A type of swine fever virus E rns Porcine monoclonal antibodies against proteins and their applications

Abstract

This invention belongs to the field of biotechnology, specifically relating to a classical swine fever virus E. rns A porcine monoclonal antibody against a protein. The amino acid sequence of the heavy chain variable region of the porcine monoclonal antibody is shown in SEQ ID NO:1, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:2, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:4. The full-length genes of the heavy and light chains of this porcine monoclonal antibody are derived from the same classical swine fever virus E. rns Obtained through protein-specific amplification in porcine B cells, this antibody retains its native structure, avoiding the affinity loss that may occur with artificial recombination. It exhibits high affinity and specificity, making it a suitable candidate for classical swine fever virus E1. rns This provides a precise molecular tool for elucidating the antigenic structure of the protein and establishing diagnostic methods for classical swine fever. The monoclonal antibody was then applied to the classical swine fever virus E... rns In the antibody blocking ELISA detection method, the established E rns Antibody blocking ELISA detection method has high sensitivity and specificity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a classical swine fever virus E. rns Porcine monoclonal antibodies against proteins and their applications. Background Technology

[0002] Classical swine fever (CSF) is a highly contagious disease caused by classical swine fever virus (CSFV) in pigs, characterized by fever, hemorrhage, and high mortality. CSFV virus particles are spherical, 40-50 nm in diameter, enveloped, and contain a single-stranded positive-sense RNA genome. Its genome encodes four structural proteins (E...). rns (E1, E2, and C) and 8 non-structural proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Among them, E rns E2 protein is the main antigenic protein, which can stimulate the body to produce protective neutralizing antibodies.

[0003] Since its first report in Ohio, USA in 1833, swine fever (CSF) has spread and become prevalent worldwide for nearly 200 years, causing enormous economic losses to the global pig industry. It is listed as a reportable animal disease by the World Organization for Animal Health (WOAH). Although some countries, such as the United States, Australia, Canada, New Zealand, France, the United Kingdom, and Germany, have successfully eradicated CSFV, it remains prevalent in most countries in Asia, Eastern Europe, South America, and Central America.

[0004] my country employs a comprehensive prevention and control model for classical swine fever (CSF) primarily based on vaccination. In the 1950s, my country's independently developed attenuated live CSF vaccine, the HCLV strain (also known as the C strain), played a crucial role in CSF control. Immunization with the C strain provides excellent immune protection against different CSFV genotypes and is highly safe and effective. Many countries, including my country, have effectively controlled CSF outbreaks with the help of the C strain vaccine. Currently, the prevalence of CSF in my country is relatively stable, with occasional sporadic cases. However, because the overall structure of the attenuated live vaccine is not significantly different from that of naturally occurring strains, it is difficult to effectively distinguish between vaccine-immunized and wild-type infected animals through serological diagnosis, hindering the eradication of CSF. Therefore, some farms have begun immunizing with the CSFV E2 protein subunit vaccine. Vaccination stimulates the body to produce good immune protection and induces antibodies only against the E2 protein, not against the E2 protein. rns Antibodies to the protein can be detected by E.rns Antibody methods are used to differentiate between animals vaccinated against classical swine fever virus and those infected with wild-type virus, but these methods have not yet been approved for detection in China. rns Commercially available antibody kits.

[0005] Monoclonal antibodies (mAbs) are immunoglobulins (Ig) produced and secreted by a single specific B cell after stimulation of the body by a specific antigen. They are specific to that antigen only. Compared to polyclonal antibodies, monoclonal antibodies have higher specificity and homogeneity, offering significant advantages in disease diagnosis and treatment. Although E rns While the protein is relatively conserved in CSFV, the variable region exposed outside the viral envelope varies significantly across different CSFV subtypes. Traditional hybridoma techniques are inefficient and time-consuming, making it difficult to screen for specific monoclonal antibodies with good application value. In recent years, single B-cell antibody technology has emerged as a new direction to overcome this bottleneck due to its high throughput and preservation of natural antibodies. Furthermore, different animal immune systems exhibit varying abilities to recognize the same antigen, potentially leading to differences in the binding capacity of the produced antibodies. Using monoclonal antibodies derived from natural hosts can more accurately elucidate the structure and structure-activity relationship of viral antigens, identifying key epitopes and rare epitopes that play crucial roles in viral replication and infection. This not only helps reveal viral antigenic variation and immune evasion mechanisms but also provides a powerful tool for vaccine design and the development of more precise and sensitive diagnostic methods. Pigs, as the natural host of CSFV, produce specific antibodies that reflect the results of their natural immune response.

[0006] Therefore, the preparation of CSFVE rns Whole-porcine monoclonal antibody against E rns Protein antigen structure analysis and CSFVE construction rns Antibody detection methods are of great significance. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a swine fever virus E rns Porcine monoclonal antibodies against proteins.

[0008] Another object of the present invention is to provide the above-mentioned swine fever virus E rns Application of porcine monoclonal antibodies against proteins in the detection of classical swine fever virus.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A type of swine fever virus E rnsThe porcine monoclonal antibody of the protein, wherein the amino acid sequence of the heavy chain variable region of the porcine monoclonal antibody is shown in SEQ ID NO:1, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:2, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:4.

[0011] Furthermore, the constant region of the porcine monoclonal antibody heavy chain is of type IgG1.

[0012] Furthermore, the light chain constant region of the porcine monoclonal antibody is of the Lambda type.

[0013] Furthermore, the variable and constant region gene fragments of the heavy and light chains of the porcine monoclonal antibody are all derived from the same classical swine fever virus E. rns The protein was obtained by PCR amplification in a single porcine B cell with a protein specificity, while retaining its native conformation.

[0014] Furthermore, the aforementioned swine fever virus E rns A method for preparing porcine monoclonal antibodies against proteins includes the following steps:

[0015] S1: Peripheral blood mononuclear cells were isolated from the peripheral blood of pigs immunized with classical swine fever vaccine strain C. Based on the characteristics of B cell surface receptors, classical swine fever virus strain E was sorted using flow cytometry. rns Protein-specific single B cell;

[0016] S2: Using the single porcine B cell isolated in step S1 as a template, the mRNA in the cell is reverse transcribed into cDNA;

[0017] S3: Using the cDNA in S2 as a template, the full-length coding genes of the porcine IgG antibody heavy chain and light chain were amplified using specific primers.

[0018] S4: The full-length coding gene fragments of the porcine IgG antibody heavy chain and light chain obtained in step S3 are ligated into the eukaryotic expression vector pCDNA3.1-Leader by homologous recombination to obtain the recombinant expression vector pCDNA3.1-SH9 containing the complete heavy chain gene of the IgG antibody and the recombinant expression vector pCDNA3.1-SL9 containing the complete light chain gene of the IgG antibody.

[0019] S5: The recombinant expression vector containing the complete heavy and light chain genes of the IgG antibody obtained in step S4 was co-transfected into mammalian expression cells. After transfection, the supernatant was collected and purified to obtain classical swine fever virus E. rns Porcine monoclonal antibodies against proteins.

[0020] This invention also protects the encoding of the classical swine fever virus E. rnsGene fragments of porcine monoclonal antibodies against proteins.

[0021] Furthermore, the gene fragment encoding the variable region of the heavy chain of the porcine monoclonal antibody is shown in SEQ ID NO:5, the gene fragment encoding the constant region of the heavy chain is shown in SEQ ID NO:6; the gene fragment encoding the variable region of the light chain is shown in SEQ ID NO:7, and the gene fragment encoding the constant region of the light chain is shown in SEQ ID NO:8.

[0022] This invention also protects a recombinant expression vector comprising a gene fragment of the porcine monoclonal antibody.

[0023] This invention also protects a host cell, which includes the above-described recombinant expression vector.

[0024] This invention protects the aforementioned swine fever virus E rns Application of porcine monoclonal antibodies against proteins in the preparation of reagents for diagnosing classical swine fever virus.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention utilizes single B-cell antibody technology to screen and prepare classical swine fever virus E for the first time. rns A protein-specific porcine monoclonal antibody was developed. The gene sequence of this monoclonal antibody is entirely derived from immunized pigs. The full-length coding genes for both the heavy and light chains were amplified from the same porcine B cell, preserving the antibody's native conformation and the natural pairing of the heavy and light chains. This avoids conformational changes or affinity loss that may occur with artificial recombination, exhibiting natural affinity and specificity. This provides a precise molecular tool for the antigenic structure analysis and diagnostic methods of classical swine fever virus (CSFV). Simultaneously, the CSFV E1 protein was... rns Porcine monoclonal antibodies against the protein were used to establish classical swine fever virus E12. rns The antibody blocking ELISA detection method has been established and exhibits good sensitivity and specificity. It does not cross-react with sera of common porcine viruses such as ASFV, FDMV, BVDV, PCV, and PRRSV, providing important technical support for the clinical diagnosis and eradication of classical swine fever and showing broad application prospects. Attached Figure Description

[0027] Figure 1 The PCR amplification results of the full-length coding genes of the porcine IgG antibody heavy and light chains are shown, where M is the standard mass of 2000 DNA molecules; 1 is the full-length gene of the porcine IgG antibody heavy chain (approximately 1400 bp); and 2 is the full-length gene of the porcine IgG antibody light chain (approximately 650 bp).

[0028] Figure 2The results of SDS-PAGE verification of eukaryotic expression and purification of porcine monoclonal antibody are shown. M is a 170 kDa Protein Marker; 1 is cell supernatant after transfection; 2 is cell lysate after transfection; 3 is purified and concentrated porcine monoclonal antibody (reduction treatment); 4 is purified and concentrated porcine monoclonal antibody (non-reduction treatment).

[0029] Figure 3 Indirect ELISA results of porcine monoclonal antibody;

[0030] Figure 4 The indirect immunofluorescence results (100X) of porcine monoclonal antibodies are shown, where HCLV, Thiverval, SM, IVDC-HeB-01 and BVDV are the IFA results of porcine monoclonal antibodies and their corresponding strains, respectively; Blank is the negative control.

[0031] Figure 5 The results of the screening for optimal antigen coating concentration are shown.

[0032] Figure 6 This shows the results of screening for the optimal enzyme-labeled monoclonal antibody dilution factor;

[0033] Figure 7 The results of the optimal blocking solution screening are shown.

[0034] Figure 8 The results of the screening for optimal reaction conditions are shown, where A represents the screening for optimal antigen coating conditions; B represents the screening for optimal blocking conditions; C represents the screening for optimal serum incubation conditions; D represents the screening for optimal enzyme-labeled monoclonal antibody incubation conditions; and E represents the screening for optimal color development conditions.

[0035] Figure 9 The ROC curve for determining the optimal critical value is shown.

[0036] Figure 10 Demonstrates the sensitivity of blocking ELISA methods;

[0037] Figure 11 This demonstrates the specificity of the blocking ELISA method. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these embodiments are commercially available.

[0039] Example 1: Classical Swine Fever Virus E rns Preparation and identification of porcine monoclonal antibodies

[0040] 1. Animal immunization

[0041] Two healthy 3-week-old Landrace pigs that were negative for both classical swine fever antigen and antibody were immunized three times, on days 0, 21, and 42. The first immunization was administered via intramuscular injection of classical swine fever C strain vaccine into the neck at a dose of 30 doses per pig. On day 21, a second immunization with classical swine fever C strain vaccine was given using the same dose and route as the first immunization. On day 42, purified CSFVE was administered. rns A booster immunization of protein was administered to the neck muscles at a dose of 100 μg / head; blood was collected from the anterior vena cava on day 52.

[0042] 2. Isolation of porcine peripheral blood mononuclear cells (PBMCs)

[0043] Collect 50 mL of blood from the anterior vena cava and add it to a heparin anticoagulant tube. Invert and mix thoroughly to prevent blood clotting. Separate PBMCs from the porcine anticoagulant blood according to the lymphocyte sorting solution instructions and count them.

[0044] 3. Swine fever virus E rns Isolation of protein-specific single B cells

[0045] Porcine PBMCs were diluted to 2 × 10⁻⁶ using cell staining buffer. 7 Calculate the concentration of cells / mL, add 500 μL to a flow cytometry tube, and add 3 μg of His-tagged CSFVE. rns Protein, 8 μL of Alexa Fluor 647-labeled anti-CD21 antibody, and 2 μL of FITC-labeled anti-pig IgM antibody were incubated at 4°C in the dark for 30 min. Simultaneously, an isotype control (without CSFVE) was prepared using the same cells. rns (protein); centrifuged at 1000 r / min, 4℃ for 5 min, washed twice with cell staining buffer, resuspended in 500 μL cell staining buffer, added 1 μL of PE fluorescently labeled anti-His tag secondary antibody, and incubated at 4℃ in the dark for 20 min; centrifuged at 1000 r / min, 4℃ for 5 min, resuspended in 500 μL cell staining buffer, and sorted CSFV-E using a BD FACSAria II flow cytometer. rns Individual B cells were specifically sorted into PCR 8-cell wells containing 5 μL of sterile, enzyme-free PBS.

[0046] 4. PCR amplification of the full-length porcine IgG antibody gene

[0047] Following the instructions of the SuperScript™ IV Single Cell / Low-Input cDNA PreAmp Kit, the mRNA in the sorted porcine B cells was reverse transcribed into cDNA. Using the cDNA as a template, and referring to the primers in the patent (Zhu Yuanyuan, Huang Yufeng, Che Siqi, et al. A method for preparing a universal whole-porcine monoclonal antibody: ZL202411077473.1[P].2024-10-15.), the full-length genes of the porcine IgG antibody heavy and light chains were amplified.

[0048] The primer sequences are shown in Table 1. The full-length heavy chain gene was amplified using primers Sus-WH-F and Sus-WH-HisR. The PCR reaction mixture consisted of 10 μL Phusion™ Plus Green PCR Master Mix, 1 μL each of the forward and reverse primers, and 1.5 μL of template, for a total volume of 25 μL. The PCR amplification program was: 98℃ for 30 s; 98℃ for 10 s, 60℃ for 10 s, 72℃ for 50 s, 35 cycles; 72℃ for 5 min. The full-length light chain gene was amplified using primers Sus-WL-F and Sus-WL-HisR. The PCR reaction mixture consisted of 10 μL Phusion™ Plus Green PCR Master Mix, 1 μL each of the forward and reverse primers, and 1.5 μL of template, for a total volume of 25 μL. The PCR amplification program was: 98℃ for 30 s; 98℃ for 10 s, 60℃ for 10 s, 72℃ for 20 s, 35 cycles; 72℃ for 5 min. The amplified products were identified by 1.5% agarose gel electrophoresis, and the results were as follows: Figure 1 As shown. Bands matching the target fragment size were recovered, purified, and sequenced for verification.

[0049] Table 1. Primer sequence list for porcine IgG antibody amplification

[0050]

[0051] 5. Construction of expression vectors for porcine IgG antibody heavy and light chains

[0052] The PCR products from step 4, which were verified by sequencing to be the complete full-length genes of the porcine IgG antibody heavy and light chains, were recovered and purified using primers listed in Table 1 (Sus-TY-HF and Sus-TY-HR for heavy chain amplification, and Sus-TY-LF and Sus-TY-LR for light chain amplification). Homologous sequences of the pCDNA3.1-Leader expression vector were added to the 5' end of the upstream primer and the 3' end of the downstream primer. The PCR program was as follows: 98℃ for 30s; 98℃ for 10s, 60℃ for 10s, 72℃ for 50s, 35 cycles; 72℃ for 5min. After identification by 1.5% agarose gel electrophoresis, bands matching the size of the target fragment were recovered and purified. By ligating homologous recombinase into the eukaryotic expression plasmid pCDNA3.1-Leader, recombinant expression plasmids pCDNA3.1-SH9 containing the complete heavy chain gene of the IgG antibody and pCDNA3.1-SL9 containing the complete light chain gene of the IgG antibody were obtained.

[0053] 6. Expression and purification of porcine monoclonal antibodies

[0054] HEK-293T cells were passaged into six-well plates and transfected when the cell density reached 60%-80%. Using a single well in a six-well plate as an example, the antibody heavy / light chain recombinant expression plasmids pCDNA3.1-SH9 and pCDNA3.1-SL9 obtained in step 5 were co-transfected. Preparation of Solution A: In a 1.5 mL centrifuge tube, add 1 μg of heavy chain plasmid, 2 μg of light chain plasmid, and 250 μL of opti-MEM, gently tap to mix, and let stand for 5 min. Preparation of Solution B: In another 1.5 mL EP tube, add 7.5 μL of PEI transfection reagent and 250 μL of opti-MEM, gently tap to mix, and let stand for 5 min. Then, slowly add Solution A to Solution B dropwise, gently tap to mix, let stand for 20 min, and then evenly drop the transfection complex onto the cells. After incubation for 4 h, add 1.5 mL of normal culture medium. Cell supernatant was collected 48-72 hours after transfection. The porcine monoclonal antibody was purified and concentrated using nickel affinity chromatography based on the 6×His-tag purification label on the antibody.

[0055] 7. SDS-PAGE identification of porcine monoclonal antibodies

[0056] Take 40 μL of cell supernatant, cell lysis buffer, and purified monoclonal antibody, add 10 μL of 5× protein loading buffer (containing DTT) to each, vortex to mix, incubate at 100℃ for 10 min, remove and briefly incubate, and identify after cooling; simultaneously, take 30 μL of purified antibody, add 10 μL of 4× non-denaturing protein loading buffer, vortex to mix; add the above protein samples to the corresponding wells according to the protein precast gel instructions, and electrophoresis at 120V for 60 min. Remove the protein gel after electrophoresis, appropriately cut off the gel blocks without samples, place the protein gel in Coomassie Brilliant Blue staining solution, and stain on a shaker for 15-20 min; after staining, transfer the protein gel to destaining solution, incubate overnight at room temperature on a shaker, and take pictures using the ChemiDoc™ XRS+ gel imaging system when the background color of the protein gel becomes lighter and the protein bands are clear.

[0057] The results are as follows Figure 2 As shown, under reducing conditions, the disulfide bonds between the antibody heavy and light chains are reduced and broken, and two bands can be observed. The heavy chain size is about 55 kDa, and the light chain size is about 25 kDa, which is consistent with the expected size. Under non-reducing conditions, the antibody molecule maintains its native conformation, and the two heavy chains and two light chains are linked together by disulfide bonds, with a band size of about 160 kDa, which is consistent with the expected size.

[0058] 8. ELISA validation of porcine monoclonal antibodies

[0059] CSFV E rns The protein was diluted to 1 μg / mL with CBS as the coating antigen, and 100 μL was added to each well of the ELISA plate. The plate was sealed with sealing film and incubated overnight at 4°C. The plate was washed three times with PBST, and 200 μL of 5% skim milk powder was added to each well. The plate was blocked at 37°C for 2 h. After washing three times with PBST, the purified porcine monoclonal antibody was diluted to 10 μg / mL and serially diluted 2-fold, with 100 μL of each dilution added to each well. Swine fever-negative serum was used as a negative control, and the plate was incubated at 37°C for 1 h. After washing three times with PBST, 100 μL of rabbit anti-porcine IgG-HRP antibody (1:5000 dilution) was added to each well, and the plate was incubated at 37°C for 1 h. After washing three times with PBST, 100 μL of TMB chromogenic solution was added to each well, and the plate was incubated at room temperature in the dark for 10 min. The chromogenic reaction was stopped by adding 50 μL of stop solution, and the OD was read using an ELISA reader. 450nm The absorbance value at wavelength will be compared with the cutoff value (OD value of the negative control group). 450nm The average value multiplied by 2.1 is used as the positive cutoff value. A value greater than the cutoff value is considered positive; otherwise, it is considered negative. Results are as follows: Figure 3 As shown, the OD of porcine monoclonal antibodies 450nm The value is significantly higher than the cut-off value, proving that this monoclonal antibody can interact with CSFVE. rns Protein-specific binding.

[0060] 9. Validation of porcine monoclonal antibodies by indirect immunofluorescence assay (IFA)

[0061] PK-15 cells were cultured in 96-well plates. When the cells reached approximately 80% confluence, the culture medium was discarded, and the cells were washed once with PBS. The HCLV, Thiverval, SM, and IVDC-HeB-01 (subtype 2.1) viral solutions were then diluted to 100-200 TCID using MEM medium. 50 At a viral titer of / mL, 100 μL was seeded into each well of PK-15 cells. After adsorption at 37°C for 1 h, the viral solution was discarded, and the cells were washed once with PBS. 100 μL of MEM culture medium containing 1% FBS was added to each well, and the cells were incubated at 37°C for another 72 h. The culture of MDBK cells and the seeding of BVDV were performed in the same manner as above, except that DMEM culture medium was used instead. The culture medium was discarded, and 100 μL of pre-chilled fixative (acetone:methanol = 1:1) was added to each well. The cells were fixed at -20°C for 30 min. The fixative was discarded, and the cells were washed three times with PBS. The purified monoclonal antibody was diluted to 10 μg / mL, and 100 μL was added to each well. The negative control was classical swine fever negative serum. The cells were incubated at 37°C for 1 h. Discard the monoclonal antibody, wash three times with PBS, add 100 μL of FITC-labeled rabbit anti-pig IgG antibody (1:200 diluted in PBS) to each well, incubate at 37°C for 1 h, wash three times with PBS, add 100 μL of PBS to each well, and observe under an inverted fluorescence microscope. The results are as follows. Figure 4 As shown, this porcine monoclonal antibody can react with the 1.1 subtype of classical swine fever HCLV, the Thiverval vaccine strain, and the SM virulent strain, and can also react with the 2.1 subtype of IVDC-HeB-01 circulating strain, but does not cross-react with BVDV, which belongs to the same genus of swine fever virus.

[0062] Example 2: Classical Swine Fever Virus E rns Establishment of antibody-blocking ELISA method

[0063] 1. HRP markers for porcine monoclonal antibodies

[0064] Following the instructions of the HRP protein labeling kit, purified classical swine fever virus E was labeled. rns HRP labeling was performed on porcine monoclonal antibodies derived from protein.

[0065] 2. Preliminary establishment of the blocking ELISA method

[0066] Coating antigen: CSFVE rnsProtein was diluted to 1 μg / mL with antigen coating buffer, and 100 μL was added to each well of an ELISA plate. The plate was incubated overnight at 4°C. The buffer was discarded, and the plate was washed three times with PBST (300 μL / well). Blocking: 200 μL of 5% skim milk powder was added to each well, and the plate was incubated at 37°C for 2 h. The buffer was discarded, and the plate was washed three times with PBST (300 μL / well). Serum incubation: Classical swine fever standard positive serum and SPF swine serum were diluted 1:10, and the serum to be tested was diluted 1:2. 100 μL of the buffer was added to each well of the ELISA plate, and the plate was incubated at 37°C for 1 h. The buffer was discarded, and the plate was washed three times with PBST (300 μL / well). ELISA-labeled monoclonal antibody incubation: HRP-labeled porcine monoclonal antibody was diluted 1:1000 with PBS, and 100 μL was added to each well of the ELISA plate. The plate was incubated at 37°C for 45 min. The buffer was discarded, and the plate was washed three times with PBST (300 μL / well). μL / well; Color development: Add 100 μL of TMB color development solution to each well and incubate at room temperature in the dark for 10 min; Termination and reading: Add 50 μL of stop solution to each well to stop color development, and read the OD using a microplate reader. 450nm Absorbance at wavelength. Percentage inhibition (PI) = (Negative control OD) 450nm Value - Sample OD 450nm Value) / Negative control OD 450nm Value × 100%.

[0067] 3. Determination of the optimal antigen coating concentration

[0068] CSFV E rns Proteins were diluted with antigen coating buffer at concentrations of 0.125, 0.25, 0.5, 1, 2, and 4 μg / mL. Blocking ELISA assays were performed following the steps described above, with each condition repeated in triplicate. The blocking rate was calculated, and the concentration with the highest blocking rate was considered the optimal antigen coating concentration for this method. Results are as follows: Figure 5 As shown, the blocking rate was highest at an antigen coating concentration of 1 μg / mL, which was 77.5%. Therefore, 1 μg / mL was selected as the optimal antigen coating concentration.

[0069] 4. Determination of the optimal dilution of enzyme-labeled monoclonal antibody

[0070] Based on the above optimized conditions, the blocking rate of the enzyme-labeled monoclonal antibody was measured at dilutions of 1:1000, 1:2000, 1:4000, 1:8000, and 1:16000. Each condition was repeated in triplicate, and the dilution with the highest blocking rate was taken as the optimal enzyme-labeled monoclonal antibody dilution for this method. The results are as follows: Figure 6 As shown, the highest blocking rate of 80.1% was achieved when the dilution ratio was 1:2000. Therefore, 1:2000 was selected as the optimal dilution of the enzyme-labeled monoclonal antibody.

[0071] 5. Determination of the optimal sealing solution

[0072] Based on the above optimized conditions, the blocking effects of four blocking solutions—5% skim milk powder, 5% BSA, 5% FBS, and 5% horse serum—were determined. Each condition was repeated in triplicate, and the blocking solution with the highest blocking rate was selected as the optimal blocking solution for this method. The results are as follows: Figure 7 As shown, the blocking rate is the highest at 93.7% when the blocking solution is 5% BSA. Therefore, 5% BSA is selected as the optimal blocking solution.

[0073] 6. Determination of optimal antigen coating conditions

[0074] Based on the above optimized conditions, the effect of coating conditions of CSFV Erns protein at 37℃ for 30 min, 37℃ for 1 h, 37℃ for 2 h, and 4℃ for 14 h on the blocking effect was determined. Each condition was repeated in triplicate, and the coating condition with the highest blocking rate was taken as the optimal antigen coating condition for this method. The results are as follows: Figure 8 As shown in Figure A, the blocking rate is the highest at 91.7% when the antigen coating condition is 4℃ for 14 h. Therefore, 4℃ for 14 h is selected as the optimal antigen coating condition.

[0075] 7. Determination of optimal closure conditions

[0076] Based on the above optimized conditions, the effect of the optimal blocking solution on the blocking effect was determined at 37℃ for 30 min, 37℃ for 1 h, and 37℃ for 2 h. Each condition was repeated in triplicate, and the blocking condition with the highest blocking rate was taken as the optimal blocking condition for this method. The results are as follows: Figure 8 As shown in Figure B, the highest blocking rate of 93.8% was achieved when the sealing condition was 37℃ for 2 hours. Therefore, 37℃ for 2 hours was selected as the optimal sealing condition.

[0077] 8. Determination of optimal incubation conditions for serum samples

[0078] Based on the above optimized conditions, the effect of serum sample incubation at 37℃ for 30 min, 37℃ for 45 min, and 37℃ for 1 h on the blocking effect was determined. Each condition was repeated in triplicate, and the serum incubation condition with the highest blocking rate was taken as the optimal serum sample incubation condition for this method. The results are as follows: Figure 8 As shown in Figure C, the highest blocking rate (92.1%) was achieved when the serum sample was incubated at 37°C for 1 h. Therefore, 37°C for 1 h was selected as the optimal incubation condition for serum samples.

[0079] 9. Determination of optimal incubation conditions for enzyme-labeled monoclonal antibodies

[0080] Based on the above optimized conditions, the blocking rate of enzyme-labeled monoclonal antibodies was measured at 37℃ for 30 min, 37℃ for 45 min, and 37℃ for 1 h. Each condition was repeated in triplicate, and the incubation conditions with the highest blocking rate were taken as the optimal incubation conditions for this method. The results are as follows: Figure 8 As shown in D, the highest blocking rate (91.6%) was achieved when the enzyme-labeled monoclonal antibody was incubated at 37℃ for 1 h. Therefore, 37℃ for 45 min was selected as the optimal incubation condition for the enzyme-labeled monoclonal antibody.

[0081] 10. Determining the optimal color development conditions

[0082] Based on the above optimized conditions, the effect of incubation of TMB chromogenic solution at 37℃ and room temperature (25℃) for 5 min, 10 min, and 15 min on the blocking effect was determined. Each condition was repeated in triplicate, and the chromogenic condition with the highest blocking rate was taken as the optimal chromogenic condition for this method. The results are as follows: Figure 8 As shown in E, the blocking rate is the highest at 92.2% when the color development condition is room temperature (25℃) for 5 min. Therefore, room temperature (25℃) for 5 min is selected as the optimal color development condition.

[0083] 11. Determination of the optimal critical value

[0084] Forty E samples were tested using the optimized blocking ELISA method. rns Antibody-positive serum samples and 60 E rns The blocking rate of antibody-negative serum samples was analyzed using SPSS 26.0 statistical analysis software. An ROC curve was plotted with sensitivity on the ordinate and (1-specificity) on the abscissa. Each point on the curve represents the sensitivity and specificity corresponding to a critical value. The point closest to the top left corner has the highest sum of sensitivity and specificity; this critical value is called the optimal critical value. The Youden index for each critical value was calculated: Youden index = sensitivity - (1-specificity). The critical value corresponding to the maximum Youden index was taken as the optimal critical value for this method.

[0085] The results are as follows Figure 9 As shown, the area under the ROC curve (AUC) was 0.972, the standard deviation was 0.014, and the asymptotic 95% confidence interval was (0.945, 0.999). The maximum Youden index was 0.875, corresponding to a sensitivity of 0.925 and a specificity of 0.950, with a blocking rate of 48.4%. Therefore, 48.4% was used as the cutoff value for this method; a serum sample blocking rate ≥ 48.4% was considered an E curve. rns A positive antibody result is considered E when the serum sample blocking rate is <48.4%. rnsAntibody negative.

[0086] 12. Sensitivity testing

[0087] Classical swine fever standard positive serum was diluted at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256, and detected using the established blocking ELISA method. Each sample was performed in triplicate, and the highest dilution yielding a positive result was defined as the limit of detection (LOD) of this method. Results are as follows: Figure 10 As shown, when the standard positive serum for classical swine fever is diluted to 1:128, the detection result of this method is still positive. Therefore, the lower limit of detection of this method is 1:128, which has good sensitivity.

[0088] 13. Specific detection

[0089] The established blocking ELISA method was used to detect standard positive sera for CSFV, ASFV (African swine fever virus), PRRSV (porcine reproductive and respiratory syndrome virus), FMDV (foot-and-mouth disease virus), PCV (porcine circovirus), BVDV (bovine viral diarrhea virus), and PPV (porcine parvovirus). Each sample was tested in triplicate to evaluate the specificity of the method. Results are as follows: Figure 11 As shown, except for CSFV standard positive serum test results which were positive, the standard positive serum test results for other porcine viruses and BVDV were all far below the critical value, indicating that this method has good specificity and does not cross-react with other viral sera.

[0090] 14. Repeatability testing

[0091] Three positive serum samples with different antibody levels and one negative serum sample were selected and subjected to intra-batch repeatability testing on ELISA plates coated with the same batch of antigen. Five replicates were set up for each sample, and the coefficient of variation (CV) was calculated. Eight serum samples were randomly selected and subjected to inter-batch repeatability testing on ELISA plates coated with antigen from three different batches, and the CV was calculated. The formula for calculating the CV is: CV = standard deviation / mean × 100%. A CV less than 10% indicates good repeatability.

[0092] The results are shown in Tables 2 and 3. The intra-batch coefficient of variation was 1.043% to 4.025%, and the inter-batch coefficient of variation was 1.617% to 7.869%, both less than 10%, indicating that the detection method has good repeatability.

[0093] Table 2. Results of intra-batch repeatability tests

[0094]

[0095] Table 3. Results of inter-batch repeatability testing

[0096]

[0097] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A porcine pestivirus E rns protein, characterized in that, The amino acid sequence of the heavy chain variable region of the porcine monoclonal antibody is shown as SEQ ID NO: 1, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO: 2; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 3, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO:

4.

2. The porcine pestivirus E protein of claim 1 rns porcine monoclonal antibody to the porcine pestivirus E protein, characterized in that, The heavy chain constant region of the porcine monoclonal antibody is of IgG1 type.

3. The porcine pestivirus E protein of claim 1 rns porcine monoclonal antibody to the porcine pestivirus E protein, characterized in that, The light chain constant region of the porcine monoclonal antibody is of Lambda type.

4. A gene segment encoding a porcine pestivirus E protein of claim 1. rns monoclonal antibody of porcine origin to the E protein of porcine pestivirus.

5. The genetic fragment of claim 4, wherein, The gene fragment encoding the heavy chain variable region of the porcine monoclonal antibody is shown as SEQ ID NO: 5, the gene fragment encoding the heavy chain constant region is shown as SEQ ID NO: 6; the gene fragment encoding the light chain variable region is shown as SEQ ID NO: 7, and the gene fragment encoding the light chain constant region is shown as SEQ ID NO:

8.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the gene fragment of the porcine monoclonal antibody according to claim 4 or 5.

7. A host cell, characterized in that, The host cell comprises the recombinant expression vector according to claim 6.

8. The porcine pestivirus E of claim 1 rns Use of porcine-derived monoclonal antibodies to the E protein in the preparation of diagnostic porcine pestivirus reagents.

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