Indirect elisa detection method based on p30 protein of african swine fever virus

Abstract

This invention relates to the field of virus detection technology, specifically to an indirect ELISA detection method based on the p30 protein of African swine fever virus (ASFV). The invention uses pCMV3 as a eukaryotic expression vector to construct the pCMV3-p30 recombinant plasmid. Eukaryotic expression of the ASFV p30 protein is performed on HEK-293F cells, and compared with HEK-293T cells. The results show that the p30 protein expression level is higher on HEK-293F cells compared to HEK-293T cells, enabling large-scale production of the p30 protein. When applied to the indirect ELISA detection of ASFV, the required concentration of coated p30 protein is low, significantly reducing detection costs. The method exhibits high sensitivity and specificity, and the detection concordance rate with commercial ELISA kits is 100%, demonstrating broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, specifically to an indirect ELISA detection method based on the p30 protein of African swine fever virus. Background Technology

[0002] African swine fever virus (ASFV) p30 protein is an early-expressed membrane protein encoded by the CP204L gene. Pigs produce p30 protein within 3-4 hours of ASFV infection and continue to produce it throughout the infection period. It is currently widely used as a target for ASF detection in serological testing. While many ASF ELISA detection methods exist based on the p30 protein, existing methods suffer from high costs, low antigen preparation and yield, unsuitability for large-scale production, low sensitivity, and low concordance with commercial ASF ELISA kits. Therefore, developing a new, low-cost ELISA product or method with high antigen yield, high sensitivity, good specificity, and consistent concordance with commercial ELISA kits is urgently needed. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide an indirect ELISA detection method based on the p30 protein of African swine fever virus.

[0004] This invention provides a method for preparing African swine fever virus p30 protein, which involves constructing a eukaryotic expression vector with the nucleotide sequence shown in SEQ ID NO:3, and then transfecting the eukaryotic expression vector into HEK-293F cells, followed by culture and purification to obtain the p30 protein.

[0005] Furthermore, the purification process also includes a concentration step.

[0006] In this invention, experimental results demonstrate that the expression level of p30 protein is high in HEK-293F cells, reaching 4.456 mg / mL. The expression level in other cells is significantly lower than that in HEK-293F cells (such as HEK-293T cells). Compared with other cells, expressing p30 protein using HEK-293F cells is simple, low-cost, and highly efficient in cell culture and protein preparation. The resulting p30 protein requires a lower concentration for ELISA detection compared to p30 protein expressed in other cells, significantly reducing detection costs.

[0007] The nucleotide sequence shown in SEQ ID NO:3 includes the Kozak sequence, which is an important sequence for initiating translation in eukaryotes and is used to improve the transcription and translation of target genes.

[0008] Furthermore,

[0009] The eukaryotic expression vector is pCMV3;

[0010] The extracted solution was PBS.

[0011] This invention provides the p30 protein prepared by the aforementioned method.

[0012] Existing methods for expressing p30 protein primarily utilize prokaryotic expression systems. Prokaryotic expression refers to gene expression occurring within prokaryotes (mainly *E. coli*). However, the expression products from these systems tend to form inclusion bodies, exhibit low activity, and are insoluble, easily leading to protein inactivation during purification. Furthermore, expressing mammalian viral proteins via *E. coli* results in low homology with mammalian proteins and can only express some unglycosylated or simply modified recombinant proteins. This invention utilizes a mammalian eukaryotic expression system for p30 protein expression, enabling the expressed protein to self-modify, exhibiting good homology with mammalian proteins. The entire process is completed under serum-free culture conditions, and the preparation method is simple and rapid. Furthermore, this invention screened host cells for p30 protein. HEK-293T cells were used, but the protein expression level was low, making large-scale production impossible. HEK-293F cells were used for eukaryotic expression of p30 protein. The results showed that large-scale production of p30 protein could be carried out using HEK-293F cells, and the purified protein reached 4.456 mg / mL. It has the advantages of high expression level, correct folding conformation, and the ability to achieve large-scale production.

[0013] The prepared p30 protein was applied to the indirect ELISA detection of African swine fever virus. Compared with other existing p30 protein-based methods, the antigen coating concentration can be as low as 0.3 μg / mL, reducing the amount of antigen used (existing technologies typically use antigen coating concentrations above 0.5 μg / mL, requiring a large amount of antigen). The sensitivity can reach 1:12800 (commercial kits have a sensitivity of 1:6400). Simultaneous detection of 342 serum samples with a commercial kit showed a high concordance rate of 100%, enabling clinical application while reducing costs. Furthermore, compared to other existing products, the detection time is significantly shortened, with color development occurring within 5 minutes.

[0014] The present invention provides a formulation comprising excipients and the p30 protein described herein.

[0015] The formulation described in this invention contains the p30 protein described in this invention, and the formulation can be used for the preparation of p30 protein antibodies and / or the detection of African swine fever virus.

[0016] In this invention, the excipients include adjuvants, buffer solutions, antioxidants, protein protectants, etc., used to maintain the activity of the p30 protein or enable it to function.

[0017] This invention provides the application of at least one of the following (I) to (II) in the preparation of African swine fever virus detection products:

[0018] I) The p30 protein described in this invention;

[0019] II) The formulation described in this invention.

[0020] This invention provides a kit for detecting African swine fever virus, comprising at least one of an antigen coating solution, a serum diluent, and / or a blocking solution, and the p30 protein described in this invention.

[0021] Furthermore, in the aforementioned kit,

[0022] The antigen coating solution is a carbonate buffer solution;

[0023] The serum diluent includes PBS and BSA;

[0024] The blocking solution includes PBS and BSA.

[0025] Furthermore, in the aforementioned reagent kit,

[0026] The antigen coating solution is a 0.05 mmol / L carbonate buffer solution with a pH of 9.6;

[0027] The serum diluent includes PBS and 1% BSA by mass.

[0028] The blocking solution comprises PBS and 2% BSA by mass.

[0029] The kit described in this invention also includes enzyme-labeled antibodies, hydrochloric acid, and detection consumables. Specifically, the enzyme-labeled antibody is HRP-goat anti-pig-IgG, and the hydrochloric acid is 1M hydrochloric acid.

[0030] This invention provides a method for detecting African swine fever virus for non-diagnostic purposes, which involves detecting samples using the preparations and / or kits described in this invention.

[0031] Furthermore, the sample is serum.

[0032] This invention uses pCMV3 as a eukaryotic expression vector to construct the pCMV3-p30 recombinant plasmid. Eukaryotic expression of the African swine fever virus p30 protein was performed on HEK-293F cells and compared with HEK-293T cells. The results showed that the p30 protein expression level was higher on HEK-293F cells compared to HEK-293T cells, enabling large-scale production of the p30 protein. When applied to indirect ELISA detection of African swine fever virus, the required concentration of coated p30 protein is low, significantly reducing detection costs. The detection sensitivity and specificity are high, and the detection concordance rate with commercial ELISA kits is 100%, demonstrating broad application prospects. Attached Figure Description

[0033] Figure 1 The results of PCR amplification of the CP204L gene (612bp) are shown, where M is the standard mass of DNA molecules; 1 and 2 are the PCR amplification results of the CP204L gene (p30 encoding gene); and 3 is the negative control.

[0034] Figure 2 This paper presents the construction and identification of the p30 recombinant plasmid, where M represents the standard mass of DNA molecules; 1 represents the PCR identification of the recombinant plasmid pCMV3-p30; and 2 represents the negative control.

[0035] Figure 3 SDS-PAGE and Western Blot analysis of p30 protein expressed in HEK-293F cells were performed. In A, SDS-PAGE analysis showed that M represents the standard mass of the protein molecule; 1 is the cell supernatant after transfection; 2 is cell debris from sonicated transfected cells; 3 is the cell lysate of transfected cells; 4 is the cell supernatant of untransfected cells; 5 is the cell debris from sonicated untransfected cells; 6 is the cell lysate of untransfected cells. In B, Western Blot analysis showed that M represents the standard mass of the protein molecule; 1 is the cell supernatant after transfection; 2 is the cell debris from sonicated transfected cells; 3 is the cell lysate of transfected cells; 4 is the cell supernatant of untransfected cells; 5 is the cell debris from sonicated untransfected cells; 6 is the cell lysate of untransfected cells.

[0036] Figure 4 The results show the SDS-PAGE and Western Blot identification of p30 protein expressed in HEK-293F cells after purification and concentration. In A, the results are SDS-PAGE identification, where M is the standard mass of the protein molecule, 1 is the p30 protein before purification and concentration, and 2 is the p30 protein after purification and concentration. In B, the results are Western Blot identification, where M is the standard mass of the protein molecule, 1 is the p30 protein before purification and concentration, and 2 is the p30 protein after purification and concentration.

[0037] Figure 5 Determination of antigen coating solution and serum diluent;

[0038] Figure 6 Determining the dilution factor between the blocking solution and the enzyme-labeled antibody;

[0039] Figure 7 The optimization of reaction conditions is shown, where A is the determination of antigen coating conditions; B is the determination of blocking conditions; C is the determination of serum reaction conditions; D is the determination of secondary antibody incubation conditions; and E is the determination of colorimetric conditions.

[0040] Figure 8 OD of 90 negative serum samples 450 nm Normal distribution plot of values;

[0041] Figure 9 OD of 90 negative serum samples 450 nm One-sample Kolmogorov-Sminov test for the value;

[0042] Figure 10 ELISA sensitivity detection of p30 protein expressed in HEK-293F cells;

[0043] Figure 11 ELISA-specific detection of p30 protein expressed in HEK-293F cells;

[0044] Figure 12 SDS-PAGE and Western Blot analysis of p30 protein expressed in HEK-293T cells were performed. In A, SDS-PAGE analysis showed that M represents the standard mass of the protein molecule; 1 is the cell supernatant after transfection; 2 is the cell lysate after transfection; 3 is the cell supernatant of untransfected cells; 4 is the cell lysate of untransfected cells. In B, Western Blot analysis showed that M represents the standard mass of the protein molecule; 1 is the cell supernatant after transfection; 2 is the cell lysate after transfection; 3 is the cell supernatant of untransfected cells; 4 is the cell lysate of untransfected cells.

[0045] Figure 13 The image shows the purified and concentrated p30 protein after expression in HEK-293T cells. In the image, M represents the standard mass of the protein molecule, 1 represents the p30 protein before purification and concentration, and 2 represents the p30 protein after purification and concentration.

[0046] Figure 14 The sensitivity of ELISA detection for p30 protein expressed in HEK-293T cells was demonstrated.

[0047] Figure 15The specificity of ELISA detection of p30 protein expressed in HEK-293T cells was demonstrated. Detailed Implementation

[0048] This invention provides an indirect ELISA detection method based on the p30 protein of African swine fever virus. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the method and application described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0049] The nucleotide sequence of p30 is: (SEQ ID NO:1);

[0050] The nucleotide sequence of the Kozak sequence is: gccgccacc (SEQ ID NO:2);

[0051] The nucleotide sequence of P30 combined with the Kozak sequence is: (SEQ ID NO:3).

[0052] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0053] The present invention will be further illustrated below with reference to the embodiments:

[0054] Example 1: Eukaryotic Expression of p30 in HEK-293F Cells and Establishment of Indirect ELISA Method 1. Construction of p30 Recombinant Plasmid

[0055] Based on the p30 full-genome sequence of strain GZ201801 in GeneBank, a Kozak sequence was added to the N-terminus and a 6×His tag was added to the C-terminus. Using strain GZ201801 as a template, the CP204L gene encoding the p30 protein was amplified (CP204L gene is 612 bp; PCR amplification results are shown below). Figure 1As shown in the figure, the target fragment was ligated into the pCMV3 vector, and the ligation product was transformed into E. coli (E. coli) DH5α competent cells. The plasmid was then extracted and sequenced, confirming the successful construction of the pCMV3-p30 eukaryotic expression plasmid (construction and identification of the p30 recombinant plasmid are shown in the figure). Figure 2 (As shown).

[0056] Eukaryotic expression and purification of 2p30 protein

[0057] 2.1 Eukaryotic expression of p30 protein

[0058] 100 μg of pCMV3-p30 recombinant plasmid was transfected into 100 mL of HEK-293F cells. After 4–5 days, the cell supernatant was collected by centrifugation. 25–30 mL of PBS was added to the cells, and the cells were lysed by sonication (4 s sonication, 5 s interval, 40% amplitude). The cell lysate and cell debris were collected by centrifugation. The cell supernatant, cell debris, and cell lysate were analyzed by SDS-PAGE and Western blot to determine p30 protein expression. The experimental results showed (e.g.) Figure 3 Cell debris and cell lysate show distinct bands at around 30 kDa, while no bands are found in cell supernatant. Therefore, p30 protein is expressed in HEK-293F cells.

[0059] 2.2 Purification and concentration of p30 protein

[0060] After confirming p30 protein expression, the expression product was bound to nickel column resin packing material. Expression products not bound to the nickel column resin packing material were filtered using an affinity chromatography column. The expression products bound to the nickel column resin packing material were then eluted with a gradient of 40 mM imidazole, 250 mM imidazole, and 500 mM imidazole to obtain purified p30 protein. This purified protein was then concentrated by ultrafiltration and analyzed using SDS-PAGE and Western blotting (e.g., [missing information]). Figure 4 It can be observed that the purified p30 protein has no visible impurities compared to the unpurified protein, and there is a more obvious band at around 30 kDa, indicating that the p30 protein has been successfully purified and concentrated. The protein concentration was determined to be 4.456 mg / mL by the BCA method, indicating a high expression level.

[0061] 3. Establishment of the Indirect ELISA Method

[0062] 3.1 Determination of antigen coating concentration and dilution factor of serum to be tested

[0063] Antigen dilution: Dilute the purified p30 protein to 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, and 6 μg / mL; Serum sample dilution: Dilute positive and negative sera to 1:50, 1:100, 1:200, 1:400, 1:800, and 1:1600, respectively; Plate coating: Coat 100 μL / well of the diluted p30 protein onto an ELISA plate, incubate at 4℃ for 12 h, then discard the solution; Blocking: 5% skim milk powder, 2 0.00 μL / well, block at room temperature (25℃) for 2 h, wash 4 times with PBST (300 μL / well, 1 min / wash); Apply primary antibody: add diluted serum to the ELISA plate, 100 μL / well, incubate at 37℃ for 1 h, wash 4 times; Apply secondary antibody: dilute HRP-goat anti-pig-IgG 1:5000, 100 μL / well, incubate at 37℃ for 45 min, wash 4 times; Color development: add TMB chromogenic solution, 100 μL / well, incubate at room temperature (25℃) in the dark for 5 min; Termination: add 1M hydrochloric acid, 100 μL / well, and immediately read the OD value. 450nm Data calculation: OD 450 nm When the value is approximately 1 and the P / N value is at its minimum, select this coating concentration and dilution concentration.

[0064] Table 1. Determination of antigen coating concentration and dilution factor of the serum to be tested

[0065]

[0066]

[0067] The above results show that when the p30 protein concentration is between 0.3 and 4 μg / mL and the serum dilution is between 1:50 and 1:800, the OD of positive serum is... 450 nm Values ​​between 1.021 and 1.997 indicate that the OD value of negative serum is... 450 nm The values ​​ranged from 0.122 to 0.528, with P / N values ​​ranging from 3.185 to 8.403. When the p30 protein concentration was 0.3 μg / mL and the serum dilution was 1:800, the OD value of the positive serum... 450 nm The value is approximately equal to 1, and the OD of negative serum is... 450 nm The value was the lowest, and the P / N ratio was the highest (i.e., 8.403). Therefore, the optimal antigen coating concentration was 0.3 μg / mL (consistent with the kit), and the serum dilution was 1:800.

[0068] 3.2 Determination of antigen coating solution and serum diluent

[0069] ELISA plates were coated with PBS and carbonate buffered saline (CBS) at a concentration of 0.3 μg / mL, respectively. Serum was diluted 1:800 using PBS and PBS containing 1% BSA, respectively. The assay was performed following the above steps.

[0070] Depend on Figure 5 It can be seen that when the antigen coating solution is CBS and the serum diluent is PBS containing 1% BSA (mass fraction), the OD of positive serum is... 450 nm Maximum value, negative serum OD 450 nm The value is the smallest, and the P / N value is the largest (i.e., 17.104). Therefore, the optimal antigen coating solution is CBS, and the serum diluent is PBS containing 1% BSA (mass fraction).

[0071] 3.3 Determination of the dilution factor between blocking buffer and enzyme-labeled antibody

[0072] The antigen was prepared at a concentration of 0.3 μg / mL using CBS and coated onto ELISA plates. Serum was diluted 1:800 with PBS containing 1% BSA. Blocking solutions were prepared using 5% skim milk powder (PBS containing 5% skim milk powder), 5% FBS (PBS containing 5% fetal bovine serum), 2% BSA (PBS containing 2% BSA), and 1% gelatin solution (PBS containing 1% gelatin). Blocking was performed at room temperature (25°C) for 2 hours. Goat anti-porcine IgG-HRP was diluted at 1:500, 1:1000, 1:2500, 1:5000, 1:7500, and 1:10000. Each condition was performed in duplicate.

[0073] Depend on Figure 6 It can be seen that when the blocking solution is PBS containing 2% BSA (mass fraction) and the enzyme-labeled antibody is diluted 1:2500, the P / N value is the largest (i.e., 9.746). Therefore, the optimal blocking solution is PBS containing 2% BSA (mass fraction) and the optimal enzyme-labeled antibody is diluted 1:2500.

[0074] Table 2. Determination of the dilution factor between blocking buffer and enzyme-labeled antibody

[0075]

[0076] 3.4 Optimization of reaction conditions

[0077] 3.4.1 Determination of antigen coating conditions

[0078] ELISA plates were coated at 4℃ for 14 h, 37℃ for 1 h, 2 h, and 4 h respectively (0.3 μg / mL, 100 μL / well), with 4 wells for each condition (containing 2 positive sera and 2 negative sera).

[0079] Depend on Figure 7 As can be seen from A, the P / N value is the largest when the coating conditions are 4℃ and 14h. Therefore, 4℃ and 14h are the optimal antigen coating conditions.

[0080] 3.4.2 Determination of Closure Conditions

[0081] Blocking was performed using PBS containing 2% BSA (mass fraction), 200 μL / well, at room temperature (25℃) for 1 h and 2 h, and at 37℃ for 1 h and 2 h, respectively.

[0082] Depend on Figure 7 As can be seen from B, the P / N value is the largest when the sealing condition is 37℃ for 2 hours. Therefore, incubation at 37℃ for 2 hours is the optimal sealing condition.

[0083] 3.4.3 Determination of serum reaction conditions

[0084] Positive and negative sera were diluted 1:800, 100 μL / well, and reacted at 37℃ for 15 min, 30 min, 45 min, and 60 min, respectively.

[0085] Depend on Figure 7 The results from the C-test indicate that the optimal serum reaction conditions are 37℃ for 60 minutes.

[0086] 3.4.4 Determination of enzyme-labeled antibody incubation conditions

[0087] Goat anti-pig IgG-HRP was diluted at a ratio of 1:2500, 100 μl / well, and incubated at 37°C for 15 min, 30 min, 45 min, and 60 min.

[0088] Depend on Figure 7 As indicated by D in the diagram, the optimal incubation conditions for the enzyme-labeled antibody are 37°C for 45 minutes.

[0089] 3.4.5 Determination of color development conditions

[0090] Add 100 μL of TMB colorimetric solution to each well and incubate at 37°C or room temperature (25°C) for 5 min, 10 min, and 15 min, respectively.

[0091] Depend on Figure 7 The results from the E test show that the P / N ratio is the highest when the color development condition is incubation at room temperature (25℃) for 5 minutes. Therefore, room temperature (25℃) for 5 minutes is selected as the optimal color development condition.

[0092] In summary, comparing the P / N values ​​under various conditions, the highest P / N values ​​were found to be 16.153, 19.012, 19.042, 20.127, and 18.932 for the following conditions: coating the p30 protein solution on an ELISA plate at 4°C for 14 h, blocking it at 37°C for 2 h, reacting it with serum at 37°C for 1 h, reacting it with the enzyme-labeled antibody at 37°C for 45 min, and finally developing the color at room temperature (25°C) for 5 min. Therefore, the above conditions are the optimal reaction conditions for this detection method.

[0093] 3.5 Determination of Critical Values

[0094] Ninety negative serum samples were tested by ELISA, with each sample tested in duplicate in two wells. The OD values ​​of the samples were measured using a microplate reader. 450 nm Value, calculate the average and standard deviation (S), when serum A positive result is determined when the serum... The result was determined to be negative at that time. The results were deemed suspicious. A normal distribution plot of the 90 negative serum samples was plotted using SPSS 26.0 statistical analysis software to evaluate the accuracy of the data and the cutoff value determination.

[0095] OD was detected in 90 negative serum samples using ELISA. 450 nm The calculated average value is 0.184 and the standard deviation is 0.050. Therefore, when the serum sample OD... 450 nm A value ≥0.335 is considered positive. 450nm A value < 0.285 is considered negative, and a value ≤ 0.285 is considered negative. 450 nm A value < 0.335 is considered suspicious. Analyze the data using SPSS 26.0 and plot a normal distribution (e.g., ...). Figure 8 Its skewness coefficient is 0.292 and its kurtosis coefficient is -0.761, both <1; the Kolmogorov-Sminoff test (also known as the one-sample Kolmogorov-Sminoff test) shows the following results: Figure 9 As shown in the figure, P = 0.200 (>0.05), indicating that the sample data has good significance. Therefore, the experimental data conforms to a normal distribution and is accurate.

[0096] 3.6 Sensitivity Testing

[0097] Positive sera were diluted at ratios of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, and 1:25600, and were tested using the ELISA method described in this study and commercially available kits. Each sample was performed in triplicate, and the OD values ​​of the samples were measured. 450 nm .

[0098] From the experimental results ( Figure 10 As can be seen, the lowest positive serum dilution that the commercial kit can detect is 1:6400, while the lowest positive serum dilution of this detection method is 1:12800. Its sensitivity is twice that of the commercial kit, demonstrating excellent sensitivity.

[0099] 3.7 Specificity Detection

[0100] Three samples of positive serology were collected for African swine fever virus (ASFV), classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), and porcine circovirus (PCV). Four replicates were set up for each group, and a negative control was also included. Results are as follows: Figure 11 As shown, this detection method has good specificity and does not cross-react with other porcine virus sera.

[0101] 3.8 Repeatability Test

[0102] Intra-batch repeatability test: Take 5 positive serum samples and 5 negative serum samples, and test them on the same batch of ELISA plates, with 5 replicates for each sample.

[0103] Inter-batch repeatability experiment: p30 protein was diluted at different time points, coated on 5 ELISA plates, and the above serum was tested without replication.

[0104] Table 3. Results of intra-batch repeatability experiments

[0105]

[0106] Table 4. Results of inter-batch repeatability experiments

[0107]

[0108]

[0109] The results above show that the intra-batch detection coefficient of variation is 2.311%–7.407%, and the inter-batch detection coefficient of variation is 3.544%–9.877%, both less than 10%. Therefore, this detection method has good repeatability. 3.9 Detection of clinical samples and concordance rate.

[0110] A total of 342 serum samples from different batches were compared with commercially available ELISA test kits, and the concordance rate was calculated.

[0111] Table 5. Compliance Rate Test

[0112]

[0113] The results above show that the positive concordance rate of this detection method with commercial kits is 100%, and the negative concordance rate is 100%. Therefore, the method established in this study has a 100% concordance rate with commercial kits and can be used for clinical sample testing.

[0114] Example 2: Expression and Application of p30 Protein in HEK-293T Cells

[0115] Expression of 1p30 protein in 293T cells

[0116] pCMV3-p30 was transfected into HEK-293T cells. After 48 hours, cell supernatant and cell lysate were collected, and SDS-PAGE and Western blot were performed to identify p30 protein expression. The experimental results showed (e.g.) Figure 12 The cell lysate showed a distinct band at around 30 kDa, indicating that p30 protein is expressed in HEK-293T cells.

[0117] Purification of 2p30 protein

[0118] After confirming p30 protein expression in HEK-293T cells, the cell lysate was purified using a nickel column as described above. SDS-PAGE was then performed to identify the purified protein (e.g.,...). Figure 13 As shown in the figure, the p30 protein was successfully purified, and the protein concentration was determined to be 0.389 mg / mL by the BCA method (Bicinchoninic acid). The final protein concentration was much lower than that obtained by HEK-293F cell preparation (i.e., 4.456 mg / mL), and its protein yield was much lower than that obtained by HEK-293F cell preparation.

[0119] 3. Sensitivity and specificity of ELISA detection

[0120] The sensitivity and specificity of ELISA detection of p30 protein expressed in HEK-293T cells were assessed using the aforementioned established ELISA method.

[0121] 3.1 Sensitivity Testing

[0122] Depend on Figure 14It can be seen that the ELISA detection sensitivity of p30 protein expressed by HEK-293T cells is the same as that of p30 protein expressed by HEK-293F cells, both being 1:12800.

[0123] 3.2 Specificity Detection

[0124] Depend on Figure 15 It can be seen that the ELISA detection of p30 protein expressed by HEK-293T cells has good specificity and does not cross-react with other viruses. However, compared with p30 protein prepared by HEK-293F cells, the OD of positive serum is lower. 450 nm The value is low.

[0125] In summary, the p30 protein prepared using HEK-293T cells has the same ELISA detection sensitivity and specificity as that prepared using HEK-293F cells. However, the protein concentration obtained by HEK-293T cells is much lower than that obtained by HEK-293F cells, and the protein expression and preparation efficiency is low, making it difficult to achieve large-scale production.

[0126] 3.3 Comparison and summary of p30 protein expression differences between HEK-293T cells and HEK-293F cells

[0127] The culture medium used for HEK-293T cells requires the addition of fetal bovine serum. Once the cells have filled the culture dish, they cannot continue to grow. They can only be expanded by "passing from one to three" (i.e., transferring all the cells in one culture dish to three culture dishes). During the culture process, the adherent cells must be digested and dislodged from the culture dish before expansion can be carried out. Moreover, the total number of cells is limited by the culture dish, resulting in low cell culture and expansion efficiency.

[0128] HEK-293F cells only require serum-free culture medium, and their cost is lower than that of HEK-293T cells. During cell culture, there is no need for digestion or pipetting; cell expansion culture can be carried out simply by adding culture medium to the cell shake flask. The total number of cells is not limited by the cell shake flask, resulting in higher cell culture and expansion efficiency.

[0129] Therefore, through extensive experiments comparing HEK-293F cells and HEK-293T cells, and considering factors such as cell culture efficiency and protein preparation efficiency, HEK-293F cells were selected for the preparation of p30 protein, thus achieving the goal of mass production of p30 protein.

[0130] Comparative Example 1: Comparison with other indirect ELISA methods based on African swine fever virus p30 protein

[0131] The method described in the article "Establishment of an indirect ELISA antibody detection method based on African swine fever virus p30 protein" was used for prokaryotic expression. The coating antigen concentration was 1 mg / L (i.e. 1 μg / mL), which is much higher than the antigen coating concentration required by this method. Moreover, the color development time is 15 min. The overall concordance rate with commercial kits was only 62.1%.

[0132] The method described in the article "Establishment and Application of Tag-Free p30-ELISA Antibody Detection Method for African Swine Fever Virus" was used for prokaryotic expression. The coating antigen concentration was 1 mg / L, the sensitivity was 1:512, and the overall concordance rate with the commercial kit was 97.78%.

[0133] The method described in "Establishment and Application of Prokaryotic Expression of African Swine Fever Virus p30 Protein and Indirect ELISA Antibody Detection Method" was used for prokaryotic expression, with a sensitivity of 1:1024 and an overall concordance rate of 98.8% with commercially available kits.

[0134] While the method described in "Establishment and Application of Indirect ELISA Detection Method for African Swine Fever Virus Antibody" achieves the same sensitivity and clinical sample concordance rate as this method, the antigen coating concentration is as high as 20 μg / mL, resulting in higher costs.

[0135] The method of "Eukaryotic expression of African swine fever virus P30 protein and establishment of indirect ELISA antibody detection method" was used for eukaryotic expression. The antigen coating concentration was 0.4 μg / mL, the sensitivity was 1:128, and the clinical concordance rate was 98.9%.

[0136] The antigen coating concentration of this method is 0.3 μg / mL, and the amount of antigen required for detection is lower than that of existing detection methods, effectively reducing detection costs. The sensitivity is as high as 1:12800, while that of commercial kits is 1:6400, making the sensitivity twice that of commercial kits, which is significantly better than existing commercial kits. Moreover, the clinical concordance rate with commercial kits is 100%, and it can be applied to clinical testing.

[0137] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for indirect ELISA detection based on African swine fever virus p30 protein, characterized in that, This includes antigen coating solution, serum diluent, blocking solution, and p30 protein; The p30 protein is obtained by constructing the nucleotide sequence shown in SEQ ID NO:3 into a eukaryotic expression vector, which is then cultured and purified after transfection of HEK-293F cells. The eukaryotic expression vector is pCMV3; The antigen coating solution is a carbonate buffer solution; The serum diluent was PBS containing 1 wt% BSA; The blocking solution is PBS containing 2 wt% BSA.

2. The reagent kit according to claim 1, characterized in that, The antigen coating solution is a 0.05 mmol / L carbonate buffer solution with a pH of 9.

6.

3. An indirect ELISA detection method based on the p30 protein of African swine fever virus for non-diagnostic purposes, characterized in that, To use the kit described in claim 1 or 2 to test the sample; The detection includes the following steps: Antigen dilution: Dilute the purified p30 protein to 0.3 μg / mL; Serum sample dilution: Dilute positive and negative sera to 1:800 respectively; Plate coating: Coat 100 μL / well of diluted p30 protein into an ELISA plate, incubate at 4°C for 14 h, then discard the solution; Blocking: PBS containing 2% BSA, 200 μL / well, incubated at 37°C for 2 h, followed by washing with PBST 4 times, 300 μL / well, 1 min / wash; Primary antibody application: Add diluted serum to the microplate, 100 μL / well, incubate at 37°C for 1 h, and wash the plate 4 times; Secondary antibody application: HRP-goat anti-pig-IgG was diluted at 1:2500, 100 μL / well, incubated at 37℃ for 45 min, and the plate was washed 4 times; Color development: Add 100 μL of TMB color development solution to each well and incubate at room temperature (25°C) in the dark for 5 minutes. Termination: Add 1M hydrochloric acid, 100 μL / well, and immediately read the OD. 450nm .

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