A kit for detecting African swine fever virus P30 antibodies and its application

Abstract

The present invention discloses a kit for detecting African swine fever virus P30 antibodies and its application. The present invention obtains a recombinant African swine fever virus protein by serially connecting several regions with relatively strong immunogenicity in the amino acid sequence of the P30 protein, and then serially constructs a trimer of the recombinant protein. Foldon is then connected to the African swine fever virus protein serial trimer to construct a trimeric recombinant protein. Based on the trimeric recombinant protein, a kit for detecting African swine fever virus P30 antibodies is prepared. The kit has a higher sensitivity than many similar products on the market, with a sensitivity of up to 1:32,000, and is highly specific. Furthermore, the molecular weight of the trimeric recombinant protein has been increased by three times, further improving the stability and half-life of the kit. This is beneficial to the detection rate of African swine fever virus antibodies and has high application value. Furthermore, the kit is simple to prepare, has low production costs, and is easy to promote.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a kit for detecting African swine fever virus P30 antibodies and an application thereof. Background Art

[0002] African swine fever (ASF) is an acute, severe, hemorrhagic infectious disease caused by the African swine fever virus (ASFV), which can cause widespread mortality in domestic pigs and wild boars. Currently, there is no effective vaccine. Currently, the ASFVs circulating in pig farms are relatively complex, categorized by virulence into highly virulent and less virulent strains; by gene type, into genotype I, genotype II, and recombinant genotype I and II strains; and by gene deletion, into CD2v-deleted, MGF-deleted, and multi-gene-deleted strains. Clinical manifestations vary significantly between pigs infected with different strains. In particular, infection with less virulent strains has a long incubation period, subtle clinical symptoms, and a low virus detection rate. This can easily lead to misdiagnosis and the breeding of infected pigs, ultimately resulting in irreparable losses. Therefore, improving the detection rate of clinically infected pigs with ASFV, so that carriers can be eliminated and cleared, is a pressing issue for intensive pig farming.

[0003] After pigs are infected with African swine fever virus, they can produce specific antibodies against multiple viral proteins in their bodies. Among them, antibodies against the structural protein P30 can be produced 8 days after infection with the virus (references 1 and 2), which is an ideal antigen for the development of early antibody diagnostic kits for African swine fever. The national standard (GB / T18648-2020) "African Swine Fever Diagnostic Technology" issued in 2020 also recommends the use of p30 protein as a coating protein in the ELISA method for African swine fever antibody detection. At present, a number of antibody detection kits for P30 protein have been launched on the market at home and abroad. However, there are still some pigs with low antibody levels in their serum that cannot be detected, and the pig farming industry is still at risk of an African swine fever outbreak. Therefore, how to improve the sensitivity of the detection method and thereby increase the detection rate of African swine fever virus antibodies is an urgent problem that needs to be solved.

[0004] References 1. Giménez-Lirola LG, Mur L, Rivera B, Mogler M, Sun Y, Lizano S, Goodell C, Harris DL, Rowland RR, Gallardo C, Sánchez-Vizcaíno JM, Zimmerman J. Detection of African Swine Fever Virus Antibodies in Serum and Oral FluidSpecimens Using a Recombinant Protein 30(p30)Dual Matrix Indirect ELISA.PLoSOne.2016Sep9;11(9):e0161230.doi:10.1371 / journal.pone.0161230.PMID:27611939;PMCID:PMC5017782.

[0005] Reference 2. Cubillos C, Gómez-Sebastian S, Moreno N, MC, Mulumba-Mfumu LK, Quembo CJ, Heath L, Etter EM, Jori F, Escribano JM, Blanco E. Africanswine fever virus serodiagnosis: a general review with a focus on the analyzes of African serum samples.Virus Res.2013Apr;173(1):159-67.doi:10.1016 / j.virusres.2012.10.021.Epub 2012Nov 3.PMID:23131491. Summary of the Invention

[0006] The first aspect of the present invention aims to provide a recombinant African swine fever virus P30 protein;

[0007] The second aspect of the present invention aims to provide a multimer constructed based on the above-mentioned recombinant protein.

[0008] The third aspect of the present invention aims to provide a fusion protein.

[0009] The fourth aspect of the present invention aims to provide biological materials related to the recombinant protein of the first aspect of the present invention, the multimer of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention.

[0010] The fifth aspect of the present invention aims to provide applications of the above-mentioned recombinant protein, polymer, fusion protein or biomaterial.

[0011] The sixth aspect of the present invention aims to provide a kit.

[0012] The purpose of the seventh aspect of the present invention is to provide a method for detecting African swine fever virus P30 antibodies.

[0013] The technical solution adopted by the present invention is:

[0014] In a first aspect of the present invention, a recombinant protein of African swine fever virus P30 is provided, wherein the amino acid sequence of the recombinant protein is any one of the following:

[0015] a) SEQ ID NO. 8; or

[0016] b) an amino acid sequence that is at least 90% homologous to SEQ ID NO. 8 and has the same or similar functions; or

[0017] c) An amino acid sequence shown in SEQ ID NO. 8 that has been modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

[0018] In a second aspect, the present invention provides an African swine fever virus P30 recombinant protein multimer, wherein the multimer is a protein multimer in which the recombinant protein described in the first aspect of the present invention is repeatedly connected in series; the number of repetitions is 2 to 4 times.

[0019] Preferably, the amino acid sequence of the polymer is any one of the following:

[0020] a) SEQ ID NO. 9; or

[0021] b) an amino acid sequence that is at least 90% homologous to SEQ ID NO. 9 and functions identically or similarly; or

[0022] c) An amino acid sequence shown in SEQ ID NO. 9 that has been modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

[0023] The third aspect of the present invention provides a fusion protein, which comprises the African swine fever virus P30 recombinant protein described in the first aspect of the present invention or the multimer described in the second aspect of the present invention.

[0024] Preferably, the fusion protein further comprises a Foldon short peptide.

[0025] Preferably, the fusion protein further comprises a linker sequence and / or a tag sequence.

[0026] Preferably, the tag sequence includes but is not limited to His, GST, Flag, HA and other tag sequences.

[0027] Preferably, the linker sequence includes (ggggs)n, (gggs)n, (ggs)n, (g)n, (gs)n, (eaaak)n, or (xp)n, where n is a natural number of 0-5.

[0028] Preferably, the amino acid sequence of the fusion protein is any one of the following:

[0029] a) SEQ ID NO. 10; or

[0030] b) an amino acid sequence that is at least 90% homologous to SEQ ID NO. 10 and functions identically or similarly; or

[0031] c) An amino acid sequence shown in SEQ ID NO. 10 that has been modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

[0032] The fourth aspect of the present invention provides a biological material related to the recombinant protein of the first aspect of the present invention, the multimer of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention; the biological material comprises any one of B1) to B4);

[0033] B1) a nucleic acid molecule encoding the recombinant protein of the first aspect of the present invention, the multimer of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention;

[0034] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0035] B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0036] B4) A recombinant cell containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

[0037] Preferably, the expression cassette refers to DNA capable of expressing the recombinant protein, multimer, or fusion protein in a host cell. This DNA may include not only a promoter for initiating transcription of the recombinant protein, multimer, or fusion protein gene, but also a terminator for terminating gene transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0038] Preferably, the vector may be a plasmid, cosmid, phage or viral vector.

[0039] Preferably, the recombinant cells include prokaryotic cells and eukaryotic cells. The prokaryotic cells include bacteria or algae. The eukaryotic cells include fungi, mammalian cells, or insect cells. The recombinant organism does not contain reproductive material.

[0040] The fifth aspect of the present invention provides the use of the African swine fever virus P30 recombinant protein described in the first aspect of the present invention, the multimer described in the second aspect of the present invention, the fusion protein described in the third aspect of the present invention, or the biological material described in the fourth aspect of the present invention in the preparation of a kit for detecting African swine fever virus P30 antibodies.

[0041] The sixth aspect of the present invention provides a kit comprising a coating plate coated with the fusion protein according to the second aspect of the present invention.

[0042] Preferably, the kit further comprises at least one of a coating buffer, a washing solution, a sample diluent, an enzyme-labeled secondary antibody, a color developing solution, a stop solution, a negative control, and a positive control.

[0043] Preferably, the coating concentration is 0.2-4 μg / mL.

[0044] Preferably, the coating buffer comprises 0.005-0.015 mol / L pH 7-7.5 PBS solution.

[0045] Preferably, the blocking solution comprises 4-7% skim milk.

[0046] Preferably, the washing solution comprises PBST with a pH of 7 to 7.5.

[0047] Preferably, the sample diluent comprises PBST containing 4-7% BSA (bovine serum albumin).

[0048] Preferably, the enzyme-labeled secondary antibody is an IgG antibody labeled with horseradish peroxidase (HRP).

[0049] Preferably, the color developing solution comprises TMB color developing solution.

[0050] Preferably, the stop solution comprises 0.5-1.5 M H2SO4.

[0051] Preferably, the negative control is normal pig serum that is not immunized with African swine fever virus P30 protein; and the positive control is normal pig serum that is immunized with African swine fever virus P30 protein.

[0052] The seventh aspect of the present invention provides a method for detecting African swine fever virus P30 antibodies, comprising using the kit described in the fifth aspect of the present invention to detect African swine fever virus P30 antibodies.

[0053] Preferably, the antibody comprises the following steps: taking a serum sample to be tested and adding it to a coated plate coated with a fusion protein after being blocked with a blocking solution, adding an enzyme-labeled secondary antibody, incubating, adding a color developing solution for color development, adding a stop solution, and then measuring the absorbance value, and judging whether African swine fever virus P30 antibodies are present in the sample according to the absorbance value.

[0054] The beneficial effects of the present invention are:

[0055] The present invention obtains a recombinant African swine fever virus protein by serially connecting several regions with relatively strong immunogenicity on the amino acid sequence of the P30 protein, and constructs a trimer by serially connecting the recombinant proteins. Then, Foldon is connected to the African swine fever virus protein serial trimer to construct a trimeric recombinant protein, and a kit for preparing antibodies against African swine fever virus P30 based on the trimeric recombinant protein. The sensitivity of the kit is higher than that of many similar products on the market, with a sensitivity of up to 1:32000, and the kit has high specificity. In addition, the molecular weight of the trimeric recombinant protein is increased by 3 times, which further improves the stability and half-life of the kit. It is beneficial to the detection rate of African swine fever virus antibodies and has high application value. In addition, the kit is simple to prepare, has low production cost, and is easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Schematic diagram of the trimer structure of the recombinant protein and its working principle. A. The protein monomers are connected to the Foldon peptide via the freely rotatable amino acid chain GGGS (Linker). The three Foldon monomers are bound together by intermolecular forces to form a stable trimer recombinant protein (3D structure from Protein Data Bank, ID: 4NCU). B. Schematic diagram of various binding modes of the trimer recombinant protein to the coated plate. The Foldon peptide has multiple hydrophobic amino acids at its carbon terminus. If bound to the hydrophobic groups on the plate, it will exhibit the first, most ideal binding mode on the left, resulting in all three protein monomers being semi-free in the serum sample. C and D show the binding modes of commonly used recombinant proteins and synthetic peptides to the coated plate, respectively.

[0057] Figure 2The expression, purification and identification process of the recombinant protein. A. 293T cells were transfected with the plasmid expressing P30 protein (P30-His) and the control plasmid pcDNA3.1, respectively. The results of the immunoblotting experiment showed that the target protein was successfully expressed (indicated by the arrow); B. The test results of the commercial kit showed that the positive serum and negative serum were successfully prepared; C. The immunogenicity of the amino acid sequence of the P30 protein was predicted, among which the amino acid sequence portion with a peak above the baseline was the immunogenic region. The horizontal line below the prediction graph represents the P30 protein, and the full length consists of 200 amino acids; D. The upper figure is a schematic diagram of the structure of the recombinant proteins P30-1 and P30-2. The gray part represents the P30 protein, and the numbers represent the serial numbers of the amino acid residues. The lower figure is the immunoblotting method for detecting the purified recombinant proteins. Histones P30-1 and P30-2; E. Indirect ELISA assay for the specificity of P30-1 and P30-2 in recognizing P30-positive serum; F. The upper figure shows the schematic structure of recombinant proteins P30-3 and P30-4. The gray portion represents the three repeat sequences of the P30 protein. The lower figure shows immunoblotting of purified recombinant proteins P30-3 and P30-4; G. Indirect ELISA assay for the specificity of P30-3 and P30-4 in recognizing P30-positive serum; H. Immunoblotting assay for P30-3. Denatured samples boiled before loading show monomers, while native samples not boiled before loading show trimers; I. Indirect ELISA assay for the specificity of P30-3 in recognizing P30-positive serum. The serum samples were diluted 1:100.

[0058] Figure 3 To optimize the antigen coating solution and concentration, A and B show the ELISA absorbance (OD450) and the ratio of positive to negative serum (P / N) of serum samples using three different coating solutions (PBS, carbonate buffer, and Tris-HCl). A larger ratio indicates higher specificity. C and D show the ELISA absorbance (OD450) and the ratio of positive to negative serum (P / N) of serum samples using different antigen coating concentrations. The serum was diluted at a 1:100 ratio.

[0059] Figure 4The specificity, sensitivity, and stability of the indirect ELISA method were tested. A. The specificity of the indirect ELISA method of the present invention was tested using positive sera for different viruses, with a serum dilution factor of 1:100 (positive sera for four different viruses were purchased from the China Veterinary Drug Administration); B. African swine fever virus-positive and negative sera were serially diluted and tested using the indirect ELISA method of the present invention, achieving a sensitivity of 32,000-fold; C. The indirect ELISA method of the present invention (Haida) was used to simultaneously test 100-fold diluted African swine fever virus-positive sera with kits produced by two different manufacturers; D. The ELISA test results of the stability test of the coating protein P30-3 at 37°C were obtained, with a serum dilution factor of 1:1000. DETAILED DESCRIPTION

[0060] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0061] The present invention connects a short peptide derived from T4 phage fiber protein - Foldon with a specific region of African swine fever virus protein and expresses it in E. coli to obtain a clover-shaped trimeric recombinant protein (such as Figure 1 As shown in A). Each monomer in this recombinant protein is connected to the Foldon short peptide through a short amino acid chain (Linker) that can rotate freely. Figure 1 As shown in Figure B, when the trimeric protein is bound to the antigen-coated plate, most of the monomers of the antigen protein can shake in multiple directions in the serum sample, and the antigen binding sites are fully exposed, thereby greatly increasing the number of captured specific antibodies. However, the commonly used recombinant proteins or artificially synthesized peptides directly bind to the coated plate, with a relatively low degree of freedom. The number of captured antibodies is limited by the number of correct binding sites exposed when the plate is coated ( Figure 1 C, 1D).

[0062] The amino acid sequences involved in the present invention are:

[0063] 1. Foldon short peptide: GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO.1);

[0064] 2. Linker: GGGS(SEQ ID NO.2);

[0065] 3. His tag: HHHHHHHH (SEQ ID NO. 3);

[0066] 4. P30-1:

[0067] MDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLST VKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNEHHH HHHH(SEQ ID NO.4)

[0068] 5. P30-2:

[0069] MEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTV QHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKISFYLTYIHHH HHHHH(SEQ ID NO.5);

[0070] 6. P30 full length:

[0071] DFILNISMKMEVI FKTDLRSSSQ VVFHAG SLYNW FSVEIINSGRIVTTAIKTLLSTVKYDI VKSARIY AGQGYTEHQAQEEWN MILHVLF EEETESSASSENIHEKN DNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHN FIQTIYGTPLKE EEKEVVRLMVIKLLKKISFYLTYI(SEQ ID NO.6);

[0072] 7. Amino acid sequence of the P30 protein inserted into the vector pcDNA3.1:

[0073] MSPLWWGFLLSCLGCKILPGAQGHHHHHHHHDFILNISMKMEVIFKTDLRSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMIL HVLFEEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKISFYLTYI(SEQ ID NO.7);

[0074] 8. The construction method of P30-3 is:

[0075] The underlined short peptides in the P30 sequence were connected in series to form SEQ ID NO.8, which was repeated three times to form SEQ ID NO.9. Then, a His tag was added to the front, and a linker and Foldon short peptide sequence were added to the back to form a fusion protein, the sequence of which is shown in SEQ ID NO.10:

[0076] FKTDLRSSQSLYNWVKSARIYAGQGYTEHQAQEEWNEEETESSASSENIHEKNF IQTIYGTPLKE(SEQ ID NO.8);

[0077] FKTDLRSSSQSLYNWVKSARIYAGQGYTEHQAQEEWNEEETESSASSENIHEKNF IQTIYGTPLKE (first repeat) FKTDLRSSSQSLYNWVKSARIYAGQGYTEHQAQEEWNEEETESSASSENIHEKNFIQTIYGTPLKE (second repeat) FKTDLRSSSQSLYNWVKSA RIYAGQGYTEHQAQEEWNEEETESSASSENIHEKNFIQTIYGTPLKE (third repeat) (SE Q ID NO.9);

[0078] The P30-3 sequence used for expression after construction is:

[0079] MHHHHHHHFKTDLRSSSQSLYNWVKSARIYAGQGYTEHQAQEEWNEEETESSASSENIHEKNFIQTIYGTPLKE (first repeat) FKTDLRSSSQSLYNWVKSARIYAGQGYTEHQAQEEWNEEETESSASSENIHEKNFIQTIYGTPLKE (second repeat) FKTDLRSSSQSLYNWVKSARIYAGQGYTEHQAQEEWNEEETESSASSENIHEKNFIQTIYGTPLKE (third repeat) GGGSGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO. 10);

[0080] 9. P30-4 (replace the Foldon short peptide in the P30-3 sequence with a His tag):

[0081] MHHHHHHHFKTDLRSSSQSLYNWVKSARIYAGQGYTEHQAQEEWNEEETESSASSENIHEKNFIQTIYGTPLKE (first repeat) FKTDLRSSSQSLYNWVKSARIYAGQGYTEHQAQEEWNEEETESSASSENIHEKNFIQTIYGTPLKE (second repeat) FKTDLRSSSQSLYNWVKSARIYAGQGYTEHQAQEEWNEEETESSASSENIHEKNFIQTIYGTPLKE (third repeat) HHHHHHH (SEQ ID NO. 11).

[0082] The African swine fever-positive serum, foot-and-mouth disease virus-positive serum, blue ear virus-positive serum, and classical swine fever virus-positive serum involved in the present invention were purchased from the China Veterinary Drug Administration.

[0083] 1. Expression and purification of recombinant proteins

[0084] Construction of recombinant expression vectors: The recombinant protein gene was His-tagged and artificially synthesized, then inserted into the expression vector pET21a+. The cells were then transformed into Escherichia coli BL21(DE3) and incubated in LB medium without antibiotics at 220 rpm and 37°C for 40 minutes. 100 μL of the bacterial solution was then spread onto an ampicillin-resistant LB plate and incubated inverted at 37°C for 12 hours.

[0085] Recombinant protein expression and purification: Single colonies from the above plates were picked and incubated in LB medium containing ampicillin resistance at 220 rpm and 37°C. When the turbidity reached an OD600 absorbance of approximately 0.6, IPTG was added to a final concentration of 1 mM and induced overnight. The cells were harvested by centrifugation and analyzed by polyacrylamide gel electrophoresis and Coomassie blue staining to visualize the expression of the target protein. The cells were then lysed by sonication and centrifuged at 10,000 rpm and 4°C for 30 minutes. The supernatant was purified using a nickel column according to conventional methods.

[0086] 2. Identification of recombinant protein

[0087] (1) Polyacrylamide gel electrophoresis (SDS electrophoresis): 15% separation gel and 5% stacking gel (SDS electrophoresis kit purchased from Beijing Solebau Technology Co., Ltd., catalog number P1200) were prepared, with a sample load of 5 μg per well; electrophoresis was performed at 80 V for approximately 30 minutes. When the protein sample reached the interface between the separation gel and stacking gel, electrophoresis was switched to 120 V until the bromophenol blue front moved to the bottom of the gel. The electrophoresis was terminated, the gel was removed, and the protein purity was checked by direct staining with Coomassie brilliant blue, or the next step was continued.

[0088] (2) Western blot: After electrophoresis, the gel containing the target protein was cut out at the appropriate position and the protein was transferred to a PVDF membrane (purchased from Thermo Fisher Scientific, Catalog No. 88518) in the traditional way. The transfer conditions were 300 mA current intensity for 1 hour and 10 minutes. Then, the PVDF membrane was removed and placed in TBST containing 5% skim milk powder and blocked at 37°C for 2 hours. The membrane was placed in the primary antibody diluted in 5% skim milk powder (mouse anti-His tag antibody, Anti-6×His Tag antibody, Catalog No. D191001, purchased from Sangon Biotech (Shanghai) Co., Ltd.) at a dilution ratio of 1:3000 and incubated at 4°C with slow shaking overnight. The sections were washed four times with TBST for 5 minutes each time, and HRP-conjugated goat anti-mouse IgG (purchased from Sangon Biotech (Shanghai) Co., Ltd., Catalog No. D110087) diluted in 5% BSA was added at a dilution of 1:7000. The sections were incubated at 37°C for 1 hour. The sections were washed four times with TBST for 5 minutes each time, and ECL color development solution (purchased from Shanghai Qihai Futai Biotechnology Co., Ltd., Catalog No. E002-100) was added. The gels were photographed using a gel imager.

[0089] 3. Preparation of African swine fever virus positive and negative sera

[0090] Piglets 30 days after birth were injected intramuscularly with a plasmid expressing the P30 protein of the African swine fever virus to prepare positive serum. The specific method is to dilute the plasmid with normal saline to a concentration of 200 μg / mL, and inject 1 ml into the muscle behind the ear of each pig at multiple points. Then, boost immunization is performed every 3 weeks for a total of 3 immunizations. On the 10th day after the last injection, blood is collected to separate the positive serum for later use. Negative serum comes from pigs injected only with normal saline. Finally, the serum antibody titer is tested using the "African Swine Fever Virus ELISA Antibody Detection Kit" produced by Beijing Jinnuo Biotech Co., Ltd. (Cat. No.: JN60912).

[0091] 4. Indirect ELISA method

[0092] The coating antigen is a recombinant protein expressed in Escherichia coli at a concentration of 1 μg / mL.

[0093] The coating buffer was 0.1 mol / L pH 8.8 Tris-HCl buffer, which was prepared by adding 12.1 g Tris, double-distilled water to 900 ml, adjusting the pH to 8.8, and then diluting the volume to 1000 mL.

[0094] The blocking solution was 5% skim milk, which was prepared by dissolving 5% skim milk in 0.01 mol / L phosphate buffered saline (PBS) with a pH of 7.2.

[0095] The washing solution was PBST with a pH of 7.2. The preparation method was to take 3.63 g of Na2HPO4·12H2O, 0.24 g of KH2PO4, 8 g of NaCl, 0.2 g of KCl, and 1 mL of Tween-20, add double-distilled water to 1000 mL, and adjust the pH to 7.2.

[0096] The sample diluent was PBST containing 5% BSA (bovine serum albumin). The preparation method was to take 3.63g of Na2HPO4·12H2O, 0.24g of KH2PO4, 8g of NaCl, 0.2g of KCl, and 1mL of Tween-20, add double-distilled water to 1000mL, and adjust the pH to 7.2. Finally, add BSA to a concentration of 5%.

[0097] The secondary antibodies were horseradish peroxidase (HRP)-labeled mouse anti-pig IgG antibody, Beijing Borsi Technology Co., Ltd., catalog number: BHR797, and horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody, Invitrogen, catalog number: 62-6520. The chromogenic substrate was TMB chromogenic solution produced by Sigma, catalog number: 34021.

[0098] The stop solution is 1M H2SO4, which is prepared by taking 54.3mL of 95% concentrated sulfuric acid and adding distilled water to 1000mL to make a H2SO4 solution with a concentration of 1mol / L.

[0099] High-binding polystyrene 96-well plates, brand NUNC, product number 468667MaxiSorp, produced by Thermo Fisher Scientific.

[0100] The specific method is:

[0101] First, the plate antigen was diluted to 1 μg / mL with coating buffer and coated on a 96-well plate (polystyrene plate (NUNC), product number 468667, MaxiSorp; purchased from Thermo Fisher Scientific). 100 μL was added to each well, covered with a sealing film, and placed at 4°C overnight.

[0102] Block, discard the coating solution on the next day, wash once, add 300 μL of 5% skim milk to each well, incubate at 37°C for 2 hours, wash three times, and pat dry;

[0103] Serum samples were added, 100 μL per well, incubated at 37°C for 1 h, and then washed four times according to the above washing method;

[0104] Secondary antibody, horseradish peroxidase (HRP)-labeled anti-IgG antibody against the corresponding species, was added at a dilution of 1:3000, 100 μL was added to each well, incubated at 37°C for 30 min, and washed four times;

[0105] For color development, prepare TMB color development solution according to the instruction manual and add 100 μL to each well of the ELISA plate. After reacting at room temperature in the dark for 10 min, add 50 μL of 1 M H2SO4 solution to each well to terminate the reaction and read the OD450 absorbance value.

[0106] 5. Purification of IgG from standard positive pig serum

[0107] Reagents:

[0108] 25 mM TBS: Dissolve 3.025 g Tris and 4.375 g NaCl in 400 mL of distilled water. Adjust the pH to 7.4 with HCl. Make up to 500 mL and store at 4°C.

[0109] 50 mM Glycine: Dissolve 0.375 g of glycerine in 80 mL of distilled water, adjust the pH to 1.9 with HCl, and dilute to 100 mL. Store at 4°C.

[0110] 1M Tris: Dissolve 12.1 g Tris in 80 mL of distilled water, adjust the pH to 9.0 with HCl, and make up to 100 mL. Store at 4°C.

[0111] Protein A+G Agarose: purchased from Beyotime Biotechnology Co., Ltd., product number P2019-10ml.

[0112] step:

[0113] Step 1: Dilute porcine serum 1:10 with TBS, filter through a 0.22 μM filter, and add it to a Protein A+G Agarose column equilibrated with TBS. Then, wash away impurities in the unbound serum with TBS.

[0114] In the second step, 10 mL of 50 mM Glycine was added dropwise to the Protein A+G Agarose column to elute the IgG, and the eluted liquid was neutralized with Tris (pH 9.0).

[0115] In the third step, a 100 kDa ultrafiltration tube (Millipore Amicon Ultra, product number R1HB04151) was washed once with 4°C pre-cooled deionized water, and then the IgG-containing liquid collected in the second step was added and centrifuged at 4000 rpm at 4°C to concentrate to about 1 mL.

[0116] Step 4: Add PBS buffer and centrifuge under the same conditions to concentrate again. Repeat this step three times. After the final concentration, remove the IgG, measure the concentration, aliquot, and store at -80°C.

[0117] Example 1

[0118] Taking African swine fever virus as an example, design, expression and verification of recombinant protein:

[0119] First, the full-length sequence of P30 protein was added with a signal peptide and a His tag, and then inserted into the pcDNA3.1 plasmid to make the plasmid pcDNA3.1-P30-His. 293T cells were transfected, and immunoblotting was used to verify that the plasmid could express P30 protein ( Figure 2 A). Then, the plasmid pcDNA3.1-P30-His was used to immunize pigs to prepare African swine fever virus positive serum, and the serum of non-immunized pigs was used as negative serum, and the antibody titer was detected using a commercial kit ( Figure 2 B). Prediction of immunogenicity of P30 protein amino acid sequence ( Figure 2 C), the prediction website is http: / / tools.iedb.org / bcell / . Then two different recombinant proteins were designed, named P30-1 and P30-2, respectively. Among them, P30-1 selected the first half of the amino acid sequence of P30 protein, and P30-2 selected the second half ( Figure 2D). The genes encoding P30-1 and P30-2 were inserted into the prokaryotic expression vector pET-21a, expressed in E. coli and purified ( Figure 2 D (Figure below).

[0120] Indirect ELISA assays were used to detect the specificity of recombinant proteins, such as Figure 2 As shown in Figure E, P30-1 can detect some positive sera of African swine fever virus, but also has non-specific binding with negative sera; while P30-2 has no binding ability to both positive and negative sera.

[0121] On this basis, combined with Figure 2 Based on the prediction results of C, several regions with strong immunogenicity in the amino acid sequence of P30 protein were connected in series and repeated three times. A His tag was added in front of the repeated sequence and GGGS (Linker) was used to connect the repeated sequence with a Foldon short peptide to design a recombinant protein named P30-3. At the same time, the Foldon short peptide in the amino acid sequence of P30-3 was replaced with a His tag to design another recombinant protein named P30-4 ( Figure 2 F (above).

[0122] The genes expressing P30-3 and P30-4 were inserted into the prokaryotic expression vector pET-21a, and the recombinant proteins P30-3 and P30-4 were successfully expressed ( Figure 2 F below). Figure 2 As shown in Figure G, the indirect ELISA results indicate that the recombinant protein P30-3 binds significantly more strongly to ASFV-positive serum than P30-4. P30-3 was then further tested by immunoblotting; the P30-3 sample was split into two portions. One portion was loaded with loading buffer and boiled for 10 minutes, followed by addition to the gel wells. The other portion was mixed with loading buffer, then immediately added to the gel wells without boiling and electrophoresis was initiated.

[0123] The experimental results show that ( Figure 2 H), the molecular weight of P30-3 in the sample without boiling depolymerization (undenatured) is about 80kDa, which is three times the molecular weight of the depolymerized (denatured) sample, indicating that P30-3 is a trimer structure. The same indirect ELISA experiment was used to detect the specificity of P30-3, such as Figure 2 As shown in Figure 1, P30-3 can specifically detect positive serum of African swine fever virus, and its nonspecific binding with negative serum is relatively low.

[0124] Example 2. Optimization of antigen coating solution and antigen coating concentration

[0125] 1. Optimize the antigen coating solution: Use PBS, carbonate buffer, and Tris-HCl as the antigen coating solution, respectively. Under the same reaction conditions, use African swine fever virus-positive serum samples purchased from the China Veterinary Drug Administration to detect the effects of different coating solutions on ELISA results.

[0126] See the results Figure 3 A. It was found that when PBS and carbonate buffer were used as antigen coating solution, the binding ability of recombinant protein P30-3 to positive serum was slightly higher than that of Tris-HCl. The P / N value was calculated ( Figure 3 B) shows that Tris-HCl can significantly reduce the binding of the recombinant protein to the antibody (p<0.05). In this example, PBS was used as the coating buffer for the recombinant protein P30-3.

[0127] The formulas of the three antigen coating solutions are as follows:

[0128] 0.01 mol / L pH 7.2 PBS: Dissolve 3.63 g of Na₂HPO₄·12H₂O, 0.24 g of KH₂PO₄, 8 g of NaCl, and 0.2 g of KCl in 900 mL of distilled water, adjust the pH to 7.2, and make up to 1 L.

[0129] 0.05 mol / L pH 9.6 carbonate buffer: Dissolve 1.59 g of Na2CO3 and 2.93 g of NaHCO3 in 900 mL of distilled water, adjust the pH to 9.6, and dilute to 1 L.

[0130] 0.1 mol / L pH 8.8 Tris-HCl: Add 12.1 g Tris, dissolve in 900 mL distilled water, adjust the pH to 8.8, and then dilute to 1 L.

[0131] 2. Optimize the antigen coating concentration: Dilute P30-3 with PBS to five concentrations of 0.25, 0.5, 1, 2, and 4 μg / mL for coating, and measure the effect of different antigen concentrations on ELISA test results.

[0132] The results showed that the OD450 value gradually increased with the increase of the protein concentration ( Figure 3 C), but when the plate protein concentration is greater than 0.5 μg / mL, the P / N value is basically stable ( Figure 3 D) Taking into account the long-term storage stability of the product, 1 μg / mL was determined as the coating concentration of the coating protein.

[0133] Example 3 An indirect ELISA kit for detecting African swine fever virus P30 antibodies

[0134] An indirect ELISA kit for detecting African swine fever virus P30 antibodies, comprising a coating plate coated with an antigen, a coating buffer, a washing solution, a sample diluent, an enzyme-labeled secondary antibody, a color developing solution, a stop solution, a negative control, and a positive control;

[0135] The coating antigen is P30-3, the coating concentration is 1 μg / mL; the coating buffer is 0.01 mol / L pH7.2 PBS solution; the blocking solution is 5% skim milk; the washing solution is PBST at pH 7.2; the sample diluent is PBST containing 5% BSA (bovine serum albumin); the enzyme-labeled secondary antibody is horseradish peroxidase (HRP)-labeled mouse anti-swine IgG antibody; the color developing solution is TMB color developing solution; the stop solution is 1M H2SO4; the negative control is normal pig serum that is not immunized with African swine fever virus P30 protein; the positive control is normal pig serum that is immunized with African swine fever virus P30 protein.

[0136] The preparation process of the antigen-coated plate is as follows:

[0137] 1. Dilute the coating antigen P30-3 to 1 μg / mL with coating buffer and coat a 96-well plate (polystyrene plate (NUNC), product number 468667, MaxiSorp; purchased from Thermo Fisher Scientific). Add 100 μL per well, cover with sealing film, and incubate at 4°C overnight.

[0138] 2. Block the plate. Discard the coating solution on the next day. Wash once, add 300 μL of 5% skim milk to each well, incubate at 37°C for 2 hours, wash three times, and pat dry to obtain the antigen-coated plate.

[0139] The method of using the above-mentioned indirect ELISA kit is as follows:

[0140] 1. Add serum sample, add 100 μL to each well, incubate at 37°C for 1 hour, and then wash 4 times according to the above washing method;

[0141] 2. Add secondary antibody, horseradish peroxidase (HRP)-labeled anti-IgG antibody of the corresponding species at a dilution of 1:3000, add 100 μL to each well, incubate at 37°C for 30 minutes, and wash four times;

[0142] 3. Color development: Prepare TMB color development solution according to the instructions and add 100 μL to each well of the ELISA plate. After reacting at room temperature in the dark for 10 minutes, add 50 μL of 1M H2SO4 solution to each well to terminate the reaction and read the OD450 absorbance value.

[0143] Example 3. Specificity, sensitivity, and stability of the indirect ELISA method

[0144] 1. First, the specificity of the indirect ELISA method in Example 3 was tested using positive pig sera against different viruses.

[0145] The results are as follows Figure 4 As shown in A, this detection method can only identify African swine fever virus-positive serum, and has no nonspecific reaction with positive serum of foot-and-mouth disease virus, classical swine fever virus, and porcine blue ear virus, as well as negative serum of African swine fever virus.

[0146] 2. Indirect ELISA was used to detect African swine fever virus positive serum purchased from the China Veterinary Drug Administration. The results showed that antibodies in virus positive serum diluted 32,000 times could be detected ( Figure 4 B).

[0147] 3. The detection method of Example 3 was compared with two other commercial kits produced by different manufacturers. Among them, "Jinnuo" is the "African Swine Fever Virus ELISA Antibody Detection Kit" (Article No.: JN60912) produced by Beijing Jinnuo Baitai Biotechnology Co., Ltd., and "Yisenbao" is the "African Swine Fever Virus Indirect ELISA Antibody Detection Kit" (Article No.: FZE024) produced by Beijing Yisenbao Biotechnology Co., Ltd.

[0148] The results showed that the sensitivity of the detection method of Example 3 in identifying P30 antibodies was much higher than that of the products of the other two manufacturers, and the OD450 absorbance value was more than twice that of the commercial kits tested ( Figure 4 C).

[0149] 4. Block the P30-3 protein-coated ELISA plate with 5% skim milk and store it at 37°C. Then, take out the ELISA plate at different time points and detect it using the indirect ELISA method.

[0150] The results showed that the ELISA plate was stored at 37°C for 14 days without significant changes in the test results ( Figure 4 D).

[0151] The above specific embodiments provide a detailed description of the present invention. However, the present invention is not limited to the above embodiments. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with each other unless there is a conflict.

Claims

1. A recombinant protein of African swine fever virus P30, characterized in that The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

8.

2. A polymer, characterized in that The multimer is formed by repeatedly connecting the recombinant protein according to claim 1 in series.

3. The multimer according to claim 2, characterized in that Repeat 2 to 4 times.

4. The multimer according to claim 2 or 3, characterized in that The amino acid sequence of the polymer is shown in SEQ ID NO.

9.

5. A fusion protein, characterized in that The fusion protein comprises: (1) Foldon peptide and the recombinant protein according to claim 1; or (2) A Foldon short peptide and a multimer according to any one of claims 2 to 4.

6. The fusion protein according to claim 5, characterized in that The fusion protein further comprises a linker sequence and / or a tag sequence.

7. The fusion protein according to any one of claims 5 to 6, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO.

10.

8. A biological material related to the recombinant protein of claim 1, the multimer of any one of claims 2 to 4, or the fusion protein of any one of claims 5 to 7; the biological material comprises any one of B1) to B4); B1) a nucleic acid molecule encoding the recombinant protein of claim 1, the multimer of any one of claims 2 to 4, or the fusion protein of any one of claims 5 to 7; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) A recombinant cell containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

9. Use of the African swine fever virus P30 recombinant protein according to claim 1, the multimer according to any one of claims 2 to 4, the fusion protein according to any one of claims 5 to 7, or the biological material according to claim 8 in detecting African swine fever virus P30 antibodies for non-diagnostic purposes or in preparing a kit for detecting African swine fever virus P30 antibodies.

10. A kit comprising a coated plate coated with the fusion protein according to any one of claims 5 to 7; the kit further comprising at least one of a coating buffer, a washing solution, a sample diluent, an enzyme-labeled secondary antibody, a color development solution, a stop solution, a negative control, and a positive control.

11. The kit according to claim 10, characterized in that The coating concentration is 0.2-4 μg / mL.

12. The kit according to claim 10, characterized in that The coating buffer is a 0.005-0.015 mol / L pH 7-7.5 PBS solution.

13. A method for detecting African swine fever virus P30 antibodies for non-diagnostic purposes, comprising using the kit according to any one of claims 10 to 12 to detect African swine fever virus P30 antibodies.

14. The method according to claim 13, characterized in that The method comprises the following steps: taking a serum sample to be tested and adding it to a plate coated with a fusion protein after being blocked with a blocking solution, adding an enzyme-labeled secondary antibody, incubating, adding a color developing solution for color development, adding a stop solution, and then measuring the absorbance value, and judging whether African swine fever virus P30 antibodies are present in the sample according to the absorbance value.

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