GTPV diagnosis antigen and monoclonal antibody and application thereof

Abstract

The invention discloses a diagnostic antigen of a goat pox virus (GTPV), and belongs to the field of molecular biology. The diagnostic antigen of the goat pox virus (GTPV) belongs to the field of molecular biology. A nucleotide sequence of a coding gene of the antigen is shown as SEQ ID NO.1. The antigen protein has immunogenicity, can be used for immunizing animals to generate a monoclonal antibody with good activity, and can react with GTPV immune serum, so that the antigen protein can be used for serological detection and vaccine immune evaluation of GTPV. The invention also discloses a monoclonal antibody for resisting the antigen, and an application of the antigen and / or the monoclonal antibody in preparation of a GTPV diagnostic kit. According to the invention, GTPV detection of goatpox virus is realized, and the kit has the advantages of high sensitivity and specificity, rapid and convenient detection and the like.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and immunology, and specifically relates to a diagnostic antigen of goat pox virus (GTPV) of the genus Capripoxvirus. The present invention also relates to a monoclonal antibody of the antigen, and the use of the antigen and / or the monoclonal antibody in the preparation of a GTPV diagnostic kit. Background Art

[0002] Bovine lumpy skin disease (LSD) is an infectious disease of cattle caused by bovine lumpy skin disease virus (LSDV). LSD is mainly characterized by the production of extensive nodules on the skin surface of the whole body. The nodules are 5 to 50 mm in diameter, hard, and flat-topped papules, which can penetrate into the subcutaneous layer and even the muscle. LSDV belongs to the Poxviridae family (Poxviridae) and the genus Capripoxvirus (CaPV). It is a double-stranded DNA virus with an envelope and a large genome containing 156 hypothetical genes. The virus is genetically stable and has only one serotype. The nucleotide sequence similarity between LSDV and the sheep and goat pox viruses of the same genus is as high as more than 96%, with high antigenic similarity and serological cross-reaction. For this reason, my country clinically uses a 5-fold dose of goat pox attenuated vaccine for sheep to prevent and control LSD.

[0003] At present, the World Organization for Animal Health (WOAH) recommends the detection of goatpox virus serological detection methods such as neutralization test (VNT), indirect immunofluorescence test (IFA) and protein immunoblot test (Western Blot), among which the virus neutralization test is the gold standard for detecting goatpox virus antibodies, but this method is complicated, time-consuming and not suitable for large-scale detection of serum samples, and it is difficult to achieve detection at the grassroots level. At present, there is no ELISA kit with an approval number for detecting goatpox virus serum antibodies at home and abroad.

[0004] In view of the current situation in my country where a 5-fold sheep dose of goatpox attenuated live vaccine is used to prevent bovine nodular dermatitis, the applicant, from the perspective of clinical prevention and control, has screened a diagnostic target from the GTPV genome by using a protein immunoblot test (Western Blot) combined with mass spectrometry sequencing. The nucleotide sequence encoding the antigen is molecularly cloned into the pET-28a vector, and a competitive ELISA serological antibody detection method is established based on the expression of the recombinant protein in a prokaryotic system and the preparation of monoclonal antibodies using the protein. This method can realize serum antibody detection of GTPV infection or immunization of the goatpox virus genus. The present invention provides a practical and high-value tool for serological detection of GTPV and vaccine immunity evaluation. Summary of the invention

[0005] The first object of the present invention is to provide a GTPV antigen. The antigen is a diagnostic target screened from the GTPV genome using a protein immunoblot test combined with mass spectrometry sequencing. The applicant expressed a recombinant protein after molecular cloning of the nucleotide sequence encoding the antigen, and named it rAXA19967.1 protein. The nucleotide sequence of its encoding gene (rAXA19967.1 gene) is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The antigen protein is immunogenic, and immunizing animals with it can produce monoclonal antibodies with good activity. The antigen protein is reactive with GTPV immune serum, and therefore, can be used for serological detection of GTPV and vaccine immunity evaluation.

[0006] The protein and its function in in vitro diagnosis were obtained and characterized from GTPV for the first time by the applicant and had not been recorded in any literature before.

[0007] The second object of the present invention is to provide a method for preparing the antigen. The PCR method is used to design primers to amplify the target gene fragment with a nucleotide sequence as shown in SEQ ID NO.1 from the GTPV genome, and then the target gene fragment is cloned into an expression vector to obtain a recombinant plasmid, and the recombinant plasmid is transformed into competent cells to induce expression to obtain a recombinant protein, which is the antigen.

[0008] The third object of the present invention is to provide a monoclonal antibody against the antigen. The antigen is used to immunize mice, and the mouse spleen cells are fused, and then hybridoma cells are obtained through subclone screening, and then the monoclonal antibody is prepared by in vivo ascites induction method. The monoclonal antibody can react specifically with the antigen, and thus can be used to detect the antigen.

[0009] Furthermore, the monoclonal antibody is secreted by hybridoma cell line 6H4, which has been deposited in the China Center for Type Culture Collection in Wuhan, Hubei Province on March 6, 2025, with a deposit number of CCTCC NO: C202576. The monoclonal antibody secreted by the hybridoma cell has the advantages of high antibody concentration, high titer and blocking activity.

[0010] The fourth object of the present invention is to provide a hybridoma cell line 6H4, which can secrete and produce the monoclonal antibody.

[0011] The fifth object of the present invention is to provide the use of the antigen and / or the monoclonal antibody in the preparation of a GTPV diagnostic kit.

[0012] The sixth object of the present invention is to provide a goat pox diagnostic kit, which contains the above-mentioned antigen and / or the monoclonal antibody. For example, an indirect ELISA test is performed on the antibodies in GTPV serum using a kit containing the antigen; or a direct ELISA test is performed on the GTPV antigen in the animal using a kit containing the monoclonal antibody; or a blocking ELISA test is performed on the antibodies in GTPV serum using a kit containing both the antigen and the antibody. For those skilled in the art, there is no technical obstacle to assembling the above kit and applying it to the detection of the corresponding antibodies or antigens when the antigens and antibodies are obtained.

[0013] Furthermore, the kit is a competitive ELISA kit for detecting GTPV antibodies, and the kit contains an ELISA plate coated with the antigen and the monoclonal antibody labeled with horseradish peroxidase.

[0014] The kit also contains diluent, washing solution, positive and negative serum, color development solution and stop solution.

[0015] The seventh object of the present invention is to provide a competitive ELISA method for detecting GTPV antibodies, the method comprising the following steps:

[0016] (1) coating an ELISA plate with the antigen;

[0017] (2) Block the ELISA plate with blocking solution;

[0018] (3) labeling the monoclonal antibody with horseradish peroxidase;

[0019] (4) diluting the serum sample and the monoclonal antibody labeled with horseradish peroxidase and adding them to the ELISA plate for reaction;

[0020] (5) Add the colorimetric solution and stop solution. 450 Reading at wavelength.

[0021] The detection principle of this method is: after the antigen is bound to the solid phase carrier, after the serum sample and enzyme-labeled monoclonal antibody are added, the antibodies in the serum sample and the enzyme-labeled monoclonal antibody competitively react with the antigen on the solid phase carrier. The more antibodies there are in the sample to be tested, the fewer complexes are formed by the enzyme-labeled monoclonal antibody binding to the solid phase antigen, and after the substrate (developing solution) is added, the less colored substances are produced by the enzyme catalysis bound to the solid phase carrier. Conversely, the fewer antibodies there are in the sample to be tested, the more complexes are formed by the enzyme-labeled monoclonal antibody binding to the solid phase antigen, and after the substrate is added, the more colored substances are produced by the enzyme catalysis bound to the solid phase carrier. Qualitative and quantitative detection of serum antibodies can be achieved by detecting absorbance at a specific wavelength.

[0022] Furthermore, the coating concentration of the antigen is 1 μg / mL; the dilution multiple of the serum sample is 1:2; and the dilution multiple of the monoclonal antibody labeled with horseradish peroxidase is 1:4000.

[0023] The above detection method can be used to accurately detect antibodies in serum, which is helpful for carrying out serological detection of GTPV and evaluating the antibody levels of cattle immunized with goatpox attenuated vaccine. It can also be used for laboratory screening and identification of pathogen types in serum for non-disease diagnosis purposes.

[0024] The beneficial effects of the present invention are:

[0025] The present invention uses protein immunoblotting combined with mass spectrometry sequencing to screen a diagnostic antigen for GTPV from the GTPV genome, providing a new target for serological detection of goat pox, which is beneficial for disease prevention and control. Based on the monoclonal antibody of the invented antigen, the present invention also successfully established a competitive ELISA method to achieve serological antibody detection and vaccine immunity evaluation of GTPV. The kit of the present invention also has the advantages of strong sensitivity and good specificity, as follows:

[0026] In terms of analytical specificity, the detection method established by this method has no cross-reaction with the positive serum of bovine viral diarrhea virus, bovine mycoplasma and bovine mycobacterium except for the specific binding reaction with the positive serum of GTPV. In terms of analytical sensitivity, when the positive serum is diluted to 1:4, the PI of this method is 52.61% (PI ≥ 51.46%, judged as positive). Therefore, the minimum detection limit of positive serum is 1:4, which meets the 1:2 dilution of serum established by the ELISA method.

[0027] For more detailed technical solutions, please refer to the specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a map of the construction of the pET28a-AXA19967.1 recombinant plasmid.

[0029] Figure 2 This is a gel image of PCR identification of the pET28a-AXA19967.1 recombinant plasmid.

[0030] Figure 3 The results of SDS-PAGE (A) and Western Blot (B) analysis of the pET28a-AXA19967.1 recombinant protein, in the figure: 1: protein molecular weight standard; 2: pET-28a; 3: pET-28a induced at 37°C for 5h; 4: rAXA19967.1 protein; 5: rAXA19967.1 protein induced at 37°C for 5h.

[0031] Figure 4The results of SDS-PAGE purification analysis of rAXA19967.1 protein are shown in the figure: 1: protein molecular weight standard; 2: uninduced rAXA19967.1 protein culture solution; 3: rAXA19967.1 protein culture solution after 5 hours of induction; 4: flow-through containing 1% imidazole; 5: 10% imidazole washing solution; 6: 25% imidazole elution solution; 7: 50% imidazole elution solution.

[0032] Figure 5 The results of the reactivity of rAXA19967.1 protein were obtained by incubating the target protein with goatpox attenuated vaccine immune serum. In the figure: 1: protein molecular weight standard; 2: rAXA19967.1 protein; 3: pET-28a empty vector.

[0033] Figure 6 To identify the SDS-PAGE results of purified ascites, in the figure: 1: protein molecular mass standard; 2: liquid before 6H4 ascites passes through the column; 3: liquid after 6H4 ascites passes through the column; 4: 6H4 antibody wash solution; 5: 6H4 antibody eluate A; 6: 6H4 antibody eluate B.

[0034] Figure 7 The scatter plot shows the PI value of 30 immune sera and 30 clinical negative sera.

[0035] Figure 8 The ROC curve is drawn. DETAILED DESCRIPTION

[0036] The present invention is described in detail below with reference to specific embodiments.

[0037] Key materials and their descriptions:

[0038] pET-28a vector: a commercial vector used to construct the pET28a-AXA19967.1 recombinant plasmid.

[0039] BL21(DE3) cells: Commercial competent cells, used to express rAXA19967.1 protein.

[0040] Serum from cattle immunized with goatpox attenuated vaccine, clinical negative serum and myeloma cells SP2 / 0 were stored in our laboratory.

[0041] SPF-grade BALB / c mice were purchased from the Experimental Animal Center of Huazhong Agricultural University, and the animal experiment ethics approval number was HZAUMO-2022-0180.

[0042] Other biological materials or reagents not described herein are conventional materials in the art.

[0043] Example 1: Preparation of rAXA19967.1 protein

[0044] 1. Construction of pET28a-AXA19967.1 recombinant plasmid

[0045] Based on the whole genome sequence of goatpox virus (AV41 strain) (GenBank NO: MH381810.1), the target gene sequence was selected using Snap gene software, and the sequence length was 513 bp (SEQ ID NO.1). Specific primers were designed to amplify the target fragment and T7 / T7er identification primers were used to identify the target fragment (Table 1). Molecular cloning into the pET28a vector obtained the recombinant plasmid pET28a-AXA19967.1 containing the target gene. For details, see the map of the pET28a-AXA19967.1 recombinant plasmid ( Figure 1 ). The PCR identified the recombinant bacteria pET28a-AXA19967.1 band size of 798bp ( Figure 2 ).

[0046] Table 1 Specific primers for amplifying AXA19967.1 gene and identifying positive recombinant bacteria

[0047]

[0048] Table 2 PCR amplification system

[0049]

[0050] The PCR amplification system is shown in (Table 2).

[0051] PCR reaction conditions: denaturation at 98°C for 30s followed by cycling: 98°C for 10s, 60°C for 5s, 72°C for 5s, 35 cycles, total extension at 72°C for 5min, storage at 4°C. PCR product electrophoresis: 1.0% agarose gel electrophoresis to identify PCR band size, while spotting DL2000 Ladder.

[0052] Table 3 PCR identification system

[0053]

[0054] The PCR identification system is shown in (Table 3).

[0055] PCR reaction conditions: denaturation at 94°C for 5 min, followed by cycling: 94°C for 30 s, 54°C for 30 s, 72°C for 1 min, 35 cycles, total extension at 72°C for 10 min, storage at 4°C. PCR product electrophoresis detection: 1.0% agarose gel electrophoresis to identify PCR band size, while spotting DL2000 Ladder.

[0056] 2. Miniprep of plasmid DNA

[0057] The small amount preparation method of plasmid DNA was carried out according to the instructions of Tiangen High Purity Plasmid Mini-Preparation Kit. The specific steps are as follows:

[0058] (1) Take 5 mL of overnight culture solution and add it to a centrifuge tube. Centrifuge at 8000 r / min for 3 min to collect the bacterial precipitate and discard the supernatant as much as possible.

[0059] (2) Add 250 μL of Buffer P1 to the centrifuge tube containing the bacterial pellet. Check to ensure that RNase A has been added. Use a 1 mL pipette or vortex oscillator to mix thoroughly to suspend the bacterial pellet.

[0060] (3) Add 250 μL of Buffer P2 to the centrifuge tube and mix by gently inverting the tube 4 to 6 times to thoroughly lyse the bacteria. The solution should become cool and viscous.

[0061] (4) Add 350 μL of Buffer P3 to the centrifuge tube and immediately and gently invert the tube 4 to 6 times to mix thoroughly. A white flocculent precipitate should appear. Centrifuge at 13,000 rpm for 10 min.

[0062] (5) Transfer the supernatant obtained in step 4 to the adsorption column Spin Column CM in the collection tube. Centrifuge at 13,000 r / min for 1 min. Discard the waste liquid in the collection tube and place the adsorption column back into the collection tube.

[0063] (6) Add 750 μL of Buffer PW to the adsorption column and check that anhydrous ethanol has been added. Centrifuge at 13,000 rpm for 1 min and discard the waste liquid in the collection tube.

[0064] (7) Place the adsorption column back into the collection tube and centrifuge at 13,000 rpm for 2 min. Discard the waste liquid and place the adsorption column at room temperature for several minutes to dry thoroughly.

[0065] (8) Place the adsorption column in a new centrifuge tube and add 50 μL of DEPC H to the middle of the adsorption film. 2 O, place at room temperature for 3 minutes, centrifuge at 13000r / min for 1 minute, collect into a centrifuge tube, and store the plasmid at -20℃.

[0066] 3. Transform the plasmid into E. coli BL21 (DE3) competent cells

[0067] (1) Take 100 μL of competent cell suspension and add 1 μL of plasmid, gently rotate to mix the contents, and place on ice for 30 min (set up an empty control without plasmid DNA in the experiment).

[0068] (2) Heat shock the centrifuge tube in a circulating water bath preheated to 42°C for 45 seconds.

[0069] (3) Quickly transfer the centrifuge tube to ice and allow the cells to cool for 2 minutes.

[0070] (4) Add 700 μL of LB liquid culture medium to each tube. Heat the culture medium to 37°C in a water bath, then transfer the centrifuge tube to a 37°C shaker and incubate for 1 hour to allow the bacteria to recover (to achieve effective transformation, the speed should not exceed 225 r / min during recovery).

[0071] (5) Take 100 μL of the transformed competent cells and transfer them to the antibiotic (Kan resistance) LB agar plate. Use a sterile curved glass rod to evenly spread the transformed cells on the surface of the agar plate. This is considered a low concentration. Centrifuge at 5000 r / min for 3 min to collect the bacteria. After discarding the culture medium, keep 100 μL of the resuspended cells and evenly spread them on the surface of the agar plate. This is considered a high concentration.

[0072] (6) Place the plate upright at 37°C until the liquid is absorbed, then invert the plate and incubate for 10 to 12 hours to observe the results.

[0073] 4. Expression of rAXA19967.1 protein in small amounts and confirmation of its expression form

[0074] (1) The correctly sequenced plasmid was transformed into the BL21 expression strain and cultured overnight on a Kan-resistant plate.

[0075] (2) Pick a single clone of bacteria containing the recombinant plasmid and place it in 5 mL of LB liquid culture medium (Kan resistance, 1:1000 dilution) and culture it at 37°C overnight; at the same time, pick the corresponding bacteria transformed with the empty vector as a later control.

[0076] (3) Inoculate the seed solution at a ratio of 1:100. Generally, 100 μL of the recombinant plasmid solution and the empty plasmid solution are added to two bacterial bottles containing 10 mL of liquid LB medium (LB should be shaken sufficiently), and cultured at 37°C until the OD 600 The value reaches 0.6-0.8 (it takes about 3 hours, pay attention to observe the turbidity of the bacteria, that is, when the bacterial solution begins to become turbid).

[0077] (4) Take 1 mL of liquid as the uninduced control, and add IPTG inducer to the rest to make the final concentration reach 0.5 mM (IPTG: liquid culture medium = 1:1000, i.e., add 10 μL to 10 mL of culture medium). Induce protein expression by shaking and culturing at 37°C for 5 h.

[0078] (5) After confirming that the protein can be stably expressed in E. coli BL21, induce 10 mL of expression bacteria again, collect the bacterial solution in batches using 2 mL centrifuge tubes, centrifuge at 12000 r / min for 2 min, wash with 1× PBS, resuspend the bacteria in 1 mL 1× PBS, break the bacteria using an ultrasonic disruptor, centrifuge at 12000 r / min for 10 min at 4°C, treat the supernatant and precipitate separately and prepare protein gel samples for SDS-PAGE detection to determine the expression form of the recombinant protein. Then use Western Blot to detect the His tag to confirm that the protein is indeed expressed ( Figure 3 ).

[0079] 5. Large-scale expression and purification of rAXA19967.1 protein

[0080] (1) Dilute the overnight bacteria at a ratio of 1:100. Generally, add 10 mL of the recombinant plasmid solution to a 1 L culture bottle containing LB medium (use a large conical bottle to shake the bacteria to ensure that the bacteria are fully shaken). Cultivate at 37°C until the OD 600 The value reaches 0.6~0.8, and the time is about 3h.

[0081] (2) Add IPTG at a ratio of 1:1000 to a final concentration of 0.5 mM and culture at 37°C with shaking for 5 h.

[0082] (3) Before high-pressure disruption, collect the bacterial liquid in a 50 mL centrifuge tube, centrifuge at 4°C, 12,000 r / min for 10 min, resuspend and wash twice with PBS. Then use high-pressure disruption to make the bacterial cells clear and transparent, and collect the liquid.

[0083] (4) Precool the high-speed centrifuge to 4°C, centrifuge at 12,000 rpm for 10 min, and collect the supernatant.

[0084] (5) Filter the supernatant after crushing with a 0.45 μm filter and place on ice until loading onto the column.

[0085] (6) Use 20 mL of 1% imidazole to equilibrate the His-tagged Ni-NTA Resin column bed.

[0086] (7) The supernatant after filtration through a 0.45 μm filter is passed through the column twice and the collected liquid after passing through the column is considered as liquid A.

[0087] (8) Wash the non-specifically bound proteins with 30 mL of 10% imidazole and collect the solution as solution B.

[0088] (9) Wash the specific binding protein with 25 mL of 25% imidazole and collect the solution as solution C.

[0089] (10) Wash the specific binding protein with 20 mL of 50% imidazole and collect the solution as solution D.

[0090] (11) The eluted target protein liquid was ultrafiltered and concentrated using a 10 kDa ultrafiltration tube, and the target protein was replaced from the high concentration of imidazole using a PBS solution. The solution was centrifuged at 4000 r / min at 4°C. The ultrafiltration time was determined according to the filtration efficiency of the ultrafiltration tube. The above liquid was subjected to SDS-PAGE electrophoresis detection ( Figure 4 ).

[0091] (12) The final protein concentration was determined by BCA kit to be 0.483 mg / mL. The aliquots were stored in a -80°C refrigerator for later use.

[0092] 6. SDS PAGE and Western Blot Verification of the Reactivity of rAXA19967.1 Protein

[0093] Two prepared SDS-polyacrylamide gels were taken out and the protein loading amount was confirmed to be consistent by SDS-PAGE, and the reactivity of rAXA19967.1 protein was verified by Western Blot.

[0094] (1) Preparation of separation gel: Select an appropriate volume of separation gel, refer to the formula table, add separation gel to a moderate volume, add isopropanol to press the gel, and after the separation gel solidifies, pour out the isopropanol, rinse lightly with pure water, and then dry with absorbent paper.

[0095] (2) Preparation of stacking gel: Select a suitable volume of stacking gel, refer to the formula in the table, add stacking gel until full, insert a comb, and remove the comb from the tank soaked in electrophoresis buffer after the stacking gel solidifies.

[0096] (3) Sample preparation: The sample was mixed with SDS-PAGE loading buffer, treated at 100°C for 10 min, immediately placed in an ice bath for 2 min, centrifuged at 10,000 rpm for 30 s, and 1× glycine buffer was used as the electrophoresis buffer.

[0097] (4) Sample loading: Generally, 10 μL of sample is added to each well, along with 5 μL of marker (mixed with 5 μL of 1× Loading Buffer).

[0098] (5) Gel running: Keep the voltage at 80V until the sample reaches the boundary between the concentrated gel and the separating gel, then adjust to 120V to ensure that bromophenol blue runs to the bottom.

[0099] (6) After the protein gel is run, place it directly in Coomassie Brilliant Blue staining solution for 4 to 6 hours, and then transfer it to destaining solution (10% glacial acetic acid, 5% ethanol) for destaining.

[0100] (7) If a Western Blot test is required, the size of the gel must be measured: the specific length and width. According to the size of the gel, cut a PVDF membrane and three filter papers slightly larger than the gel. Soak the gel and paper in transfer buffer. Soak the membrane in methanol for 5 minutes and then transfer it to transfer buffer.

[0101] (8) Transfer: On the transfer plate, lay filter paper, membrane, gel, and filter paper in order, starting from the white layer. Cover each layer with a sufficient amount of transfer buffer and remove bubbles. Insert it into the transfer tank and set a constant voltage of 70V for 1 hour.

[0102] (9) Membrane washing: Rinse the membrane three times with 1× TBST buffer, 5 min each time.

[0103] (10) Blocking: Incubate with 5% skimmed milk powder blocking buffer at room temperature for 2 to 3 h.

[0104] (11)Same as (9).

[0105] (12) Primary antibody incubation: dilute serum with 1× TBST (immune serum dilution is 1:200) and incubate overnight at 4°C on a horizontal shaker.

[0106] (13)Same as (9).

[0107] (14) Enzyme-labeled secondary antibody incubation: horseradish peroxidase-labeled rabbit anti-bovine IgG (H+L) was diluted in 1× TBST at a dilution of 1:5000 and incubated on a horizontal shaker at room temperature for no more than 2 h.

[0108] (15)Same as (9).

[0109] (16) Color development: Equal amounts of chemiluminescent color development solution A and solution B were mixed on the membrane for development. The purified rAXA19967.1 protein is about 23 kDa in size. There are obvious target bands in the immune serum incubation, but no bands in the negative control ( Figure 5 ).

[0110] Example 2: Preparation of monoclonal antibodies against rAXA19967.1 protein

[0111] 1. Animal immunization

[0112] (1) Four female SPF BALB / c mice aged 4 to 6 weeks were selected. In this experiment, the purified protein was used as an antigen to immunize the mice. The immunization dose was 200 μg / mouse. At the same time, one mouse was selected as a negative control.

[0113] (2) During basic immunization, the antigen is mixed with equal amounts of Freund's adjuvant / Freund's incomplete adjuvant and emulsified using a three-channel stopcock-assisted syringe push method. Place a drop of the emulsified antigen on the water surface. If it does not disperse and dissolve within 30 seconds, the emulsification is complete. If it disperses quickly, emulsification needs to be continued.

[0114] (3) After the third immunization, on the 7th day, about 50 to 100 μL of blood was collected from the base of the mouse tail and placed in a PCR tube. The tube was placed in a 37°C constant temperature incubator for 30 min and centrifuged at 2000 rpm for 5 min. The serum was stored at -20°C and the indirect ELISA method was used to detect the titer of the mouse serum to determine whether a fourth immunization was needed.

[0115] (4) 3 to 5 days before cell fusion, mice with high titers were boosted with an intraperitoneal injection of 100 to 200 μg of the immune antigen without adjuvant or emulsification into each BALB / c mouse.

[0116] (5) Specific immunization procedures (Table 3).

[0117] (6) The serum titer was tested by indirect ELISA method. The results showed that the serum titer of four SPF-grade BALB / c mice immunized with rAXA19967.1 protein reached the fusion standard, and the serum titer ratio of 1# immunized mouse was the highest, reaching 1:409600. Therefore, this mouse was selected for booster immunization for cell fusion.

[0118] Table 3 Procedure for immunizing mice with rAXA19967.1 protein

[0119]

[0120] 2. The specific method of indirect ELISA operation is as follows

[0121] (1) Antigen coating: Dilute the purified protein with carbonate buffer (CBS) at pH 9.6 to a concentration of 0.25-8 μg / mL. Coat each well of the ELISA plate with 100 μL of the diluted protein and incubate at 4°C for at least 14 h.

[0122] (2) Washing: Discard the antigen and wash three times with 1× PBST, 3 min each time, spin dry, 300 μL per well.

[0123] (3) Blocking solution (1% fish gelatin): Add 1 g of fish gelatin to 100 mL of 1× PBS diluent and incubate at 37°C for 2 h.

[0124] (4) Washing: Wash three times with 1× PBST, 3 min each time, spin dry, add 300 μL per well, and store the protein-coated 96-well ELISA plate at -20°C for subsequent detection.

[0125] (5) Adding the serum to be tested: Add the serum to be tested in appropriate dilution, dilute it in multiples and add it to the reaction wells. The negative control is the serum of non-immunized mice from the same batch, 100 μL per well, and incubate at 37°C for 1 h.

[0126] (6) Repeat step 2 to wash and spin dry.

[0127] (7) Add 100 μL of horseradish peroxidase-labeled goat anti-mouse IgG (H+L) diluted to the working concentration to each well, dilute the enzyme-labeled secondary antibody to 1:5000 with 1× PBS, and incubate at 37°C for 1 h.

[0128] (8) Discard the secondary antibody, repeat step 2 to wash, and spin dry.

[0129] (9) Add 100 μL / well of color developing solution and incubate at 37°C in the dark for 10 min.

[0130] (10) Add 50 μL / well of stop solution and read the OD value on a microplate reader. 450 Values ​​and keep records.

[0131] 3. SP2 / 0 cell recovery and activation

[0132] After taking out the SP2 / 0 myeloma cells frozen in liquid nitrogen, quickly place them in a 42°C water bath and shake them quickly to completely melt. In a sterile operating table, place the thawed cell suspension in a 1.5mL sterile EP tube, centrifuge at 1000r / min for 3-5min, discard the supernatant, add 1mL of complete 1640 medium and gently resuspend, centrifuge at 1000r / min for 3-5min, discard the supernatant to eliminate the effect of DMSO in the freezing solution on the cells, and then resuspend the cells with complete 1640 and place them in a cell bottle, 5% CO 2 , 37℃ incubator. Observe the cell status the next day. If the cells are round, translucent, and adherent to the wall, they are in good condition. Subculture and expand according to the cell quantity and cell status. If some cells are floating or abnormal in morphology, gently aspirate and discard the culture medium, replace with new culture medium, and wait for the cells that have already adhered to the wall to gradually divide and grow.

[0133] 4. Preparation of Immune Splenocytes

[0134] (1) A mouse with the highest serum antibody titer was selected for three booster immunizations, with an interval of 3 to 5 days. After 3 days, the immunized mouse was bled from its eye sockets and killed by cervical dislocation after the blood was collected. The collected serum was stored as positive serum, and the serum of the unimmunized mouse was collected as negative control.

[0135] (2) Mice killed by cervical dislocation were soaked in 75% alcohol for 5 minutes for disinfection. After being taken out, they were suspended vertically for a few seconds to drain away excess alcohol. The mice were fixed in a supine position on a dissecting board. The fur layer was cut from the middle of the two hind legs using clean and sterilized scissors and tweezers. The fur layer was peeled off from bottom to top to expose the peritoneum.

[0136] (3) Replace a new set of scissors and forceps, open the peritoneum from the lower left, cut it upward and to the right, and then completely reverse the cut peritoneum from the lower left to the upper right to expose the abdominal cavity.

[0137] (4) Replace another set of scissors and forceps, carefully cut off the spleen and surrounding connecting tissues, place the spleen into a pre-prepared cell dish containing a filter mesh and basal 1640 culture medium, and use a disposable grinding rod to grind the spleen.

[0138] (5) Grind the spleen until yellow substance is visible to the naked eye. Use a disposable Pasteur pipette to transfer the ground cell suspension into a 50 mL centrifuge tube and gently stir to absorb any connective tissue that may be present.

[0139] (6) Centrifuge the spleen cell suspension at 1500 rpm for 5 min, which can be repeated once. Resuspend and count the cells in 20 mL of basal 1640 culture medium.

[0140] 5. Preparation of Feeder Cells

[0141] (1) The growth of hybridoma cells depends on a certain amount of cell density. In addition, feeder cells can secrete some cell growth factors to help the growth of hybridoma cells.

[0142] (2) Take a SPF 8-10 week old female BALB / c mouse, remove its eyeballs and bleed it to death, collect blood and separate serum, which is the negative serum; soak it in 75% alcohol for 5 minutes, move it into a sterile operating table, and follow the same operating steps as the immune spleen cell preparation method.

[0143] (3) Make a small cut in the middle of the abdomen, tear open the skin, use a 5-mL syringe to draw 5 mL of complete 1640 medium containing HAT, gently lift the peritoneum with sterilized tweezers, carefully insert the needle, inject the medium, massage and withdraw, repeat once, mix the two aspirated solutions, and observe the peritoneal macrophages.

[0144] (4) Then, mix the spleen cells obtained after grinding and add HAT complete medium to 80-100 mL, put it into a sterilized 250 mL blue-cap bottle, and place it at 37°C with 5% CO. 2 The incubator is ready for use.

[0145] 6. Cell Fusion

[0146] (1) Myeloma cells and immune spleen cells were mixed and centrifuged at a ratio of 1:5. Myeloma cells (1-2×10 7 cells / mL) and immune spleen cells (1×10 8 cells / mL) in a 50 mL centrifuge tube, mix well, and centrifuge at 1200 r / min for 10 min.

[0147] (2) Use sterile filter paper to absorb the residual culture medium liquid on the wall of the centrifuge tube to avoid affecting the concentration of the fusion agent. Tap the bottom of the centrifuge tube to loosen the cell clumps slightly. The mixed clumps of spleen cells and myeloma cells after centrifugation need to be patted into a cloud-like state. Wait until they are completely patted into a cloud-like state. Preheat the 50% PEG4000 fusion agent in a 37°C incubator in advance.

[0148] (3) The fusion process should be carried out in a 37°C water bath. Take out 0.8 mL of 50% PEG4000 preheated at 37°C and add it to the cell mass at the bottom of the centrifuge tube within 1 min. Try to minimize the collision between the cell tip and the cell. Add the solution while rotating at a constant speed. After adding, gently stir with a disposable rubber tip for 30 seconds, and then let it stand for 30 seconds to allow the fusion agent to fully take effect.

[0149] (4) Slowly add basal 1640 medium (preheated at 37°C) to terminate the fusion reaction. Add 2 mL in the first 2 minutes (i.e. 1 mL for 60 seconds), 3 mL in the 3rd to 4th minute, 5 mL in the 5th minute, and finally add about 30 mL to terminate the reaction. The entire operation should be completed within 10 minutes.

[0150] (5) Centrifuge at 1000 r / min for 8 min, discard the supernatant, and place the cell pellet in a 37°C incubator for 5-8 min. Gently resuspend the cell pellet to 120-150 mL with HAT complete medium and pre-prepared HAT medium for feeder cells (preheated at 37°C in advance), and plate the mixed cells in 6 96-well cell plates for culture, with 200-250 μL per well.

[0151] (6) Place in an incubator (37°C, 5% CO 2 ) were cultivated.

[0152] 7. Screening of positive hybridoma cell lines

[0153] (1) On the first day after fusion, take out one of the plates and observe the cell status. Move the cell culture plate as little as possible within 3 days and observe the growth status of the hybridoma cells every day to check for contamination and colony growth.

[0154] (2) On the fourth day after fusion, 50 μL of HAT complete 1640 medium was added to each well. Starting from the fifth day, HT complete 1640 medium was replaced. The wells where cells grew into clusters were marked.

[0155] (3) When the cell colonies have grown to about 1 / 4 to 1 / 3 of the culture well, the cell culture supernatant is detected by indirect ELISA in method 2 above.

[0156] (4) When performing indirect ELISA to detect the titer of cell culture supernatant, 50 μL was aspirated from each well as the primary antibody, and the secondary antibody was goat anti-mouse IgG (H+L) labeled with horseradish peroxidase.

[0157] (5) Using rAXA19967.1 protein and broken pET-28a empty load as coating antigens, indirect ELISA was performed to screen wells with high positive values ​​and low negative values, and the P / N values ​​were calculated. Wells with higher P / N values ​​were regarded as positive hybridoma cell wells.

[0158] (6) Label the corresponding positive hybridoma cells and add HT medium to prepare for expansion and subcloning.

[0159] 8. Subcloning of positive hybridoma cells

[0160] Since the fused hybridoma cells are randomly and evenly spread in the 96-well plate, it cannot be guaranteed that each well contains only one hybridoma cell that secretes antibodies against a single antigen epitope. Therefore, the cells in the positive wells should be monocloned, and the cells in each well should be divided from one cell to form a monoclonal cell colony. This experiment uses the limiting dilution method to clone positive cells. By counting the cell dilution, try to keep 1 to 2 cells in each cell well, and then subculture to form a single cell colony in the well. At the same time, through several cloning, some cells that cannot stably secrete antibodies after fusion can be eliminated. Ensure that a monoclonal cell line that stably secretes antibodies is obtained.

[0161] The steps of limiting dilution cell cloning are as follows:

[0162] (1) Screen the wells with cells that are positive in the indirect ELISA test, and gently blow up and resuspend the cells with a 200 μL pipette tip. If the cell density is high, dilute the cells 10-fold before counting. Dilute the cells to a concentration of about 100 to 200 cells per 10 mL of HT medium (i.e., about 1 to 2 cells per 100 μL).

[0163] (2) Add the diluted hybridoma cells to a 96-well plate pre-coated with feeder cells. The feeder cell concentration should be just enough to cover the entire well. Too much or too little feeder cells may affect the growth of the hybridoma cells.

[0164] (3) On the first day after cloning, observe the growth of hybridoma cells and check for contamination. When the cell colonies become large, add a drop of HT culture medium (50 μL per well).

[0165] (4) After the cell colonies grew to an appropriate size, indirect ELISA detection was performed, and subcloning was performed three times in succession. Finally, the hybridoma cell line 6H4 that could stably secrete antibodies was screened and the monoclonal antibody cell line was expanded and frozen in time.

[0166] 9. Cryopreservation of Positive Fusion Cell Lines

[0167] (1) Positive fusion cell lines were expanded and cultured in 24-well cell culture plates.

[0168] (2) During the logarithmic growth phase, the cell suspension was collected and centrifuged at 1000 rpm for 5 min, and the cell supernatant was discarded.

[0169] (3) Resuspend the cells in fetal bovine serum containing 10% dimethyl sulfoxide and dispense them into cell cryopreservation tubes, 1 mL / tube, freeze at -80°C under the protection of isopropanol, and store in a liquid nitrogen tank the next day.

[0170] 10. Large-scale preparation of monoclonal antibodies

[0171] (1) The established positive cell line is expanded and cultured for 3 to 4 generations. The titer of the cell supernatant is detected by indirect ELISA. If the titer is still high, it is used to prepare ascites.

[0172] (2) Pre-stimulation: Female BALB / c mice aged 8 to 10 weeks were selected and first injected intraperitoneally with 500 μL of Freund's incomplete adjuvant per mouse.

[0173] (3) After 5 to 7 days, hybridoma cells were injected intraperitoneally. The expanded hybridoma cells were resuspended in basal 1640 medium and centrifuged at 1000 rpm for 10 min. Approximately 5×10 5 ~1×10 6 The injection volume for each mouse was about 300 μL.

[0174] (4) Begin observing the mouse’s abdominal cavity to see if it is significantly enlarged around the 7th day after injection. When the mouse becomes unable to move, first wipe the mouse’s abdomen with alcohol cotton and then insert a 10 mL syringe needle into the mouse’s abdomen to facilitate the outflow of peritoneal fluid. At the same time, be aware that needle holes may cause infection to the mouse, so be sure to disinfect the mouse properly.

[0175] (5) Centrifuge at 10,000 rpm and 4°C for 15 min. Aspirate the supernatant, which is the ascites. Discard the fat component on the top and any impurities such as red blood cells on the bottom. Frozen at -80°C.

[0176] 11. Purification of Ascites

[0177] The monoclonal antibody ascites was purified using rProtein G Beads 4FF column. The specific steps are as follows:

[0178] (1) Load rProtein G Beads 4FF into a suitable chromatography column and balance the column with 5 times the volume of binding buffer so that the filler is in the same buffer system as the target protein to protect the protein.

[0179] (2) Add the sample to the equilibrated rProtein G Beads 4FF (to ensure that the target protein is in full contact with rProtein GBeads4FF and to improve the recovery rate of the target protein), and collect the effluent.

[0180] (3) Wash with 10 to 15 column volumes of wash buffer to remove non-specifically adsorbed proteins and collect the wash buffer.

[0181] (4) Use 5 to 10 column volumes of elution buffer and collect the eluate, which is the target protein fraction.

[0182] (5) Use 3 column volumes of binding buffer and 5 column volumes of pure water to equilibrate the packing material, and finally equilibrate it with 5 column volumes of 20% ethanol. Then store it in an equal volume of 20% ethanol and store it at 4°C to prevent the packing material from being contaminated by bacteria.

[0183] (6) The eluate was dialyzed in PBS (pH 7.0, 0.01 M) for 24 h and the eluate with higher purity was collected.

[0184] (7) SDS-PAGE electrophoresis to detect the effect of purified antibodies ( Figure 6 ).

[0185] (8) The BCA protein concentration was used to determine the 6H4 antibody concentration. The results showed that the antibody concentration of the purified rAXA19967.1 monoclonal antibody 6H4 strain was 1.020 mg / mL.

[0186] 12. Identification of monoclonal antibody subtypes

[0187] (1) Return the reagent kit to room temperature and prepare the cleaning solution into working solution with pure water.

[0188] (2) Take out the ELISA plate. Each sample needs 8 wells, 8 wells for positive control, and 8 wells for negative control. Store the excess in a ziplock bag and remember to put in a desiccant.

[0189] (3) Add 50 μL of cell culture supernatant (or specific affinity purified antibody) to the enzyme-labeled microplate, add 8 wells for each sample, and add 50 μL per well for each positive control and negative control. Then add 50 μL of sample diluent to each well. Apply sealing film and incubate at 37°C for 30-40 minutes.

[0190] (4) Discard the liquid in the plate, wash the plate 5 times, and then pat dry on a non-fibrous absorbent material or machine wash 5 times. Then add 100 μL of each of the 8 enzyme-labeled secondary antibodies (IgG1a, IgG2a, IgG3, IgG2b, IgA, IgM, κ, λ) to each of the 8 wells of each specimen, and the same for the 8 wells of the universal positive control. Mark the sample addition map or the ELISA plate. Apply a sealing film and incubate at 37°C in the dark for 30 to 40 minutes.

[0191] (5) Aspirate the liquid in the plate, wash the plate 5 times, and then pat dry on a non-fibrous absorbent material or machine wash 5 times. Add 100 μL of TMB single-component colorimetric solution to each well, replace a new sealing film, and place the plate in the dark at 37°C for 15 min.

[0192] (6) The results can generally be observed with the naked eye. The Ig class or subclass of the sample can be determined by looking at the enzyme-labeled secondary antibody corresponding to the well that shows blue color. After adding the reaction stop solution (50 μL per well), the reaction is terminated and the OD value is measured using an ELISA reader. 450 Measure the absorbance.

[0193] The results showed that the heavy chain subtype of monoclonal antibody 6H4 against rAXA19967.1 protein of goatpox virus was IgG1a, and the light chain type was κ.

[0194] 13. Monoclonal Antibody Titer Determination

[0195] The monoclonal antibody 6H4 before and after purification was eluted at a ratio of 1:2 0 ×10 2 Dilute to 1:2 15 ×10 2 The antibody titer was tested by indirect ELISA test. The results showed that the titer of 6H4 antibody before purification reached 2 15 ×10 2 Above; the titer of the purified 6H4 antibody reached 2 9 ×10 2 Above (Judgment criteria: OD was measured after diluting the antibody in multiples 450 When the value reaches or is closest to 1, the titer of the monoclonal antibody is the corresponding dilution ratio).

[0196] 14. Blocking Activity Analysis

[0197] The competitive ELISA method was used to calculate the inhibition rate (PI) to analyze the antibody blocking activity. The specific operation is as follows:

[0198] (1) Antigen coating: The purified rAXA19967.1 protein was diluted to 1 μg / mL with coating solution (CBS) and coated on the ELISA plate at 37°C for 2 h.

[0199] (2) Washing: Washing solution 1× PBST, 300 μL per well, let stand for 3 min each time, wash twice.

[0200] (3) Blocking: Blocking solution: 5% (w / v) skim milk powder, 200 μL per well, incubate at 37°C for 1 h.

[0201] (4) Washing: Washing solution 1× PBST, 300 μL per well, let stand for 3 min each time, wash 3 times.

[0202] (5) Add immune serum and clinical negative serum diluted with PBS to 1:2. Dilute the purified 6H4 monoclonal antibody with 1× PBS to 1:5000, 1:10000 and 1:20000. Add 50 μL of the diluted serum and monoclonal antibody to each well and incubate at 37°C for 1 h.

[0203] (6)Same as (4).

[0204] (7) Add HRP-goat anti-mouse IgG (H+L) diluted with 1× PBS at a ratio of 1:5000, 100 μL per well, and incubate at 37°C for 1 h.

[0205] (8)Same as (4).

[0206] (9) Substrate: 100 μL of TMB single-component colorimetric solution per well, incubate at 37°C in the dark for 10 min.

[0207] (10) Stop solution: 50 μL / well.

[0208] (11) Reading: OD is read by microplate reader 450 Numeric value.

[0209] (12) Calculate the PI value according to the following formula: PI = (1-OD of the sample well) 450 Value / OD of negative control well 450 value)×100%.

[0210] The results showed that: Preliminarily, monoclonal antibody 6H4 had specific blocking activity, and the blocking effect was best after dilution of 1:20000, and the calculated PI value was 48.72%.

[0211] 15. Enzyme-labeled monoclonal antibody and titer determination

[0212] Horseradish peroxidase (HRP) labeled the purified rAXA19967.1 monoclonal antibody 6H4. The results showed that the titer of the purified monoclonal antibody after enzyme labeling was 1:2 7 ×10 2 (Judgment criteria: positive serum OD 450 value greater than or close to 1).

[0213] Example 3: Application of proteins and antibodies in ELISA detection and verification of analytical results

[0214] 1. Optimization of ELISA detection conditions

[0215] 1) Determination of antigen coating concentration and serum dilution multiple: dilute rAXA19967.1 protein to 4μg / mL, 2μg / mL, 1μg / mL, and 0.5μg / mL, and coat the ELISA plate at 37℃ for 2h. Coat two columns of 12 wells for each concentration. Dilute the negative and positive sera with diluent PBS at a ratio of 1:2, 1:4, 1:8, and 1:16, respectively. Add the negative and positive sera of each dilution to the gradient protein concentration wells. Follow the routine ELISA steps and measure the OD with an ELISA reader. 450 Numerical values, comparing the PI values ​​corresponding to different concentrations of antigen and serum, negative serum OD 450 The antigen coating concentration and serum dilution multiple corresponding to a value close to 1 were regarded as the optimal conditions (Table 4), that is, the antigen coating concentration and serum dilution multiple were 1 μg / mL and 1:2, respectively, and the PI value was 61.39%.

[0216] Table 4 Determination of antigen coating concentration and serum dilution multiple

[0217]

[0218] Note: + indicates positive serum, - indicates negative serum.

[0219] 2) Optimization of the optimal working concentration of enzyme-labeled monoclonal antibody: According to the conditions determined above, other experimental conditions were fixed, and the enzyme-labeled monoclonal antibody was diluted at 1:4000, 1:6000, 1:8000 and 1:10000 respectively. According to the conventional ELISA operating steps, the optimal enzyme-labeled monoclonal antibody working concentration was selected according to the PI value (Table 5). The results showed that when the optimal dilution of HRP-6H4 antibody was 1:4000, the PI value was 64.30%.

[0220] Table 5 Optimization of the optimal working concentration of enzyme-labeled monoclonal antibodies

[0221]

[0222] 3) Optimization of the optimal substrate action time: other experimental conditions were fixed, and the substrate was allowed to act at 37°C for 5 min, 10 min, 15 min, 20 min and 25 min respectively. According to the routine ELISA operation steps, the optimal substrate action time of 10 min was determined based on the PI value.

[0223] Therefore, the detailed steps for finally determining the detection method are as follows:

[0224] (1) Coating: The purified protein was coated on the ELISA plate at a concentration of 1 μg / mL using carbonate buffer (CBS, pH 9.6), 100 μL / well, and incubated at 37°C for 2 h.

[0225] (2) Washing: 1% PBST washing solution 300 μL / well, wash twice, each time for 3 minutes.

[0226] (3) Blocking: 5% (w / v) skim milk powder blocking solution 200 μL / well, incubate at 37°C for 1 h.

[0227] (4) Washing: 300 μL / well of washing solution, wash three times, 3 min each time.

[0228] (5) Add serum (positive serum and negative serum were diluted at 1:2) and enzyme-labeled monoclonal antibody (diluted at 1:4000), 50 μL / well each, and incubate at 37°C for 1 h.

[0229] (6)Same as (4).

[0230] (7) Substrate: TMB single-component colorimetric solution 100 μL / well, incubate at 37°C in the dark for 10 min.

[0231] (8) Stop solution: 50 μL / well.

[0232] (9) Reading: OD is read by microplate reader 450 Numeric value.

[0233] (10) Result determination: Calculate the OD of the negative and positive control wells and each serum well to be tested on the same ELISA plate. 450 The inhibition rate (PI) was calculated according to the following formula: PI = (1-OD of the sample well) 450 Value / OD of negative control well 450 value)×100%.

[0234] 2. Determination of critical value of ELISA test kit

[0235] Detect 30 goat pox attenuated vaccine (AV41 strain) cattle serum samples immunized for 60 days with known background and 30 clinical negative cattle serum samples with known background. Positive and negative controls were set at the same time. After repeated detection of OD 450 The PI value is calculated ( Figure 7 ).

[0236] The critical value judgment standard of this method is determined according to the ROC curve as follows: The critical value PI = 51.46% ( Figure 8 ). Therefore, the results of the serum samples to be tested are determined as follows: when the positive control serum PI>51.46%, the negative control OD 450When the value is ≥0.793 and the blank control PBST is <0.1, the test result is established; read the OD of the serum to be tested 450 If the calculated PI is ≥51.46%, it is judged as positive; if PI is <51.46%, it is judged as negative (PBST of the blank group is <0.1, otherwise the plate is invalid).

[0237] 3. ELISA analysis sensitivity and specificity test

[0238] In order to test whether the ELISA method based on rAXA19967.1 protein has good analytical specificity, positive serum from cattle immunized with goat pox attenuated vaccine, positive serum from bovine viral diarrhea virus, positive serum from bovine Pasteurella multocida and positive serum from bovine Mycoplasma were selected and diluted 1:2, and three samples were repeated, and the OD values ​​were read. 450 To test the analytical sensitivity of the ELISA method based on rAXA19967.1 protein, positive serum from cattle immunized with goat pox attenuated vaccine and clinical negative serum were diluted to 1:64 at a ratio of 1:2, and the OD was read. 450 Numerical value, calculate the PI value.

[0239] The results showed that except for the specific binding reaction with the positive serum of cattle immunized with goat pox attenuated vaccine, there was no cross reaction with the positive serum of bovine viral diarrhea virus, bovine Pasteurella multocida and bovine Mycoplasma, indicating that the ELISA kit has good specificity. The ELISA method established based on rAXA19967.1 protein, when the positive serum was diluted to 1:4, PI = 61.46% (PI ≥ 51.46%, judged as positive), so the minimum positive serum detection limit is 1:4, which is higher than the 1:2 established by the ELISA method.

[0240] 4. Repeatability test of ELISA detection method

[0241] In order to test whether the ELISA method based on rAXA19967.1 protein has good repeatability, 6 serum samples were taken, including 3 positive serum samples and 3 negative serum samples. Each serum sample was repeated 3 times in the same ELISA plate. The intra-batch coefficient of variation (CV,%) was calculated to determine the intra-batch repeatability of ELISA. In addition, 3 batches of ELISA plates were used to test the above serum. Finally, the inter-batch coefficient of variation (CV,%) was calculated to determine the inter-batch repeatability of ELISA. The results showed that the intra-batch repeat coefficient of variation was 3.26%-9.78%, less than 10%; the inter-batch repeat coefficient of variation was 1.05%-6.40%, less than 10%.

[0242] 5.37℃ stability test

[0243] The ELISA plates coated with rAXA19967.1 protein from the same batch were placed at 37°C for 1, 3, 5 and 7 days, and three samples of GTPV positive and negative serum were repeated. The results showed that the OD of serum samples in the same group was 0.0447 W / m2 at 37°C for 5 days. 450 The differences in the values ​​were all less than 0.2 and the PI value of the positive serum was >51.46%, indicating that the protein-coated ELISA plate could withstand high temperature damage for 5 days.

[0244] 6.ELISA test of clinical serum

[0245] 81 samples of goat pox attenuated vaccine immunized cattle serum after 60 days were tested, and GTPV immune serum (positive control) and clinical healthy cattle serum (negative control) were set up to read OD 450 The inhibition rate (PI) was calculated based on the judgment criteria, and the positive rate of immune antibodies was calculated to be 60.49% (95% CI: 49.00-71.20). This shows that the competitive ELISA method based on rAXA19967.1 protein has a good diagnostic effect and clinical application value.

Claims

1. A diagnostic antigen of goat pox virus (GTPV) of the genus Capripoxvirus, the nucleotide sequence of the encoding gene of which is shown in SEQ ID NO.

1.

2. A method for preparing the antigen according to claim 1, characterized in that: Primers are designed using the PCR method to amplify the target gene fragment with a nucleotide sequence such as SEQ ID NO.1 from the GTPV genome, and then the target gene fragment is cloned into an expression vector to obtain a recombinant plasmid. The recombinant plasmid is transformed into competent cells to induce expression to obtain a recombinant protein, which is the antigen.

3. A monoclonal antibody against the antigen according to claim 1.

4. The monoclonal antibody according to claim 3, characterized in that: The monoclonal antibody is secreted by the hybridoma cell line 6H4, and the hybridoma cell line 6H4 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C202576.

5. A hybridoma cell line 6H4, deposited in China Center for Type Culture Collection with the deposit number CCTCCNO: C202576.

6. Use of the antigen according to claim 1 and / or the monoclonal antibody according to claim 3 or 4 in the preparation of a GTPV diagnostic kit.

7. A GTPV diagnostic kit, comprising the antigen according to claim 1 and / or the monoclonal antibody according to claim 3 or 4.

8. The GTPV diagnostic kit according to claim 7, which is a competitive ELISA kit for detecting GTPV antibodies, and the kit contains an ELISA plate coated with the antigen and the monoclonal antibody labeled with horseradish peroxidase.

9. A competitive ELISA method for detecting GTPV antibodies for non-diagnostic purposes, characterized in that The following steps are involved: (1) coating an ELISA plate with the antigen according to claim 1; (2) Block the ELISA plate with blocking solution; (3) labeling the monoclonal antibody according to claim 3 or 4 with horseradish peroxidase; (4) diluting the serum sample and the monoclonal antibody labeled with horseradish peroxidase and adding them to the ELISA plate for reaction; (5) Add the colorimetric solution and stop solution. 450 Reading at wavelength.

10. The competitive ELISA method for detecting GTPV serum antibodies according to claim 9, characterized in that: The coating concentration of the antigen is 1 μg / mL; the dilution multiple of the serum sample is 1:2; and the dilution multiple of the monoclonal antibody labeled with horseradish peroxidase is 1:4000.

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