A method for rapid testing of the efficacy of bovine nodule skin disease inactivated vaccine

Abstract

The application discloses a specific goatpox virus P32 truncated protein and application thereof in vaccine efficacy detection. The amino acid sequence of the protein is shown as SEQ ID NO: 3. The specific truncated form (aa1-aa238) has significantly better soluble expression in E. coli than the full-length and other truncated forms (such as aa1-aa277). A competitive ELISA method for quantitatively detecting the efficacy of bovine nodular skin disease inactivated vaccine is established by taking the specific truncated form as a coating antigen. The method is systematically optimized, and has high sensitivity, strong specificity and good repeatability. Key is that the relative efficacy (RP) value measured by the method is highly correlated with animal challenge protection results, and when the RP value is greater than or equal to 1.0, complete immune protection can be predicted, so that the traditional animal challenge test can be reliably replaced. Compared with the immune challenge method, the detection period is greatly shortened (from 63 days to 2 days), target animals are not used, the detection cost is saved, and the method does not need P3 laboratory conditions and has no biosafety risk.

Description

Technical Field

[0001] This invention belongs to the field of vaccine testing technology, specifically relating to a method for rapidly testing the efficacy of an inactivated vaccine against bovine nodular dermatitis. Background Technology

[0002] Lump skin disease (LSD), caused by bovine nodular skin disease virus (LSDV), is a highly contagious bovine viral disease. LSD is primarily transmitted through blood-sucking insects, with an incubation period of 3–12 days. Its main characteristic is the appearance of nodules of varying sizes on the skin and mucous membranes, leading to decreased hide quality, reduced milk production in dairy cows, abortion in pregnant animals, infertility in male animals, and severe secondary infections, thus having a serious impact on the cattle industry. The World Organisation for Animal Health (WOAH) lists LSD as a reportable disease, and my country classifies it as a Class II animal disease.

[0003] LSDV belongs to the family Poxviridae, subfamily Vertebraviirinae, and genus Capunpoxvirus. It shares over 97% genomic homology with capunpoxvirus (GPV) and is a cytoplasmic, enveloped, double-stranded DNA virus. Its genome is approximately 150 kb long, encoding 147 open reading frames. The encoded protein ranges from 53 to 2027 amino acids, including conserved poxvirus replication regions, structural genes, and genes determining virulence and host range. GPV P32, located at 64-65 kb in the GPV genome, shares 100% homology with the LSDV P32 protein. GPV P32 is antigen-specific, an enveloped protein with a relative molecular mass of approximately 32,000, and a transmembrane domain at its carboxyl terminus, exhibiting cytotoxicity. Located on the capunpoxvirus membrane, GPV P32 is a highly specific structural protein shared by all capunpoxvirus strains isolated and identified worldwide. It contains the main antigenic determinant sites of capunpoxvirus and induces antibody production in the early stages of viral infection. A single serum sample targeting this protein can neutralize sheep pox virus, indicating that the P32 protein possesses a neutralizing epitope that strongly induces cellular immunity. Therefore, the P32 protein has potential research value for the diagnosis and immunization of goatpoxviruses, and can be used to develop novel vaccines for the prevention of sheep pox and bovine nodular skin disease, as well as for the development of diagnostic kits for goatpoxviruses.

[0004] Currently, my country mainly uses live and inactivated goatpox vaccines to control bovine nodular dermatitis. Two inactivated bovine nodular dermatitis vaccines have been approved for marketing in China. Product efficacy testing is a crucial part of the manufacturing process. However, unlike live vaccines, the efficacy of inactivated bovine nodular dermatitis vaccines cannot be evaluated by detecting the viral load of live virus in the finished product. Instead, it often requires an evaluation using a target animal "immunization-challenge" method. The entire immunization-challenge test cycle is approximately 63 days (21 days for the first immunization + 21 days for the second immunization + 21 days of observation after challenge), and because a virulent bovine nodular dermatitis virus is required, it must be conducted in a P3 animal facility. Using alternative animals for product testing is a common alternative method for efficacy testing. In recent years, a method using guinea pigs instead of cattle for efficacy evaluation has been developed, namely, evaluating efficacy by "injecting guinea pigs with the vaccine and measuring the level of bovine nodular dermatitis virus antibodies in the guinea pig serum." Using alternative animals (such as guinea pigs) to evaluate the efficacy of inactivated vaccines against bovine nodular dermatitis is a significant advancement. However, this method requires the use of laboratory animals and a P3 laboratory, has a long testing cycle (generally 42 days), and places high demands on the testing facilities. Furthermore, it raises ethical concerns regarding laboratory animals. Therefore, finding faster, lower-cost testing methods that do not require the use of laboratory animals has been a continuous pursuit for researchers.

[0005] Therefore, developing an in vitro efficacy detection method that requires no laboratory animals (including alternative animals) and has a shorter detection cycle is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method for testing the potency of a bovine nodular dermatitis inactivated vaccine using a competitive enzyme-linked immunosorbent assay (ELISA). Specifically, the antigen content is detected using a competitive ELISA method, and the potency of the finished vaccine is evaluated by comparing the relative antigen content in the tested vaccine and a reference vaccine. Compared with testing using cattle or guinea pigs, the method of the present invention not only significantly shortens the testing cycle but also eliminates the need for animals and P3 laboratories, posing no biosafety risks and saving time and economic costs, demonstrating significant advantages.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a rational design and preparation of a specific truncated P32 protein. Instead of simply removing the transmembrane region, this invention creatively distinguishes and verifies different truncated forms through extensive screening and in-depth research on protein spatial structure. Unexpectedly, the P32 protein truncated to amino acid position 238 (SEQ ID NO:3) showed significantly better expression levels and solubility than the full-length protein (SEQ ID NO:1) and another truncated form (aa1-aa277, SEQ ID NO:2). Cloning this optimized gene into the pET-30a(+) vector and inducing expression in *E. coli* achieved efficient and soluble expression of this specific truncated P32 protein. After purification, a high-purity (≥92%) and highly reactive recombinant antigen was obtained.

[0008] Furthermore, based on the goatpox virus P32 truncated protein prepared by the above screening, this invention has screened and obtained a good monoclonal antibody against the goatpox virus P32 truncated protein. This monoclonal antibody has good specificity and sensitivity, providing a good antibody for the subsequent development of detection kits and detection methods.

[0009] Furthermore, the present invention also provides a method for rapidly testing the efficacy of an inactivated vaccine against bovine nodular dermatitis, comprising the following steps: a) The truncated P32 protein was coated onto a 96-well enzyme-linked reaction plate; b) Shake the vaccine to be tested and the reference vaccine well and freeze them at -70°C for at least 24 hours. After taking them out and demulsifying them according to the demulsification method, extract the lower layer of liquid as the antigen of the vaccine to be tested and the antigen of the reference vaccine. c) After serially diluting the reference vaccine antigen and the vaccine antigen to be tested by 2 times, transfer them to the corresponding wells of the 96-well ELISA plate prepared in step a); add 50 μl of the monoclonal antibody against the p32 truncated protein of goatpox virus diluted with PBS to each well, wherein the concentration of the diluted monoclonal antibody is 1 μg / ml. d) Prepare negative and positive sera diluted at appropriate ratios as negative and positive controls, respectively; prepare PBS as a blank control. e) Place the reaction plate on a shaker at 100 r / min and 37°C for 1.5 h; discard the liquid in the plate and wash it 3 times with PBST; f) Add 100 μL of HRP-labeled rabbit anti-mouse IgG diluted 1:17500 to each well. Place the reaction plate on a shaker and react at 37°C for 1 h at 100 rpm. Discard the liquid in the plate and wash three times with PBST. g) Add 100 μL of TMB substrate solution to each well, develop color at room temperature for 15 min, add 100 μL of stop solution to each well, and then read the OD450 nm absorbance value of each well. h) Result calculation: The OD450nm reading of each well should be subtracted from the OD450nm reading of the blank control; the RP value of the antigen contained in the vaccine should be calculated using biostatistical software; if the RP value of the vaccine to be tested is not less than 1.0 compared with the reference vaccine, the vaccine efficacy test is considered qualified; otherwise, the vaccine efficacy test is considered unqualified.

[0010] Preferably, in step a) of the present invention, the coating concentration of the P32 truncated protein is 16 μg / mL, and the coating conditions are coating in carbonate buffer at pH 9.6 at 4°C for 16 hours; step a) is followed by a step of blocking with 5% skim milk powder at 37°C for 1.5 hours.

[0011] Preferably, in step b) of the present invention, the reference vaccine is an inactivated bovine nodular dermatitis vaccine whose immunogenicity has been confirmed through animal challenge experiments.

[0012] Preferably, in step b) of the present invention, the demulsification method is as follows: take out the frozen vaccine to be tested and the reference vaccine, place them in a 37°C incubator for 20 minutes, then add them to a centrifuge tube, add n-butanol at a volume ratio of vaccine to n-butanol = 9:1, mix thoroughly, and centrifuge at 5000g for 10 minutes.

[0013] Preferably, in step d) of the present invention, the positive control is a mouse-derived positive serum diluted 1:400, and the negative control is a mouse-derived negative serum diluted 1:200.

[0014] Furthermore, the present invention also discloses a kit for detecting the efficacy of an inactivated vaccine against bovine nodular dermatitis, the kit comprising an effective amount of the aforementioned truncated P32 protein and an effective amount of the aforementioned monoclonal antibody against the truncated P32 protein of goatpox virus; the kit further comprises a positive control, a negative control, an enzyme-labeled secondary antibody, a chromogenic substrate, and a stop solution.

[0015] In another aspect, the present invention also discloses the use of the aforementioned goatpox virus P32 truncated protein and / or the aforementioned monoclonal antibody against goatpox virus P32 truncated protein in the preparation of a kit for detecting the efficacy of an inactivated vaccine for bovine nodular dermatitis.

[0016] Furthermore, the present invention also discloses the use of the method or the kit described therein in animal challenge efficacy tests as an alternative to bovine nodular dermatitis vaccine.

[0017] Compared with the prior art, the present invention achieves the following technical effects: 1. The expression level (approximately 15 mg / L) and soluble percentage (approximately 40%) of the truncated P32 protein (aa1-238) in *E. coli* provided by this invention represent an order-of-magnitude improvement compared to the full-length protein (<1 mg / L, soluble <1%) and other truncated forms, solving a fundamental problem in the large-scale preparation of this diagnostic antigen. Furthermore, based on the prepared truncated P32 protein, this invention screened and obtained a good monoclonal antibody against the truncated P32 protein of goatpox virus. This monoclonal antibody exhibits good sensitivity and specificity, making it suitable for diagnosis.

[0018] 2. The detection method possesses excellent analytical performance: (1) High sensitivity and wide linear range: The method has a minimum detection limit of 2 μg / mL and good linearity in the range of 2-512 μg / mL (R²=0.9867).

[0019] (2) High specificity: It reacts only with goatpox virus antigen and has no cross-reaction with common disease antigens such as goat infectious pleuropneumonia inactivated antigen and foot-and-mouth disease virus.

[0020] (3) High repeatability: The intra-batch and inter-batch coefficients of variation are as low as 3.65% and 6.30%, respectively, which meet the precision requirements of quantitative detection.

[0021] 3. A revolutionary alternative to animal testing: The most significant benefit of this invention lies in the fact that rigorous correlation experiments have demonstrated a high degree of parallelism between the vaccine RP value measured by this method and the animal challenge protection results. Data shows that vaccines with an RP value ≥ 1.0 provide 5 / 5 complete immune protection. This establishes for the first time a reliable and quantitative in vitro indicator (RP value) to directly predict the actual protective efficacy of a vaccine in animals, thus completely replacing traditional, costly animal challenge tests.

[0022] 4. Immense Industrial Application Value: This invention provides vaccine manufacturers and quality control institutions with a rapid (results in 1-2 days), economical, standardized, and highly automated efficacy testing tool. It not only significantly reduces testing costs and time, and improves quality control efficiency, but also substantially reduces the use of laboratory animals, aligning with animal welfare (3R) principles and industry development trends, thus possessing extremely important social and economic benefits.

[0023] In summary, this invention provides a rapid method for testing the potency of an inactivated bovine nodular dermatitis vaccine. The relative potency test results using this method are consistent with those obtained using the immune challenge method. Compared to in vivo potency testing using cattle or guinea pigs, this method is not only simpler and less time-consuming, but also eliminates the need for animals and P3 laboratories, posing no biosafety risks. It reduces testing costs and labor intensity, and enables high-throughput vaccine potency testing, demonstrating significant advantages. Therefore, it has excellent application value for evaluating the potency of inactivated bovine nodular dermatitis vaccines, which have a huge market potential. Attached Figure Description

[0024] For ease of explanation, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0025] Figure 1 The results are the transmembrane region analysis of the complete P32 protein amino acid sequence, where 1-282 is the extracellular region, 283-305 is the transmembrane region, and 306-324 is the intracellular region.

[0026] Figure 2 The results are the amino acid sequence signal peptide analysis results of the complete P32 protein.

[0027] Figure 3 SDS-PAGE analysis (left) and Western blot analysis (right, anti-His antibody) of P32 protein (aa1-aa277) expression in BL21 (DE3) cells. In the figure, M1: protein marker; M2: Western marker; NC: uninduced whole-cell lysate; 1: whole-cell lysate induced at 15 °C for 16 hours; 2: whole-cell lysate induced at 37 °C for 4 hours; NC1: uninduced cell lysate supernatant; NC2: uninduced cell lysate pellet; 3: cell lysate supernatant induced at 15 °C for 16 hours.

[0028] Figure 4 SDS-PAGE analysis (left) and Western blot analysis (right, anti-His antibody) of P32 truncated protein (aa1-aa238) expression in BL21 (DE3) cells. M1: protein marker; M2: Western marker; PC1: BSA (1 μg); PC2: BSA (2 μg); NC: uninduced whole cell lysate; 1: whole cell lysate induced at 15 °C for 16 h; 2: whole cell lysate induced at 37 °C for 4 h; NC1: uninduced cell lysate supernatant; NC2: uninduced cell lysate precipitate; 3: cell lysate supernatant induced at 15 °C for 16 h.

[0029] Figure 5 The image shows the SDS-PAGE analysis of purified P32 truncated protein (aa1-aa238) expressed in vitro. In the image, 1: BSA (2 μg); 2: purified P32 truncated protein (aa1-aa238) (2 μg); M1: protein marker.

[0030] Figure 6 The Western blot analysis results for the purification of P32 truncated protein (aa1-aa238) are shown in the figure. 3: Purified P32 truncated protein (aa1-aa238) (2 μg); M2: Protein Marker.

[0031] Figure 7 To create a standard curve for the ELISA standard. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Main solutions and reagents Phosphate buffer (0.01 mol / L, pH 7.2-7.4): Weigh 8.0 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate dodecahydrate, and 0.2 g potassium dihydrogen phosphate, dissolve them in ultrapure water, bring the volume to 1 L, adjust the pH to 7.2, filter to sterilize, and store at 2-8 °C for later use.

[0034] Carbonate buffer (0.1 mol / L, pH 9.6): Weigh 5.3 g sodium carbonate and 4.2 g sodium bicarbonate, dissolve in ultrapure water, bring the volume to 1 L, adjust the pH to 9.6, filter to sterilize, and store at 2–8 °C for later use.

[0035] Washing buffer (PBST): Weigh PBS buffer (dry powder) and dissolve it in ultrapure water, bring the volume to 1L, adjust the pH to 7.4, add Tween-20 to a final concentration of 0.05%, mix well, filter to sterilize, and store at 2-8℃ for later use.

[0036] The coating antigen was a truncated p32 protein of goatpox virus prepared using an E. coli expression system.

[0037] Positive serum for goatpox virus (mouse-derived, agar amplification titer not less than 1:8).

[0038] Negative serum from healthy mice.

[0039] Reference vaccine: Inactivated bovine nodular dermatitis vaccine whose immunogenicity has been verified through this animal challenge experiment, stored frozen at -70°C or below. Prepared and tested by the China Institute of Veterinary Drug Control.

[0040] Vaccine to be tested: Commercially available inactivated bovine nodular dermatitis vaccine (goatpox virus AV41 strain, suspension culture).

[0041] The PBST buffer containing 5% skim milk, enzyme-labeled antibody dilution solution (PBST buffer containing 5% skim milk), chromogenic solution (single-component TMB chromogenic solution), and stop solution (2M H2SO4) involved are all conventional solutions in existing technologies.

[0042] Example 1: Preparation of coating antigen The P32 protein is a highly specific immunogenic structural protein in goatpox virus strains. Therefore, this study first analyzed the amino acid sequence of the complete P32 protein (GenBank: ADN44125.1), and the results are as follows: Figure 1 and Figure 2 As shown, this protein lacks a signal peptide sequence, but it is a transmembrane protein with an extracellular region consisting of amino acids 1-282 (aa1-aa282) at the N-terminus. Therefore, those skilled in the art generally prefer to directly express the sequence of this extracellular region as a recombinant protein. In this study, it was named the full-length P32 protein (its amino acid sequence is shown in SEQ ID NO:1). However, during the preparation of this protein, we found that it mainly existed in the form of inclusion bodies and its expression level was extremely low; moreover, the full-length P32 protein exhibited significant cytotoxic effects.

[0043] Based on the above research, and through extensive screening and analysis of protein spatial structures, this study selected to truncate the full-length goatpox virus P32 protein, naming it P32 protein (aa1-aa277) and truncated P32 protein (aa1-aa238) for further research. It should be noted that this example only demonstrates data from these two treatments and does not indicate that this study only performed these two treatments. P32 protein (aa1-aa246) and P32 protein (aa1-aa260) were also studied in this research, and their expression levels in this system were essentially equivalent to those of the P32 protein (aa1-aa277) described below. Therefore, selecting a suitable truncation position is a key and challenging technical issue that needs to be addressed for the P32 protein. The gene sequences of the above-mentioned P32 proteins (aa1-aa277, whose amino acid sequences are shown in SEQ ID NO:2) and truncated P32 proteins (aa1-aa238, whose amino acid sequences are shown in SEQ ID NO:3) were codon-optimized and artificially synthesized. The synthesized genes were cloned into pET-30a(+) expression vectors, and after antibiotic selection and sequencing identification, recombinant positive clones were obtained, successfully constructing goat poxvirus recombinant pET30-p32 and pET30-p32-238 expression vectors. IPTG-induced expression showed extremely low expression levels of P32 protein (aa1-aa277), and the expression product mainly existed in the form of inclusion bodies. Figure 3 The recombinant protein is approximately 33.1 kDa, with an expression level below 1 mg / L and a solubility of less than 1%. Therefore, in vitro expression of both the full-length P32 protein and the P32 protein (aa1-aa277) is unsuitable for subsequent studies. In contrast, pET30-p32-238 can stably and efficiently express the truncated P32 protein (aa1-aa238), and the expression product is mainly in a soluble form. Figure 4 The recombinant protein is approximately 28.5 kDa in size, with an expression level of approximately 15 mg / L and a solubility of 40%. The truncated goatpox virus P32 protein (aa1-aa238) expressed in vitro was purified using nickel agarose gel FF, with a purity of approximately 92%. Figure 5 Western blotting results showed that the expressed P32 truncated protein (aa1-aa238) reacted with goatpox virus-positive serum (goat-derived). Figure 6 This indicates that the product obtained from E. coli expression has good reactivity. The purified P32 truncated protein (aa1-aa238) is the coating antigen for the in vitro potency competition ELISA method of bovine nodular dermatitis inactivated vaccine (goatpox virus AV41 strain, suspension culture).

[0044] Example 2: Preparation and testing of monoclonal antibodies against the p32 truncated protein of goatpox virus As described in Example 1, this invention successfully obtained a truncated goatpox virus P32 protein (aa1-aa238) with good solubility, high expression level, and good reactivity. Based on this, this study used it as an immunogen to prepare and screen specific monoclonal antibodies against this epitope. Compared with the prior art (such as Liu Y et al., 2024), which uses inactivated virus primary immunization and yields monoclonal antibody titers with limited efficacy (>1:12800), this study adopted a strategy of direct immunization with high-purity soluble recombinant P32 truncated protein, aiming to obtain antibodies with higher affinity and stronger specificity. The purified P32 truncated protein was emulsified with adjuvant and subcutaneously immunized BALB / c mice at multiple sites. Spleen cells from mice with serum antibody titers reaching the target were fused with SP2 / 0 myeloma cells and screened using HAT medium. Using the truncated P32 protein as a coating antigen, positive hybridoma cell lines were screened by high-sensitivity indirect ELISA and subcloned by limiting dilution method. Finally, a hybridoma cell line that can stably secrete monoclonal antibody against the truncated P32 protein was obtained and named mAb-2F3.

[0045] Monoclonal antibodies were prepared using an in vivo ascites in mouse model and purified by Protein G affinity chromatography. Antibody subtype identification showed that mAb-2F3 was IgG1 / κ. Indirect ELISA assays showed that the titers of this monoclonal antibody were all above 1:1×10⁻⁶. 6 The levels were significantly higher than those reported in the literature (Liu Y et al., 2024), and it could specifically recognize the goatpox virus P32 protein (including truncated and full-length fragments). The heavy chain variable region (VH) amino acid sequence of mAb-2F3 is shown in SEQ ID NO:4, and the light chain variable region (VL) amino acid sequence is shown in SEQ ID NO:5. These hypervariable sequences determine its unique antigen-binding properties.

[0046] Further functional validation demonstrated that the monoclonal antibody obtained in this study possesses superior binding properties. Western blotting analysis confirmed that the monoclonal antibody mAb-2F3 specifically recognizes recombinant p32 truncated protein (approximately 28.5 kDa), p32 protein (aa1-aa277, approximately 33.1 kDa), and native viral proteins in cell lysates from goatpox virus infection, all expressed in prokaryotes. More importantly, compared to existing reports where only some monoclonal antibodies (such as 3E8) can recognize native viruses (Liu Y et al., 2024), the monoclonal antibodies mAb-2F3 obtained in this study specifically recognized intracellular goatpox virus and bovine nodular dermatosis virus in immunofluorescence assays (IFA), showing broader application potential. The affinity constant (Kaff) of mAb-2F3, measured using a non-competitive ELISA method, is approximately 2.5 × 10⁻⁶. 8 The L / mol ratio indicates that it has high affinity.

[0047] In summary, this invention, through optimized immunization and screening strategies, successfully prepared a monoclonal antibody with higher titer, stronger affinity, and the ability to simultaneously and efficiently recognize recombinant proteins and natural viruses. This monoclonal antibody, mAb-2F3, provides a high-performance core reagent for establishing highly sensitive and specific goatpox virus detection methods (such as competitive ELISA for vaccine efficacy evaluation), solving the problems of low antibody titer and limited ability to recognize natural viruses in existing technologies.

[0048] Example 3: Establishment of an Indirect ELISA Method 1. Determination of the optimal antigen coating concentration and the optimal concentration of negative and positive sera for use. Following the matrix checkerboard method, the coated antigen was diluted to 2, 4, 8, 16, 32, 64, 128, 256, and 512 μg / ml; positive serum was diluted to 1:100, 1:200, 1:400, and 1:800; and negative serum was diluted to 1:50, 1:100, and 1:200. OD values ​​were measured at 450 nm to determine the optimal antigen coating concentration and the optimal usage concentrations for both positive and negative sera. The results are shown in Table 1. When the antigen concentration was 16 μg / mL, the OD values ​​of positive serum at each dilution were... 450nm The value reaches a plateau; when the positive serum dilution is 1:400, OD 450nm The value is close to 1.0; when the negative serum dilution is 1:200, the P / N value is relatively large, and the OD value is also high. 450nm Value compared to blank control OD 450nm The values ​​were high, therefore, the optimal antigen coating concentration was determined to be 16 μg / mL, the optimal dilution for positive serum was 1:400, and the optimal dilution for negative serum was 1:200.

[0049] Table 1. Results of ELISA guideline titration tests for coated antigen and negative / positive serum (OD) 450nm )

[0050] 2. Antigen coating conditions The antigen was diluted to the optimal coating concentration using 0.1 mol / L pH 9.6 carbonate buffer and 0.01 mol / L pH 7.2 phosphate buffer, respectively, and coated onto the reaction plate. The plates were incubated at 4℃ for 16 hours, 20 hours, and 37℃ for 1 hour, 3 hours, respectively. OD values ​​were measured at 450 nm, and the P / N ratio was calculated to determine the optimal antigen coating conditions. The results are shown in Tables 2 and 3. The results showed that after coating at 4℃ for 16 hours, the P / N ratio for carbonate buffer (pH 9.6) was 12.35, and the P / N ratio for phosphate buffer (pH 7.2) was 10.98. Therefore, carbonate buffer (0.1 mol / L, pH 9.6) was selected as the coating solution, and the coating time was determined to be 4℃ for 16 hours.

[0051] Table 2. Coating time and OD under conditions for phosphate buffer (pH 7.2) 450nm Value measurement results

[0052] Table 3. Coating time and OD under conditions for carbonate buffer (pH 9.6) 450nm Value measurement results

[0053] 3. Determination of sealing solution and sealing time The antigen was diluted to the optimal coating concentration using coating buffer and coated onto the reaction plate. Blocking was performed using three blocking solutions: 1.0% gelatin, 1.0% BSA, and 5.0% skim milk powder, 200 μL / well. Incubation was performed at 37°C for 1, 1.5, and 2 hours, respectively. OD values ​​were measured at 450 nm, and the P / N ratio was calculated to determine the optimal blocking solution and blocking time. The results are shown in Tables 4 and 5. The results showed that the blocking effect of the three blocking solutions was, in descending order: 5% skim milk powder > 1.0% BSA > 1.0% gelatin. The highest P / N ratio (11.26) was observed at a blocking time of 1.5 hours. Therefore, 5% skim milk powder was selected as the blocking solution, and a blocking time of 1.5 hours was considered the optimal blocking condition.

[0054] Table 4 OD at different closure times 450nm Value measurement results

[0055] Table 5 OD of different sealing solutions 450nm Value measurement results

[0056] 4. Determination of working concentration and reaction time of enzyme-labeled secondary antibody The enzyme-labeled antibody conjugate of rabbit anti-mouse IgG was diluted with phosphate buffer at dilutions of 1:5000, 1:10000, 1:17500, and 1:40000, respectively. The reaction conditions were 37°C for 0.5, 1, and 2 hours, respectively. OD values ​​were measured at 450 nm, and the P / N ratio was calculated to determine the optimal dilution and incubation time for the enzyme-labeled secondary antibody. The results are shown in Tables 6 and 7. The results showed that the highest P / N ratio (11.32) was observed at a dilution of 1:17500. The highest P / N ratio (10.58) was observed at an incubation time of 1 hour. Therefore, the optimal dilution for the enzyme-labeled secondary antibody was determined to be 1:17500, and the optimal incubation time was determined to be 1 hour.

[0057] Table 6. OD at different working concentrations of enzyme-labeled secondary antibodies 450nm Value measurement results

[0058] Table 7 OD at different working times of enzyme-labeled secondary antibodies 450nm Value measurement results

[0059] 5. Determination of substrate color development time The substrate solution was incubated at room temperature for 5, 10, 15, and 20 minutes, respectively. The reaction was terminated with 2M sulfuric acid. The OD value was measured at 450 nm, and the P / N ratio was calculated to determine the substrate incubation time. The results are shown in Table 8. The results indicate that the P / N ratio was highest (10.02) when the substrate solution was incubated at room temperature for 15 minutes. Therefore, the incubation time was determined to be 15 minutes at room temperature.

[0060] Table 8 OD at different color development times 450nm Value measurement results

[0061] 6. Determination of negative and positive serum criteria Positive serum was diluted 1:400, and negative serum was diluted 1:200. Following an indirect ELISA assay, 28 tests were performed on both positive and negative sera. The OD values ​​of the 28 positive and negative sera were then analyzed. 450nm Statistical analysis of OD values ​​was performed to determine the OD values ​​of positive and negative serum. 450nm scope.

[0062] The results of determining the positive serum criteria are shown in Table 9. The results indicate that, for the same positive serum sample, 28 tests were conducted, with an average OD value (X) of 1.261 and a standard deviation (S) of 0.218. Among the 28 tests, the probability of OD values ​​falling within the range of X±s (1.043–1.479) was 64.29%; the probability of falling within the range of X±2s (0.825–1.697) was 96.43%; and the probability of falling within the range of X±3s (0.607–1.915) was 100%, conforming to a normal distribution and demonstrating good stability. Therefore, the OD values ​​of positive serum... 450nm The absorbance value is set at 0.8~2.0.

[0063] Table 9. Results of OD value determination in 28 positive serum samples.

[0064] The results of determining the negative serum standard are shown in Table 10. The results indicate that, for the same negative serum sample, 28 tests were conducted, with an average OD value (X) of 0.190 and a standard deviation (S) of 0.039. Among the 28 tests, the probability of OD values ​​falling within the range of X±s (0.151~0.229) was 71.43%; the probability falling within the range of X±2s (0.112~0.268) was 92.86%; and the probability falling within the range of X±3s (0.073~0.307) was 100%, conforming to a normal distribution and demonstrating good stability. Therefore, the OD value of negative serum... 450nm The absorption value is set to be less than 0.3.

[0065] Table 10 Results of 28 negative serum OD value measurements

[0066] Example 4: Establishment of a Competitive ELISA Method Building upon the established indirect ELISA method, a competitive ELISA method was subsequently developed.

[0067] 1. Vaccine antigen extraction Before conducting efficacy testing, shake the vaccine to be tested well and freeze it at -70°C for at least 24 hours; take out the frozen vaccine to be tested and the reference vaccine, incubate them at 37°C for 20 minutes, and then add them to centrifuge tubes, 9 ml per tube. Add n-butanol at a ratio of vaccine:n-butanol = 9:1 (V / V), mix thoroughly, centrifuge at 5000g for 10 minutes, and collect the lower layer liquid as the antigen of the vaccine to be tested and the reference vaccine.

[0068] 2. Preparation of antigen-coated plates Dilute the coating antigen to 16 μg / mL with carbonate buffer (pH 9.6) and coat 100 μl per well of a 96-well reaction plate. Incubate overnight at 4°C. Remove the reaction plate, allow it to return to room temperature, wash three times with PBST, add 200 μl of blocking buffer per well, incubate at 37°C for 1.5 hours, and wash three times with PBST.

[0069] 3. Competitive ELISA test procedure (1) In the dilution plate, add 200 μl of the treated reference vaccine antigen to each well of A3, A4, and A5, and add 200 μl of the treated test vaccine antigen to each well of A6, A7, A8, A9, A10, and A11. Add 100 μl of PBS to the remaining wells (B3-B11 to H3-H11). Serially dilute the reference vaccine antigen and the test vaccine antigen (vertically) twofold, and then transfer 50 μl from each well to the corresponding well of a 96-well ELISA plate coated with sheep pox virus P32 truncated protein antigen. (2) Add 100 μl of negative serum diluted 1:200 to each well in column 1 as a negative control; add 100 μl of positive serum diluted 1:400 to each well in column 2 as a positive control; add 100 μl of PBS to each well in column 12 as a blank control; add 50 μl of monoclonal antibody against goatpox virus P32 truncated protein diluted with PBS to each well in wells A3-A11 and H3-H11, where the concentration of the diluted monoclonal antibody is 1 μg / ml, and cover the reaction plate with a biofilm; (3) The reaction plate was placed on a shaker (100 rpm) at 37°C for 1.5 hours; the reaction plate was washed 3 times with PBST. (4) Add 100 μl of peroxidase-labeled rabbit anti-mouse IgG diluted 1:17500 to each well, place the reaction plate on a shaker (100 rpm) at 37°C for 1 hour; wash the reaction plate 3 times with PBST; (4) Add 100 μl of TMB substrate solution to each well and incubate at room temperature for 15 minutes; add 100 μl of stop solution to each well; read the absorbance of each well at 450 nm using an automatic plate reader (OD). 450nm ) (5) Calculation results of OD of each hole 450nm All readings should be subtracted from the blank control OD. 450nm Readings are taken; blank readings are the average of well readings from A12 to H12. The OD value of the sample to be tested is calculated using RelPot 4.0 software or bioassay statistics to determine the relative potency unit (RP value) of the antigen contained in the vaccine.

[0070] 4. Establishment of the standard curve Goat pox standard antigen (P32 truncated protein) was diluted with 0.01 mol / L pH 7.2 phosphate buffer to a series of standard concentrations: 256, 128, 64, 32, 16, 8, 4, 2, and 0 μg / mL. 50 μl of each concentration was added to pre-coated ELISA plates, with three replicates per well. 50 μl of 1 μg / mL monoclonal antibody against goat pox virus P32 truncated protein was added to each well. Positive, negative, and blank controls were also included. The plates were incubated at 37°C for 1.5 hours on a shaker (100 rpm). The plates were washed three times with PBST and blotted dry. 100 μl of 1:17500 diluted peroxidase-labeled rabbit anti-mouse IgG was added to each well. The plates were incubated at 37°C for 1.0 hour on a shaker (100 rpm). The plates were washed three times with PBST and blotted dry. 100 μl of peroxidase-labeled rabbit anti-mouse IgG was added to each well. TMB substrate was incubated at room temperature for 15 minutes; finally, 100 μl of stop solution was added to each well, and the OD value was measured at 450 nm. The OD value was then measured using a competitive antigen concentration of 0. 450nm The value is B0, representing the OD values ​​of the corresponding antigen concentrations. 450nmThe value is B. A standard curve was plotted with the logarithm of the competing antigen concentration on the x-axis and the binding rate (B / B0) on the y-axis. The results of the competitive ELISA standard curve plotting are shown in Table 11. Figure 7 Table 11 and Figure 7 This indicates that the antigen-antibody competitive reaction established by using different concentrations of competitive antigen exhibits a good linear relationship (R0). 2 =0.9867), the linear regression equation is Y=-33.449X+96.394, the detection range is 2μg / mL~512μg / mL, and the limit of detection is 2μg / mL.

[0071] Table 11 Results of the competitive antigen standard curve assay

[0072] 5. Repeatability test a. Five vials of vaccine from batch 201901 were randomly selected for intra-batch repeatability testing. The relative efficacy of the vaccine was tested according to the competitive ELISA test procedure, and the intra-batch coefficient of variation was calculated.

[0073] b. For the inter-batch repeatability test, two vials of vaccine from batches 201901, 201902 and 201903 were randomly selected, and the relative antigenic potency of the vaccine was determined according to the competitive ELISA method, and the inter-batch coefficient of variation was calculated.

[0074] The results of intra-batch and inter-batch repeatability tests are shown in Tables 12 and 13. The results show that, using the competitive ELISA method, the intra-batch coefficient of variation (RP) for detecting the relative potency (RP) of the 201901 batch of test vaccines five times was 3.65%; and the inter-batch coefficient of variation (RP) for detecting the relative potency (RP) of the three batches of test vaccines was 6.30%, indicating that the method has good intra-batch and inter-batch repeatability.

[0075] Table 12 Results of Intra-Batch Repeatability Testing

[0076] Table 13 Results of inter-batch repeatability testing

[0077] 6. Specificity test The RP values ​​of inactivated antigens from caprine contagious pleuropneumonia (CPP) vaccine, foot-and-mouth disease (FMD) type O, and Asia I bivalent inactivated vaccines were determined according to the competitive ELISA procedure to verify the specificity of the established competitive ELISA method. The results are shown in Table 14. The results indicate that the competitive ELISA method established in this invention did not detect RP values ​​for caprine contagious pleuropneumonia (CPP) inactivated antigen, FMD type O, and Asia I inactivated antigen. The RP value for caprine pox virus inactivated antigen was 1.35, indicating that the established competitive ELISA method has good specificity.

[0078] Table 14 Specificity Results of Competitive ELISA Methods Note: "-" indicates that no value was detected.

[0079] 7. Correlation test between relative vaccine potency (RP) and vaccine antigen content Following a competitive ELISA procedure, vaccines with different antigen contents, batch numbers K001, K002, and K003, had a pre-inactivation viral load of 10... 5.0 TCID 50 / mL, 10 4.0 TCID 50 / mL, 10 3.0 TCID 50 The relative potency of the antigen was determined by measuring / mL to examine the correlation between the vaccine antigen content and the relative potency of the vaccine antigen. The results are shown in Table 15. The results show that the RP value of batch K001 was 0.87; the RP value of batch K002 was 0.79; and the RP value of batch K003 was 0.61, indicating that the relative potency (RP) value of the vaccine is correlated with the antigen content.

[0080] Table 15 Relative Potential (RP) Values ​​of Vaccines with Different Antigen Contents

[0081] 8. Correlation test between relative vaccine potency (RP) value and immune challenge protection Three batches of laboratory-produced vaccines and three batches of vaccines with different antigen contents were tested using a competitive ELISA assay to determine the relative antigenic potency of the vaccines. Simultaneously, a challenge protection test was conducted, and the correlation between the relative antigenic potency (RP) values ​​detected by the competitive ELISA and immune challenge protection was compared. The results are shown in Table 16. Table 16 indicates that the relative antigenic potency (RP) values ​​of the three batches of tested vaccines and the three batches of vaccines with different antigen contents showed a good parallel relationship with immune challenge protection, and can replace the challenge test.

[0082] Table 16. Relative potency (RP) values ​​and immune challenge protection results of six batches of vaccines.

[0083] 9. Criteria for determining the relative efficacy of vaccines: Based on the above test results, an RP value of 0.87 provides more than 4 / 5 immune protection, meeting the requirements of vaccine quality standards. Since the reference vaccine has been proven to provide 5 / 5 immune protection through immune challenge protection tests (i.e., an RP value ≥ 1.0 indicates that the vaccine provides 5 / 5 complete immune protection), to ensure product quality, it is stipulated that the RP value of the vaccine under test should not be lower than 1.0 for the vaccine to be considered qualified; otherwise, the vaccine is considered unqualified.

[0084] Example 5: Competitive ELISA method to test the efficacy of inactivated vaccine for bovine nodular dermatitis The efficacy of a batch of bovine nodular dermatitis inactivated vaccine (batch number: 202101) was tested using the competitive ELISA method of this invention. The specific operating steps are as follows: (1) Antigen treatment: Before efficacy testing, shake the vaccine to be tested well and freeze it at -70°C for at least 24 hours; take out different batches of the vaccine to be tested and the reference vaccine frozen, incubate them at 37°C for 20 minutes, and then add them to centrifuge tubes, 9 ml per tube. Add n-butanol at a ratio of vaccine:n-butanol = 9:1 (V / V), mix thoroughly, centrifuge at 5000g for 10 minutes, and take the lower layer liquid as the antigen of the vaccine to be tested and the reference vaccine.

[0085] (2) Coating: Dilute the goatpox virus P32 protein coating antigen to working concentration with carbonate buffer at pH 9.6, add 100 μl to each well of a 96-well ELISA plate, and coat overnight at 4°C; remove the plate, allow it to return to room temperature, wash three times with PBST, and pat dry. (3) Blocking: Add 5% skim milk to block, 200 μl per well, block at 37℃ for 2 h; remove, discard the blocking solution, wash 3 times with PBST, and pat dry.

[0086] (4) Competitive ELISA test a. In the dilution plate, add 200 μl of the treated reference vaccine antigen to each well (A3, A4, A5), and 200 μl of the treated test vaccine antigen to each well (A6, A7, A8). Add 100 μl of PBS to the remaining wells (B3-B8 to H3-H8). Serially dilute the reference vaccine antigen and the test vaccine antigen (vertically) twofold, then transfer 50 μl from each well to the corresponding well of a 96-well ELISA plate coated with goatpox virus P32 protein antigen. b. Add 100 μl of appropriately diluted negative serum (1:200 dilution) to each well in column 1 as a negative control; add 100 μl of appropriately diluted positive serum (1:400 dilution) to each well in column 2 as a positive control; add 100 μl of PBS to each well in column 9 as a blank control; for wells A3–A8 and H3–H8, add 50 μl of monoclonal antibody against goatpox virus P32 truncated protein diluted to a working concentration (1 μg / ml) with PBS to each well. Cover the reaction plate with a biofilm; place the reaction plate on a shaker and incubate at 100 rpm and 37°C for 1.5 h; the sample addition diagram for each well is shown in Table 17.

[0087] Table 17 Schematic diagram of sample loading for 96-well plate

[0088] c. Discard the liquid in the plate and wash it 3 times with PBST; Add 100 μl of HRP-labeled rabbit anti-mouse IgG diluted to the working concentration with enzyme conjugate diluent to each well. Place the reaction plate in an incubator at 37 °C for 30 min; Discard the liquid in the plate and wash it 3 times with PBST; d. Add 100 μl of TMB substrate solution to each well and develop the color at 37 °C for 15 min; Add 50 μl of stop solution to each well; The absorbance value of each well is read by an automatic microplate reader at 450 nm (OD 450nm ).

[0089] (5)Calculation of results a. Result calculation method The OD 450nm reading of each well should be subtracted from the OD 450nm reading of the blank control. The blank reading is the average value of the readings of wells A12 - H12. The OD 450nm value detected for the test sample is used to calculate the relative potency unit (RP value) of the antigen contained in the vaccine by RelPot 4.0 software or biological assay statistical method. When calculating the RP value, the arithmetic mean of 3 replicates is taken.

[0090] b. Standard for the test to be valid The absorbance value of the positive control should be within the range of 0.8 - 2.0; The absorbance value of the negative control should be less than 0.3.

[0091] c. Standard for judging the potency of the vaccine Set the RP value of the reference vaccine as 1.0. If the RP value of the test vaccine is not less than 1.0, the vaccine is judged to be qualified; If the RP value of the test vaccine is less than 1.0, it should be retested once. If the RP value of the retest result is not less than 1.0, the vaccine is judged to be qualified, otherwise it is judged to be unqualified.

[0092] (6)Test results The OD 450nm value reading results of each well in the competitive ELISA test are shown in Table 18. The absorbance values of the positive controls are all within the range of 0.8 - 2.0; The absorbance values of the negative controls are all less than 0.3, and the test is valid. The relative potency unit (RP value) of the antigen contained in the vaccine is calculated by RelPot 4.0 software. The RP values of the 3 replicates are 1.57, 1.48 and 1.65 respectively, and the arithmetic mean is 1.57 (Table 19). Therefore, the RP value of the test vaccine of batch 202101 is 1.57 and it is judged to be qualified.

[0093] Table 18 OD 450nm value reading results of each well in the competitive ELISA test

[0094] Table 19 Relative potency RP value of the test vaccine calculated by RelPot 4.0 software

[0095] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A truncated P32 protein of goatpox virus, characterized in that, The amino acid sequence of the truncated P32 protein is shown in SEQ ID NO:3, and the protein is expressed in a soluble form in the Escherichia coli expression system, with a soluble expression ratio of 40%.

2. A monoclonal antibody against goatpox virus P32 truncated protein prepared using the goatpox virus P32 truncated protein according to claim 1, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively.

3. A competitive ELISA method for detecting the efficacy of an inactivated vaccine against bovine nodular dermatitis, characterized in that, The method includes the following steps: a) Coating the P32 truncated protein of claim 1 into a 96-well enzyme-linked reaction plate; b) Shake the vaccine to be tested and the reference vaccine well and freeze them at -70°C for at least 24 hours. After taking them out and demulsifying them according to the demulsification method, extract the lower layer of liquid as the antigen of the vaccine to be tested and the antigen of the reference vaccine. c) After serially diluting the reference vaccine antigen and the vaccine antigen to be tested by 2 times, transfer them to the corresponding wells of the 96-well ELISA plate prepared in step a); add 50 μl of monoclonal antibody against goatpox virus P32 truncated protein as described in claim 2, diluted with PBS, to each well, wherein the concentration of the diluted monoclonal antibody is 1 μg / ml. d) Prepare negative and positive sera diluted at appropriate ratios as negative and positive controls, respectively; PBS should also be prepared as a blank control. e) Place the reaction plate on a shaker at 100 r / min and 37°C for 1.5 h; discard the liquid in the plate and wash it 3 times with PBST; f) Add 100 μL of HRP-labeled rabbit anti-mouse IgG diluted 1:17500 to each well. Place the reaction plate on a shaker and react at 37°C for 1 h at 100 rpm. Discard the liquid in the plate and wash three times with PBST. g) Add 100 μL of TMB substrate solution to each well, develop color at room temperature for 15 min, add 100 μL of stop solution to each well, and then read the OD450 nm absorbance value of each well. h) Result calculation: The OD450nm reading of each well should be subtracted from the OD450nm reading of the blank control; the RP value of the antigen contained in the vaccine is calculated using biostatistical software; if the RP value of the vaccine to be tested is not less than 1.0 compared with the reference vaccine, the vaccine efficacy test is deemed qualified; otherwise, the vaccine efficacy test is deemed unqualified; the RP determination results are consistent with the immune protection results obtained in animal challenge experiments.

4. The method according to claim 3, characterized in that, In step a), the coating concentration of the P32 truncated protein is 16 μg / mL, and the coating conditions are coating at 4°C for 16 hours in carbonate buffer at pH 9.6; step a) is followed by a step of blocking with 5% skim milk powder at 37°C for 1.5 hours.

5. The method according to claim 3, characterized in that, In step b), the reference vaccine is an inactivated bovine nodular dermatitis vaccine whose immunogenicity has been confirmed through this animal challenge experiment.

6. The method according to claim 3, characterized in that, In step b), the demulsification method is as follows: take out the frozen vaccine to be tested and the reference vaccine, place them in a 37°C incubator for 20 minutes, then add them to a centrifuge tube, add n-butanol at a volume ratio of vaccine to n-butanol = 9:1, mix thoroughly, and centrifuge at 5000g for 10 minutes.

7. The method according to claim 3, characterized in that, In step d), the positive control is a mouse-derived positive serum diluted 1:400, and the negative control is a mouse-derived negative serum diluted 1:

200.

8. A reagent kit for detecting the potency of an inactivated vaccine against bovine nodular dermatitis, characterized in that, The kit contains an effective amount of the P32 truncated protein of claim 1, an effective amount of the monoclonal antibody against the P32 truncated protein of goatpox virus of claim 2, as well as a positive control, a negative control, HRP-labeled rabbit anti-mouse IgG, TMB chromogenic substrate, and 2MH2SO4 stop solution.

9. Use of a monoclonal antibody against goatpox virus P32 truncated protein as described in claim 1 and / or against goatpox virus P32 truncated protein as described in claim 2 in the preparation of a kit for detecting the efficacy of an inactivated vaccine against bovine nodular dermatitis.

10. Use of the method described in any one of claims 2 to 7 or the kit described in claim 8 in an animal challenge efficacy test as an alternative to bovine nodular dermatitis vaccine.

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