a single positive recombinant colony and inoculating it into LB liquid culture medium containing kanamycin and culturing it overnight, inoculating it into 200 mL LB culture medium at a ratio of 1:100 and culturing it for 8h to 10h, adding IPTG (final concentration of 1 mmol / L), and culturing it in a 16°C incubator overnight; collecting the bacterial liquid by centrifugation, resuspending the bacterial cells in PBS, and collecting the supernatant by centrifugation after ultrasonic disruption, and purifying the 122 recombinant protein by Ni-NTA to obtain the recombinant 122 protein;

[0021] Preferably, the method for preparing the polyclonal antibody is as follows: the prepared recombinant 122 protein is immunized subcutaneously at multiple points on the back of New Zealand white rabbits, blood is collected from the heart after the third immunization, serum is separated by centrifugation, IgG in the serum is purified using a GE Healthcare Protein G SepharoseTM antibody affinity chromatography purification column to obtain a polyclonal antibody to the recombinant 122 protein, and the concentration is measured by BCA and stored in a -80°C refrigerator for future use.

[0022] Preferably, the preparation method of the gold label pad is as follows: slowly add 0.1 mol / L K2CO3 to the colloidal gold solution until the pH is 7, add recombinant 122 protein with a final concentration of 0.24 mg / ml, stir for 20 minutes, add bovine serum albumin BSA with a final concentration of 10%, stir for 15 minutes and then centrifuge, discard the supernatant, and redissolve the solution according to one tenth of the volume of the original solution to obtain a recombinant 122 protein-colloidal gold marker, spray the recombinant 122 protein-colloidal gold marker on the gold label pad, and dry at 37°C for 1 hour to obtain a colloidal gold pad;

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

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

[0025] Preferably, the method for preparing the quality control line is as follows: the polyclonal antibody of the recombinant 122 protein is diluted to 2 mg / mL with PBS and then the line is drawn, and the coating volume is 1 μL / cm.

[0026] Preferably, the rotation speed of centrifugal separation during the preparation of the gold label pad is 8000 rpm / min, and the centrifugation is performed for 15 minutes.

[0027] Preferably, the rehydration solution in the preparation process of the gold label pad is 10 mM Tris-Hcl, 1% BSA, 10% sucrose, 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 TritonX-100.

[0029] In a fourth aspect, the present invention provides the use of the colloidal gold test strip described in the third aspect in the detection of sheeppox virus, bovine lumpish dermatosis virus and goatpox virus antibodies for non-disease diagnosis purposes.

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

[0031] (1) Sample processing: Collect blood samples from the animals to be tested, place them at room temperature or 4°C, centrifuge them at 3000 rpm for 10-15 min, and dilute the serum with PBS at a ratio of 1:10;

[0032] (2) Sample testing: Add 100 μL of serum sample to the sample pad of the test strip and leave at room temperature for 10 to 15 minutes to observe the results;

[0033] (3) Result determination:

[0034] Positive: When the gold-labeled recombinant 122 protein can bind to the recombinant 122 protein polyclonal antibody on the quality control line C, a red strip will appear on the quality 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 to the recombinant 122 protein labeled on the test line T to form a double antigen sandwich. At this time, a red strip will appear on the T line, which is considered positive.

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

[0036] Invalid: When the gold-labeled recombinant 122 protein does not bind to the recombinant 122 protein polyclonal antibody on the quality control line C, there will be no red band on the quality 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 fuses the 122 gene with the pET-SUMO vector, expresses it in prokaryotes and purifies it to obtain a recombinant 122 protein. It is verified by Western blot that the recombinant 122 protein can react with sheep pox virus, bovine nodular skin disease virus and goat pox virus, and can be used as a good target for diagnosing sheep pox virus.

[0039] (2) The present invention prepares a colloidal gold test strip for detecting sheep pox virus antibodies, wherein a sample pad, a gold label pad, a nitrocellulose membrane and a water-absorbing pad are arranged in sequence on the PVC bottom plate of the test strip, and the gold label pad is coated with a gold-labeled recombinant 122 protein; the test strip of the present invention can simultaneously detect antibodies to sheep pox virus, bovine nodular skin disease virus and goat pox virus, with sensitivities of 1:128, 1:128 and 1:64, respectively, and has no cross reaction with bovine viral diarrhea virus, foot-and-mouth disease type O virus, foot-and-mouth disease type A virus, sheep sore virus, brucellosis and Escherichia coli disease, has good specificity, and a diagnostic compliance rate of 93%;

[0040] (3) The present invention uses polyclonal antibodies as quality control lines, shortening preparation time and reducing 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) The present invention is suitable for rapid on-site detection, which is helpful for the early detection and prevention and control of sheep pox virus;

[0043] (6) The preparation process of the present invention is simple and easy to industrialize and promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 ORF122 gene analysis results;

[0045] Figure 2 Schematic diagram of the test strip structure provided by the present invention; wherein 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 a water absorbent pad.

[0046] Figure 3 SDS-PAGE analysis of the induced expression and purification of the recombinant protein, where M is the relative molecular mass of the protein; lane 1 is the uninduced bacterial solution of pET-SUMO-122; lane 2 is the induced bacterial solution of pET-SUMO-122; lane 3 is the supernatant of the ultrasonic fragmentation of the induced bacterial solution of pET-SUMO-122; lane 4 is the precipitate of the ultrasonic fragmentation of the induced bacterial solution of pET-SUMO-122; lane 5 is the purified recombinant 122 protein.

[0047] Figure 4 Identification of recombinant protein reactivity, 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; lane 6 is GTPV-negative serum.

[0048] Figure 5 The test strip judgment standard provided by the present invention.

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

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

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

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

[0053] Fig.10 Repeatability test of colloidal gold test strips, where A is LSDV-positive serum and B is LSDV-negative serum. DETAILED DESCRIPTION

[0054] The technical scheme of the present invention is described in detail below by specific embodiments. It should be understood that these embodiments are only used to illustrate 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 according to the teachings of the present invention without departing from the spirit and scope of the present invention.

[0055] The present invention selected 17 strains of CaPV, including 4 strains of GTPV, 9 strains of LSDV and 4 strains of SPPV for homology analysis. Gene sequence comparison analysis showed that the homology between the 17 strains of GTPV, LSDV and SPPV was not less than 93.4%, confirming the high conservation of the ORF122 gene (such as Figure 1 shown).

[0056] Example 1 Preparation of colloidal gold test strips 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 bottom plate (1), a sample pad (2), a gold label pad (3), a nitrocellulose membrane (4), and a water absorbent pad (7). The sample pad is provided at one end of the PVC bottom plate, a gold label pad is provided under the sample pad, a nitrocellulose membrane is provided under the gold label pad, and a water absorbent pad is provided at the other end of the PVC bottom plate and on the upper layer of the nitrocellulose membrane. A detection line (5) and a quality control line (6) are provided on the nitrocellulose membrane. The specific preparation includes the following steps:

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

[0059] The 122 gene (accession number: OP508345.1) was searched from the GenBank database and truncated. The truncated 122 gene (shown in SEQ ID No. 2) was sent to Wuhan Jinkairui Bioengineering Co., Ltd. for synthesis and constructed 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 culture medium containing kanamycin.

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

[0061] A single positive recombinant bacterium was selected and inoculated into LB liquid medium containing kanamycin and cultured overnight. After inoculation into 200 mL LB culture medium at a ratio of 1:100 and cultured for 8 to 10 hours, IPTG (final concentration of 1 mmol / L) was added and cultured in a 16°C incubator overnight.

[0062] The bacterial liquid was collected by centrifugation, the bacteria were resuspended in PBS, and the supernatant was collected by centrifugation after ultrasonic disruption. The recombinant 122 protein was purified by Ni-NTA as follows:

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

[0064] b. Cell disruption and protein extraction: The cells expressing the recombinant 122 protein were disrupted by ultrasonication, and the cell debris and insoluble substances were removed by centrifugation, and the supernatant was collected.

[0065] c. Loading: Slowly add the collected supernatant to the balanced Ni-NTA resin column, control the flow rate, and collect the flow-through.

[0067] d. Washing: Wash the resin column with a washing buffer containing 8M urea and collect the washing solution.

[0068] e. Elution: Use 8M urea and 250mM imidazole eluent to elute the resin column

[0069] f. Collect the eluate and perform SDS-PAGE analysis to determine the purity of the eluted protein.

[0070] g. Protein storage: After the protein concentration of the protein is determined by the BCA method, it is packaged and stored at -80°C.

[0071] The results of SDS-PAGE analysis of the induced expression and purification of the recombinant protein are as follows Figure 3 As shown in the figure, after the 122 recombinant protein was induced to express in E. coli at 16°C overnight, the SDS-PAGE test results showed that there was an obvious expression band at about 35kDa, which was consistent with the expected size, indicating that the protein was successfully induced to express. Protein products were present in the supernatant and precipitate after ultrasonic disruption of the recombinant bacteria, but were 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 that the target protein was successfully purified.

[0072] Western blot identification was performed using standard positive and negative sera of SPPV, LSDV, and GTPV as primary antibodies (1:200 dilution), and HRP-labeled rabbit anti-Mianyang / goat IgG and goat anti-bovine IgG (1:5000 dilution) as secondary antibodies; the Western blot identification steps were as follows:

[0073] a. Electrophoresis: Load the treated recombinant 122 protein sample into SDS-PAGE gel and perform electrophoresis under constant current until the bromophenol blue indicator reaches the appropriate position.

[0074] b. Transfer: Place the SDS-PAGE gel and membrane in the transfer device and use the wet transfer method to transfer the membrane under constant current.

[0075] c. Membrane treatment: After transfer, wash with TBST and place the membrane in 5% skimmed milk powder blocking solution. Shake gently at room temperature for 2 hours.

[0076] d. Primary antibody incubation: After washing the membrane with TBST, add specific primary antibody (1:200 dilution) and incubate at 4°C overnight.

[0077] e. Secondary antibody incubation: Wash the membrane with TBST, add HRP-labeled secondary antibody (1:5000 dilution), and incubate at room temperature for 1 hour.

[0078] f. Color development: Mix luminescent solution A and solution B evenly and add them onto the nitrocellulose membrane in a dark place. After they are fully combined, place the membrane into a membrane scanner for scanning and take a photo for preservation.

[0079] The results of recombinant protein reactivity identification are as follows Figure 4 As shown, the 122 recombinant protein was identified by Western blot, and a single, specific and clear target band appeared after color development, indicating that the 122 recombinant protein had a good reaction with each CaPV-positive serum.

[0080] (3) Preparation of polyclonal antibodies

[0081] The 122 recombinant protein was immunized subcutaneously at multiple points on the back of New Zealand white rabbits. After the third immunization, blood was collected from the heart, and the serum was separated by centrifugation. The polyclonal antibody titer was determined by indirect ELISA method; IgG in the serum was purified by GE Healthcare Protein GSepharoseTM antibody affinity chromatography purification column, and the concentration was measured by BCA and stored in a refrigerator at -80℃ for future use. The steps for purifying IgG in serum by antibody affinity chromatography purification column are as follows:

[0082] a. Sample loading: Load the treated serum sample onto the Protein G Sepharose column that has been pre-equilibrated with equilibration buffer.

[0083] b. Washing: Wash the column with equilibration buffer (20 mM Na2HPO4, 0.15 M NaCl, pH 7.0) to remove non-specifically bound proteins.

[0084] c. Elution: Elution of specifically bound IgG was performed using elution buffer (0.1 M glycine, pH 2.5).

[0085] d. Neutralization and storage: The eluted IgG needs to be adjusted to a neutral pH using a neutralization buffer (1M Tris) to restore antibody activity. The purified IgG can be stored in an appropriate buffer and kept at low temperature.

[0086] (4) Preparation of recombinant 122 protein-colloidal gold marker

[0087] Slowly add 0.1 mol / L K2CO3 to the colloidal gold solution until the pH is 7, add recombinant 122 protein with a final concentration of 0.24 mg / ml, stir for 20 minutes, add bovine serum albumin BSA with a final concentration of 10%, stir for 15 minutes, centrifuge at 8000 rpm / min for 15 minutes, discard the supernatant, and redissolve the solution (10 mM Tris-Hcl, 1% BSA, 10% sucrose, PH=8.5) at one-tenth the volume of the original solution to obtain recombinant 122 protein-colloidal gold marker, spray the recombinant 122 protein-colloidal gold marker on the gold label pad, and dry at 37°C for 1 hour to obtain the colloidal gold pad.

[0088] (5) Preparation of colloidal gold test strips for detecting sheep pox virus antibodies

[0089] The sample pad and the gold label pad were soaked with blocking solution (PBS solution of 0.75% trehalose, 0.5% sucrose, 2% BSA, 0.5% Tween-20 and TritonX-100) in advance, and then placed in an oven for drying. Then, the PVC base plate, the sample pad, the gold label pad, the nitrocellulose membrane and the absorbent pad were assembled together to obtain the colloidal gold test strip for detecting sheep pox virus antibodies.

[0090] (6) Test strip judgment criteria

[0091] The criteria for judging the test strips are as follows Figure 5 As shown, where:

[0092] Positive: When the gold-labeled recombinant 122 protein can bind to the recombinant 122 protein polyclonal antibody on the quality control line C, a red strip will appear on the quality 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 to the recombinant 122 protein labeled on the test line T to form a double antigen sandwich. At this time, a red strip will appear on the T line, which is considered positive.

[0093] Negative: When the gold-labeled recombinant 122 protein can bind to the recombinant 122 protein polyclonal antibody on the quality control line C, a red band appears on the quality control line. If there is no 122 protein antibody in the serum, no red band appears on the T line, and the result is considered negative.

[0094] Invalid: When the gold-labeled recombinant 122 protein does not bind to the recombinant 122 protein polyclonal antibody on the quality control line C, there will be no red band on the quality control line C, indicating that the test strip is invalid.

[0095] Example 2 Determination of the optimal pH and optimal amount of labeled protein for colloidal gold labeled recombinant 122 protein

[0096] (1) Method for determining the optimal pH of colloidal gold-labeled recombinant 122 protein in this example

[0097] a. Take 6 1.5mL sterile centrifuge tubes, add 1mL of colloidal gold solution to each tube, and adjust the pH to 4-9 with 0.1mol / L K2CO3.

[0098] b. Add a fixed amount of protein to each pH gradient gold solution, let stand at room temperature for 2 hours, and then centrifuge at 4°C, 12,000 rpm / min for 30 minutes.

[0099] c. Collect the supernatant and coat the 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). Use the indirect ELISA method to detect the protein content in the supernatant.

[0100] d. Read OD with a microplate reader 450nm value, and take pH as the horizontal axis, OD 450nm Draw a scatter plot with the value as the vertical axis, OD 450nm When the value is the lowest, it means that the amount of unlabeled protein in the supernatant of the gold solution is the least at this pH, which is the optimal pH.

[0101] The results are as follows Figure 6 As shown, the optimal pH for colloidal gold labeling of recombinant 122 protein is 7.

[0102] (2) Method for determining the amount of protein labeled by colloidal gold-labeled recombinant 122 protein in this example

[0103] a. Dilute 122 protein with PBS to 0.8, 0.4, 0.2, 0.1, 0.05, 0.02, 0.01 and 0.005 mg / mL.

[0104] b. Take 0.1 mL of the above solution and add it to the gold solution with adjusted pH, mix thoroughly, and let it stand at room temperature for 8 to 10 minutes.

[0105] c. Add 0.1 mL of 10% NaCl solution, mix thoroughly, and observe the color change after 2 hours.

[0106] d. The lowest amount of recombinant 122 protein added for the solution to remain red. An increase of 20% on this basis is the optimal amount of labeled protein.

[0107] The results are as follows Figure 7 As shown, the amount of colloidal gold labeled recombinant 122 protein was 0.24 mg / mL.

[0108] Experimental Example 3 Characteristic Detection of Colloidal Gold Test Strip Detection Method

[0109] 1. Specificity

[0110] The test strips prepared as above were used to detect BVDV, FMDV-O, FMDV-A, ORFV, brucellosis and Escherichia coli positive sera, with LSDV positive sera as control, thereby identifying its specificity.

[0111] The results are as follows Figure 8 As shown, the test strip prepared by the present invention detected BVDV, FMDV-O, FMDV-A, ORFV, brucellosis and Escherichia coli positive serum and the results were all negative, indicating that the test strip has good specificity.

[0112] 2. Sensitivity

[0113] SPPV, LSDV, and GTPV positive sera were diluted from 1:2 to 1:512 with sample diluent, and 100 μL of each was added to the sample pad. The samples were placed at room temperature for 10 to 15 minutes and the results were observed to detect their sensitivity and compare them with the test results of the commercially available Biovetest sheeppox antibody test kit.

[0114] The results are as follows Fig. 9 As shown, the positive and negative are determined according to the appearance of the T line. The results show that the minimum dilutions of SPPV, LSDV and GTPV in this method are 1:64, 1:128 and 1:128, respectively, and the minimum dilutions of SPPV, LSDV and GTPV detected by the Biovetest sheeppox antibody detection kit are 1:128, 1:256 and 1:256, respectively. Compared with the ELISA results, the test strip prepared in this study has good sensitivity.

[0115] 3. Repeatability

[0116] Three different batches of the test strips prepared by the present invention were respectively taken for repeatability tests, and the repeatability of the test strips was determined based on the output line conditions.

[0117] The results are as follows Fig.10 As shown, the results within and between batches are relatively consistent, indicating that the test strips have good repeatability.

[0118] 4. Stability

[0119] The test strips prepared by the present invention were stored at room temperature (18° C. to 25° C.) and 4° C., and stability tests were performed on the test strips at 0, 1, 3, 6, 9, 12, and 15 months.

[0120] The results are shown in Table 1. The test strips can be stably stored for 6 months at room temperature and for 9 months at 4°C.

[0121] Table 1 Colloidal gold test strip stability test results

[0122]

[0123] 5. Clinical sample testing and compliance comparison, the method is as follows

[0124] The colloidal gold test strips for sheeppox virus antibodies prepared in this study and the Biovetest sheeppox virus indirect ELISA antibody test kit were used to simultaneously test 130 clinical serum samples. The test results were statistically analyzed and the compliance rates of the two methods were compared.

[0125] The results are shown in Table 2. Among the 130 clinical serum samples, the Biovetest test kit detected 45 positive sera and 85 negative sera; the test strip prepared by the present invention detected 41 positive sera and 89 negative sera. The test strip and the antibody detection kit of Biovetest had 126 consistent test results, and the Kappa value of the two was 0.93, indicating that the colloidal gold test strip had good accuracy.

[0126] Table 2 Comparison results of detection methods

[0127]

[0128] In summary, the present invention firstly prepares recombinant 122 protein, and the recombinant 122 protein has significant antigenicity and immunogenicity, and can be used as a good target for diagnosing sheep pox virus; secondly, the present invention prepares a colloidal gold test strip for detecting sheep pox virus antibodies, and a sample pad, a gold label pad, a nitrocellulose membrane and a water-absorbing pad are arranged in sequence on the PVC bottom plate of the test strip, and the gold label pad is coated with gold-labeled recombinant 122 protein; the test strip of the present invention can simultaneously detect antibodies of sheep pox virus, bovine nodular skin disease virus and goat pox virus, and the sensitivity can reach 1:64, 1:128 and 1:128 respectively, and has no cross reaction with bovine viral diarrhea virus, foot-and-mouth disease type O virus, foot-and-mouth disease type A virus, sheep sore virus, brucellosis and Escherichia coli disease, has good specificity, and the diagnosis compliance rate reaches 93%.

[0129] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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. Use of the recombinant 122 protein as claimed in claim 1 in the preparation of a reagent for detecting antibodies against capripoxvirus.

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 label pad, a nitrocellulose membrane, a water-absorbing pad, a test line and a quality control line; characterized in that: The gold label pad is fixed with the recombinant 122 protein of claim 1 labeled with colloidal gold.

4. The colloidal gold test strip as claimed in claim 3, characterized in that: The sample pad is arranged at one end of the PVC bottom plate, a gold label pad is arranged under the sample pad, a nitrocellulose membrane is arranged under the gold label pad, the absorbent pad is arranged at the other end of the PVC bottom plate and on the upper layer of the nitrocellulose membrane, and a detection and quality control line is arranged on the nitrocellulose membrane between the gold label pad and the absorbent pad.

5. The colloidal gold test strip as claimed in claim 4, characterized in that: The detection line is marked with the recombinant 122 protein according to claim 1, and the quality control line is marked with a polyclonal antibody against the recombinant 122 protein.

6. The colloidal gold test strip as claimed 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 usage is 5 μL / cm.

7. The colloidal gold test strip as claimed in claim 6, characterized in that: The test 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 claimed in claim 7, characterized in that: The quality 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 a colloidal gold test strip as claimed in any one of claims 3 to 8, characterized in that: The method comprises the following steps: (1) preparing recombinant 122 protein and streaking on a nitrocellulose membrane to obtain a detection line; (2) preparing polyclonal antibodies against the recombinant 122 protein and streaking on a nitrocellulose membrane to obtain a quality control line; (3) preparing colloidal gold-labeled recombinant 122 protein, and fixing the colloidal gold-labeled recombinant 122 protein on a gold label pad; (4) After the sample pad and the gold label pad are soaked with the blocking solution, they are placed in an oven for drying, and then the PVC base plate, the sample pad, the gold label pad, the nitrocellulose membrane and the absorbent pad are assembled together to obtain a colloidal gold test strip for detecting sheep pox virus antibodies.

10. Use of the colloidal gold test strip according to any one of claims 3 to 8 in the detection of antibodies to sheeppox virus, bovine lumpish skin disease virus and goatpox virus for non-disease diagnosis purposes.

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