An African swine fever virus antibody detection method and its application

Abstract

The present invention relates to the field of diagnostic detection of animal virus antibodies, and specifically provides a method for detecting African swine fever virus antibodies and its application. First, the present invention screens the dominant epitope regions of the structural proteins p30, p54, and p72 of African swine fever, tandemly expresses and purifies the three dominant epitope regions using an Escherichia coli expression system, and uses the purified protein as a coating antigen to establish an indirect ELISA method for detecting clinical swine sera. The present invention makes up for the deficiencies of existing domestic and foreign detection methods, and will be more conducive to the monitoring of antibody levels and prevalence generated by ASFV vaccine immunization and wild virus infection.

Description

Technical Field

[0001] The present invention relates to the technical field of animal virus antibody detection, and particularly relates to a method for detecting African swine fever virus antibody and its application. Background Art

[0002] African swine fever (English name: Infection with African swine fever virus, abbreviation: ASF) is an acute, hemorrhagic, and highly contagious disease caused by the African swine fever virus (English name: African Swine fever virus, abbreviation: ASFV) infecting domestic pigs and various wild boars (such as African wild boars, European wild boars, etc.). The World Organization for Animal Health (OIE) lists it as a notifiable animal disease, and this disease is also a type of animal epidemic that China focuses on preventing. Its characteristic is a short disease process, with the mortality rate of the most acute and acute infections reaching up to 100%. The clinical manifestations are fever (up to 40 - 42 °C), increased heart rate, difficulty breathing, partial coughing, serous or mucopurulent secretions in the eyes and nose, cyanosis of the skin, obvious bleeding in the lymph nodes, kidneys, and gastrointestinal mucosa. The clinical symptoms of African swine fever are similar to those of classical swine fever, and it can only be diagnosed by laboratory monitoring. Currently, countries are actively conducting research and development of effective vaccines, and there are also relevant reports on the development of vaccines with good effects.

[0003] Due to the absence of commercial vaccines and effective drugs at present, the prevention and control of ASF mainly rely on regular detection, and once detected, immediate culling is carried out. Detection at the serological level is essential in routine monitoring. The ELISA method is a classical antibody detection method. Most of the current ELISA methods use single proteins such as ASFV structural proteins p30 and p72 for coating. Compared with the relatively large genome of ASFV, there are few effective antigenic epitopes, which is likely to cause false negative results. At the same time, the antibody level is an important indicator for evaluating the vaccine effect, and the research and development of vaccines require corresponding efficient antibody detection methods.

[0004] At present, there are also reports that the combination of three proteins of African swine fever virus p72, p30 and p54 or antigenic epitope polypeptides of three proteins is used as an antigen to detect African swine fever virus antibodies by ELISA (CN114088940A, CN110618279A). However, when the three proteins or antigenic epitope polypeptides of three proteins are used as antigens, most of the viral antigens used for coating are obtained by using an Escherichia coli expression system. The three proteins need to be expressed and purified three times when they are combined for coating, which will cause more Escherichia coli proteins to exist in the purified proteins. The pigs themselves will be exposed to a large number of different Escherichia coli during their growth and produce anti-Escherichia coli protein antibodies, which will cause a high background value or even a false positive when clinically detecting pig serum. Using the artificially synthesized antigenic epitope polypeptides of the three proteins as coating antigens cannot ensure that the coated polypeptides can induce the body to produce antibodies. At the same time, the synthesis cost of the polypeptides is high, which is not suitable for the detection of a large number of serum samples routinely used in clinical practice. In addition, for truncated proteins, even if different regions of the same protein are truncated, the detection antigens formed are different, and the detection effect of the final ELISA test kit is also different. Therefore, it is still necessary to further develop highly sensitive and specific ASFV antibody detection methods in clinical practice. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a method for detecting antibodies to African swine fever virus and its application. The present invention first screens the dominant epitope regions on the p30, p54, and p72 proteins of the African swine fever virus, clones the nucleotide sequences corresponding to these dominant epitope regions in series into one gene using molecular biological techniques, and to ensure the structural integrity of each epitope, a 15-amino acid linker is added between each two epitopes to express the p30-54-72 recombinant protein, and then the p30-54-72 recombinant protein is used as a coating antigen to establish an ELISA method for detecting antibodies to African swine fever virus. The present invention greatly improves the sensitivity and specificity of the detection on the basis of removing redundant sequences on the antigen as much as possible without missing effective antigenic epitopes.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a p30-54-72 recombinant protein is provided, the amino acid sequence of which is shown in SEQ ID NO.1; specifically, as follows:

[0008] ETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVTKDSKLYMLAQKTVQHIGGGGSGGGGSQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLGGGGSGGGGSRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTMSAIENLRQRNTYGGGGSGGGGSDGKADKIILAQDLLNSRISNIKNVNKSYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNKLGGGGSGGGGSRNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYYQGGGGSGGGGSHTNNNHHDEKLMSALKWPIEYMFIGLKPTWNISDQNPHQHRDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSSISD。

[0009] In the second aspect of the present invention, there is provided a gene encoding the above-mentioned p30-54-72 recombinant protein, and its nucleotide sequence is shown in SEQ ID NO.2; specifically as follows:

[0010]

[0011]

[0012] In the third aspect of the present invention, there is provided a recombinant expression vector or a genetically engineered bacterium containing the gene encoding the above-mentioned p30-54-72 recombinant protein.

[0013] In the fourth aspect of the present invention, there is provided the application of the p30-54-72 recombinant protein, the gene encoding the p30-54-72 recombinant protein, the recombinant expression vector or the genetically engineered bacterium containing the gene encoding the p30-54-72 recombinant protein in the preparation of a kit for detecting African swine fever virus antibodies.

[0014] Preferably, the kit is an ELISA detection kit.

[0015] In the fifth aspect of the present invention, an ELISA kit for detecting African swine fever virus antibodies is provided, and the ELISA kit uses p30-54-72 recombinant protein as the coating antigen.

[0016] Preferably, in the ELISA kit, the coating amount of p30-54-72 recombinant protein is 300-500 ng / well.

[0017] Furthermore, the ELISA kit further includes: enzyme-labeled antibody, sample diluent, washing solution, negative control serum, positive control serum, TMB chromogenic solution and termination solution.

[0018] More preferably, the enzyme-labeled antibody is a horseradish peroxidase-labeled goat anti-pig antibody.

[0019] In the sixth aspect of the present invention, an ELISA detection method for African swine fever virus antibodies is provided, including the following steps:

[0020] (1) Coating: Using p30-54-72 recombinant protein as the coating antigen, coat overnight at 4 °C in coating buffer; the coating buffer contains 0.356 g / L NaH2PO4, 2.772 g / L Na2HPO4·12H2O, 8.5 g / L NaCl, pH = 7.2;

[0021] (2) Washing: Wash with phosphate buffer (PBST) with a pH of 7.2 containing 0.5% (v / v) Tween-20, discard after gently shaking for 1 min, and repeat 5 times;

[0022] (3) Blocking: Block with a blocking solution containing 2.5% skim milk powder in PBST buffer with a pH of 7.2, incubate at 37 °C for 1 h;

[0023] (4) Washing: Wash with phosphate buffer (PBST) with a pH of 7.2 containing 0.5% (v / v) Tween-20, discard after gently shaking for 1 min, and repeat 5 times;

[0024] (5) Serum reaction conditions: Add the serum dilution mixture to be tested (serum:PBS = 1:40) to each well, incubate at 37 °C for 1 h, then drain, and wash with PBST;

[0025] (6) Adding enzyme-labeled antibody: Dilute the horseradish peroxidase-labeled goat anti-pig antibody 1:5000 with PBST containing 2.5% skim milk powder with a pH of 7.2, add 100 μl to each well, incubate at 37 °C for 1 h, then drain, and wash with PBST;

[0026] (7) Substrate chromogenic reaction: Add 100 μL of TMB substrate chromogenic solution to each well, and develop color at room temperature in the dark for 15 min;

[0027] (8) Termination reaction: Add 50 μL of termination solution to each well to terminate the color reaction; read the data at an absorbance of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0028] (9) Determination of the positive and negative cut-off value: According to the formula: positive and negative cut-off value = OD of negative samples 450 average value + standard deviation × 3SD, the positive and negative cut-off value is obtained; when OD 450 is above 0.371, it is determined as positive.

[0029] Advantages of the present invention:

[0030] The present invention first expresses the antigens of p30, p54, and p72 of African swine fever virus (ASFV) using a prokaryotic expression system, and then uses antigenic epitope prediction software to identify all possible antigenic epitope regions of these three proteins, and performs truncated expression respectively. Among them, p30 is divided into 3 segments, p54 is divided into 1 segment, and p72 is divided into 4 segments. The purified full-length p30, p54, and p72 proteins are used to immunize rabbits and mice respectively to obtain rabbit polyclonal antibodies and mouse polyclonal antibodies. Western blot is used to detect and screen the epitopes of the truncated proteins using clinical porcine positive sera (the ASFV positive and negative sera used in the present invention are all provided by the China Animal Health and Epidemiology Center), rabbit polyclonal antibodies, and mouse polyclonal antibodies. Using sera from three species is to avoid false negatives. Porcine sera are ASFV naturally infected sera, but the immune responses to different epitopes vary at different infection times. Sera from rabbits and mice are used to screen for possible epitopes to avoid omission of antigenic epitopes. The purpose of using sera from different species is that the immune responses of different species to the same antigen also vary. Detection in sera from two different species, rabbits and mice, indicates the authenticity of the antigenic epitope.

[0031] Then, the gene sequences corresponding to the regions of 78 - 132 aa and 134 - 174 aa in p30, 54 - 124 aa in p54, and 12 - 89 aa, 139 - 324 aa, and 445 - 524 aa in p72 obtained by screening are concatenated, and a glycine linker (GGGGSGGGGS) is added between each epitope. The ligated gene is ligated into a prokaryotic expression vector for prokaryotic expression and purification. An indirect ELISA using the p30 - 54 - 72 recombinant protein as the coating antigen is established, and the detection results are compared with a commercial ASFV antibody detection reagent using p30 and p72 as the main coating proteins and an ELISA established in this laboratory using p54 protein as the antigen. The results show that the ELISA using p30 - 54 - 72 as the coating antigen has high specificity. The present invention makes up for the deficiencies of existing domestic and foreign detection methods, provides a more effective method for the prevention and control detection of ASF, and provides a material basis for the evaluation of the effectiveness of future vaccines. Description of the Drawings

[0032] Figure 1 : Verification of the expression of African swine fever virus p30, p54, and p72 proteins by SDS-PAGE; A: M: Protein Marker; 1: Supernatant of p30 protein inclusion bodies before passing through the column; 2: Supernatant of p30 protein inclusion bodies after passing through the column; 3: Washing solution of p30 protein inclusion bodies; 4: Eluted and purified p30 protein - 1; 5: Eluted and purified p30 protein - 2; 6: Eluted and purified p30 protein - 3; 7: Supernatant of p54 protein before passing through the column; 8: Supernatant of p54 protein after passing through the column; 9: Washing solution of p54 protein supernatant; 10: Eluted and purified p54 protein - 1; 11: Eluted and purified p54 protein - 2; 12: Eluted and purified p54 protein - 3;

[0033] B: M: Protein Marker; 1: Supernatant of p72 inclusion bodies before passing through the column; 2: Supernatant of p72 inclusion bodies after passing through the column; 3: Washing solution of p72 inclusion bodies; 4: Eluted and purified p72 protein - 1; 5: Eluted and purified p72 protein - 2; 6: Eluted and purified p72 protein - 3.

[0034] Figure 2 : Western blot electrophoresis diagram of the recombinant proteins of African swine fever virus p30, p54, and p72 proteins; In the figure: M: Marker, 1: p30 protein; 2: p54 protein; 3: p72 protein.

[0035] Figure 3: Screening for the dominant epitope regions of p30, p54, and p72 using Western blot; In the figure, A: Screening for the dominant epitope regions of p30, p54, and p72 using porcine positive serum; 1 - 3 are respectively: p30 1 - 66aa, 78 - 132aa, and 134 - 174aa; 4: p54 54 - 124aa; 5 - 8 are respectively: p72 12 - 89aa, 139 - 324aa, 445 - 524aa, and 552 - 647aa; Figure B: Screening for the dominant epitope regions of p30, p54, and p72 using polyclonal antibodies of p30, p54, and p72 mice; 1 - 4 are respectively: full - length p30, p30 1 - 66aa, 78 - 132aa, and 134 - 174aa; 5 - 6: full - length p54, p54 54 - 124aa; 7 - 10 are respectively: p72 12 - 89aa, 139 - 324aa, 445 - 524aa, and 552 - 647aa; Figure C: Screening for the dominant epitope regions of p30, p54, and p72 using polyclonal antibodies of p30, p54, and p72 rabbits; 1 - 4 are respectively: full - length p30, p30 1 - 66aa, 78 - 132aa, and 134 - 174aa; 5 - 6: full - length p54, p54 54 - 124aa; 7 - 12 are respectively: full - length p72, p72 12 - 89aa, p72 12 - 89aa, 139 - 324aa, 445 - 524aa, and 552 - 647aa.

[0036] Figure 4 : SDS - PAGE verification of the expression of p30 - 54 - 72 recombinant protein; A: M: Protein Marker; 1: Supernatant of p30 - 54 - 72 recombinant protein inclusion bodies before passing through the column; 2: Supernatant of p30 - 54 - 72 recombinant protein inclusion bodies after passing through the column; 3: Washing solution of p30 - 54 - 72 recombinant protein inclusion bodies; 4: Eluted and purified p30 - 54 - 72 recombinant protein - 1; 5: Eluted and purified p30 - 54 - 72 recombinant protein - 2; 6: Eluted and purified p30 - 54 - 72 recombinant protein - 3.

[0037] Figure 5 : Western - blot verification diagram of the purification of p30 - 54 - 72 recombinant protein; M: Protein Marker; 1: Recombinant p30 - 54 - 72 protein. Detailed implementation manners

[0038] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0039] As mentioned above, African swine fever is an acute, hemorrhagic, and highly contagious disease. There is currently no commercial vaccine or effective drug. The prevention and control of ASF mainly relies on regular testing, and once found, the pigs are immediately slaughtered. Therefore, there is an urgent need to develop highly sensitive and specific ASFV antibody detection methods in clinical practice.

[0040] Based on this, the present invention uses clinical pig positive serum, rabbit polyclonal antibody and mouse polyclonal antibody to screen and verify the dominant epitope regions of p30, p54 and p72, and then uses molecular biological technology to clone the nucleotide sequences corresponding to these dominant epitope regions into one gene in series, express them into one protein, and purify them to establish an ELISA method as a coating antigen. The advantage of this method is that it does not miss effective antigen epitopes on the basis of removing redundant sequences on the antigen as much as possible, which greatly improves the sensitivity of detection. In addition, only one protein needs to be purified in this method, which greatly reduces the probability of the occurrence of foreign proteins, reduces the background of serum, and improves the specificity of the detection method, thus proposing the present invention.

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0042] The experimental materials used in the examples of the present invention but not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels.

[0043] If no specific experimental conditions and methods are specified in the examples of the present invention, conventional conditions are generally used, such as J. Sambrook et al., ed., Molecular Cloning Experiment Guide (3rd edition), Science Press, 2002; DL Spector et al., ed., Cell Experiment Guide, Science Press, 2001; or the conditions recommended by the manufacturer.

[0044] Example 1: Prokaryotic expression of African swine fever virus p30, p54, p72 proteins and truncated proteins thereof

[0045] (1) Expression of p30, p54, and p72 proteins:

[0046] The genes encoding p30, p54, and p72 proteins (the nucleotide sequence of the gene encoding p30 protein is shown in SEQ ID NO.3, the nucleotide sequence of the gene encoding p54 protein is shown in SEQ ID NO.4, and the nucleotide sequence of the gene encoding p72 protein is shown in SEQ ID NO.5) were respectively connected to the pET-28a plasmid to construct recombinant expression plasmids pET28a-p30, pET28a-p54, and pET28a-p72.

[0047] The recombinant expression plasmids pET28a-p30, pET28a-p54, and pET28a-p72 were transformed into the competent cells of the expression strain BL21(DE3)pLysS. The recombinant transformants were screened using kanamycin resistance. Single colonies were picked and activated overnight in LB medium, then transferred to fresh LB medium containing 50 mg / mL kanamycin at a ratio of 1:1000 and cultured until the OD 600 reached approximately 0.4 - 0.6, and then IPTG with a final concentration of 1 mM was added. The culture was shaken at 220 rpm and 37°C for 4.5 h for induction. The cells were collected by centrifugation at 3000 r for 5 min at 4°C, resuspended in PBS, sonicated, and centrifuged at 12000 rpm for 15 min at 4°C. The supernatant and precipitate were collected for SDS-PAGE identification.

[0048] The results were as Figure 1 shown. A specific band appeared at 31 KDa for p30; a specific band appeared at 20 KDa for p54; a specific band appeared at 70 KDa for p72, indicating that the target proteins were highly expressed.

[0049] (2) Purification of p30, p54, and p72 proteins:

[0050] After the competent cells of the expression strain BL21(DE3)pLysS transformed with pET28a-p30, pET28a-p54, and pET28a-p72 were cultured in large quantities, they were sonicated and the supernatant was collected by centrifugation. The proteins were purified using a nickel column purification kit from Nanjing Genscript Biotech Corporation under non-denaturing conditions.

[0051] (3) Identification of the purified proteins:

[0052] The purified p30, p54, and p72 proteins were subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. Using the His-tag mouse antibody as the primary antibody and the HRP-labeled goat anti-mouse IgG antibody as the secondary antibody, western blot verification was performed.

[0053] The results were as Figure 2 shown, indicating that highly pure p30, p54, and p72 proteins were obtained.

[0054] In this example, the expression of p30, p54, and p72 proteins was mainly used to immunize mice and rabbits to prepare polyclonal antibodies for antigen epitope screening.

[0055] Use antigen epitope prediction software to predict all possible antigen epitope regions of p30, p54, and p72 proteins, and perform truncated expression separately. Among them, the p72 protein is divided into 4 segments, namely: 12 - 89aa, 139 - 324aa, 445 - 524aa, and 552 - 647aa; p30 is divided into 3 segments, namely: 1 - 66aa, 78 - 132aa, and 134 - 174aa; p54 is divided into 1 segment, which is 54 - 124aa.

[0056] Example 2: Screening of Dominant Antigen Epitope Regions

[0057] 1. Preparation of mouse polyclonal antibodies:

[0058] (1) Use 6 - 7 - week - old female BALB / C mice as experimental animals, and immunize the mice with the p30, p54, and p72 proteins prepared in Example 1 as antigens. Calculate the concentration of the protein. The calculated concentration is mixed with Freund's adjuvant at a ratio of 1:1 by volume of 100 μg / protein per mouse using a medical three - way tube for emulsification. When emulsification is complete, drop a drop on deionized water. If the emulsified protein does not disperse for a long time, it indicates that emulsification is complete.

[0059] (2) For the second and third immunizations, the protein is mixed with incomplete Freund's adjuvant at a ratio of 1:1. The concentration of the protein for each immunization under the abdomen of the mice is 100 μg / mouse. The time interval between each immunization is 14 days. 14 days after the third immunization interval, collect blood from the tail vein of all immunized mice and detect the serum antibody level.

[0060] (3) If the antibody level is low, a fourth booster immunization can be carried out, and the serum antibody level is detected again after a 14 - day interval; if the antibody level meets the requirements, collect blood from the heart of the mice 3 days after detection. At the same time, centrifuge the collected blood at 6,000 rpm for 5 minutes, take the supernatant and collect it into a 1.5 mL centrifuge tube, and store it in a - 80 °C refrigerator. The collected serum is the mouse polyclonal antibody.

[0061] 2. Preparation of rabbit polyclonal antibodies:

[0062] (1) Use six - to seven - week - old female New Zealand white rabbits as experimental animals, and immunize the rabbits with the p30, p54, and p72 proteins prepared in Example 1 as antigens. The protein is mixed with Freund's adjuvant at a ratio of 1:1 by volume of 1,000 ng / protein per rabbit using a medical three - way tube for emulsification. When emulsification is complete, drop a drop on deionized water. If the emulsified protein does not disperse for a long time, it indicates that emulsification is complete.

[0063] (2) Blood was collected from the marginal ear vein of rabbits during the first immunization as the negative control for subsequent detection. During the first immunization, the protein was mixed with complete Freund's adjuvant at a ratio of 1:1; the second immunization was carried out 21 days later, and the third immunization was carried out 14 days after the second immunization. For the second and third immunizations, the protein was mixed with incomplete Freund's adjuvant at a ratio of 1:1. Multiple subcutaneous injections were performed.

[0064] (3) All immunized rabbits were bled from the marginal ear vein 14 days after the third immunization, and the serum antibody level was detected. If the antibody level is low, a fourth booster immunization can be carried out, and the serum antibody level was detected again 14 days later; if the antibody level meets the requirements, the rabbits were fixed in a supine position 3 days after the detection and then bled from the heart. At the same time, the collected blood was centrifuged at 6,000 rpm for 5 minutes, and the supernatant after centrifugation was collected into a 1.5 mL centrifuge tube and stored in a -80 °C refrigerator. The collected serum is the polyclonal antibody of the rabbit.

[0065] 3. Screening of the dominant antigenic epitope region:

[0066] (1) The p30, p54, and p72 proteins were truncated and expressed, and the corresponding truncated expressed proteins (p72 12-89aa, p72 139-324aa, p72 445-524aa, p72 552-647aa, p30 1-66aa, p30 78-132aa, p30 134-174aa, and p54 54-124aa) were purified.

[0067] (2) Verification was carried out by western blot. The prepared polyclonal antibodies of mice and rabbits were diluted 1:300 as the primary antibody, and HRP-goat anti-mouse / HRP-goat anti-rabbit diluted 1:10,000 was used as the secondary antibody for verification.

[0068] At the same time, porcine positive serum was used as the primary antibody, and HRP-goat anti-mouse / HRP-goat anti-rabbit diluted 1:10,000 / HRP-goat anti-pig diluted 1:5000 was used as the secondary antibody for verification.

[0069] The results are as Figure 3 shown. The results show that: through the verification of mouse polyclonal antibody and porcine positive serum, the dominant epitope regions of p30 can be screened out as 78-132aa and 134-174aa; the dominant epitope region of p54 is 54-124aa; the dominant epitope regions of p72 are 12-89aa, 139-324aa, and 445-524aa.

[0070] In this example, sera from three species were used to screen for the dominant antigenic epitope regions to avoid false negatives of antigenic epitopes. The porcine serum was ASFV natural infection serum, but the immune responses to different epitopes varied at different infection times. Polyclonal antibodies can recognize multiple epitopes of the same antigen. Therefore, in immunoassays, they can recognize more antigens and are less affected by antigen conformational changes. Sera from rabbits and mice were used to rescreen for possible epitopes to avoid missing antigenic epitopes. The purpose of using sera from different species is that the immune responses of different species to the same antigen also vary. Detection in sera from two different species, rabbits and mice, indicates the authenticity of the antigenic epitope.

[0071] Example 3: Tandem expression of the dominant epitope region

[0072] 1. Tandem of the antigenic dominant epitope regions of p30, p54, and p72:

[0073] Using genetic engineering techniques, the antigenic dominant epitope regions of p30, p54, and p72 screened in Example 2 were tandemly linked, with a glycine linker added between each epitope sequence to prevent steric hindrance between epitopes. The ligated gene was ligated into a prokaryotic expression vector for prokaryotic expression and purification to obtain the p30-54-72 recombinant protein. The specific process is as follows:

[0074] (1) First, the two dominant epitope regions of p30 (78-132aa and 134-174aa) were tandemly linked:

[0075] Related primers:

[0076] p30(78-132aa)-F: 5’-ccgaattcgagctccgtcgaGAGACCGAAAGCAGCGCG-3’(SEQ ID NO.6)

[0077] p30(78-132aa)-R: 5’-tgatcctccacctcctgatccacctccaccGATGTGTTGAACGGTTTTCTGC-3’(SEQ ID NO.7)

[0078] p30(134-174aa)-F: 5’-gatcaggaggtggaggatcaCAGTATGGCAAGGCGCCG-3’(SEQ ID NO.8)

[0079] p30(134-174aa)-R: 5’-agaaccaccgccacccgagccgccaccgccCAGTTTGATAACCATCAGAC-3’(SEQ ID NO.9)

[0080] The nucleotide sequence after tandem of two dominant epitope regions of p30 is shown in SEQ ID NO.10, as follows:

[0081]

[0082] (2) Then tandem the two dominant epitope regions of p30 with the dominant epitope region of p54 protein:

[0083] Primers involved:

[0084] p54(54-124aa)-F: 5’-ggcggtggcggctcgggtggcggtggttctCGTCCGGCGACCAACCGTC-3’ (SEQ ID NO.11)

[0085] p54(54-124aa)-R: 5’-cgatccgcctccaccggaacctccgcctccATAGGTGTTACGTTGACGC-3’ (SEQ ID NO.12)

[0086] p30(78-132aa)-F: 5’-ccgaattcgagctccgtcgaGAGACCGAAAGCAGCGCG-3’ (SEQ IDNO.6)

[0087] The nucleotide sequence after tandem of the two dominant epitope regions of p30 with the dominant epitope region of p54 protein is shown in SEQ ID NO.13, as follows:

[0088]

[0089]

[0090] (3) Finally, tandem the two dominant epitope regions of p30, the dominant epitope region of p54 protein with the dominant epitope of p72:

[0091] Primers involved:

[0092] p72(12-89aa)-F: 5’-ggaggcggaggttccggtggaggcggatcgGATGGGAAGGCCGACAAG-3’ (SEQ ID NO.14)

[0093] p72(12-89aa)-R: 5’-ggagcctccgccgccagatccgcctcccccAAGCTTGTTTCCCAAGGTG-3’ (SEQ ID NO.15)

[0094] p72(139 - 324aa)-F: 5’-ctggcggcggaggctccCGCAACGGATATGACTGGGA-3’(SEQ ID NO.16)

[0095] p72(139 - 324aa)-R: 5’-agaaccaccgccacccgagccgccaccgccCTGATAGTATTTAGGGGTTTG-3’

[0096] (SEQ ID NO.17)

[0097] p72(445 - 524aa)-F: 5’-cgggtggcggtggttctCACACCAACAATAACCACCAC-3’(SEQ ID NO.18)

[0098] p72(445 - 524aa)-R: 5’-cgagtgcggccgcaagcttgATCCGATATAGATGAACATGCGTC-3’(SEQ ID NO.19)

[0099] p30(78 - 132aa)-F: 5’-ccgaattcgagctccgtcgaGAGACCGAAAGCAGCGCG-3’(SEQ ID NO.6)

[0100] The nucleotide sequence after concatenating the two dominant epitope regions of p30, the dominant epitope region of p54 protein and the dominant epitope of p72 (p30 - 54 - 72) is shown in SEQ ID NO.2.

[0101] (4) Amplification was carried out by the method of homologous recombination. The nucleotide sequence after concatenating the dominant epitope region of p30, the dominant epitope region of p54 protein and the dominant epitope of p72 was ligated with the pET28a empty vector. The primer design of pET28a vector is as follows.

[0102] pET28a - F: 5’-CAAGCTTGCGGCCGCACT-3’(SEQ ID NO.20)

[0103] pET28a - R: 5’-TCGACGGAGCTCGAATTCG-3’(SEQ ID NO.21)

[0104] Homologous recombination is a method of gene cloning. That is, the dominant epitope sequences of p30, p54, and p72 are designed with primers, and the primers are equipped with homologous arms to the pET28a vector. The vector and the dominant epitope sequences of p30, p54, and p72 are respectively amplified by PCR. After nucleic acid gel recovery, they are recombined under the action of homologous recombinase to form a complete recombinant plasmid. The recombinant expression plasmid pET28a-p30-54-72 is constructed by homologous recombination.

[0105] 2. Induced expression of p30-54-72 recombinant protein

[0106] The recombinant expression plasmid pET28a-p30-p54-p72 was transformed into the competent cells of expression bacterium BL21(DE3)pLysS. The recombinant transformants were screened using kanamycin resistance. A single colony was picked and activated overnight in LB medium, and then transferred to fresh LB medium containing kanamycin resistance at a ratio of 1:1000 (volume ratio). The culture was carried out until the OD 600 was about 0.4 - 0.6, then IPTG with a final concentration of 1 mM was added, and the culture was shaken at 220 rpm and 37 °C for 4.5 h for induction.

[0107] The bacterial cells before and after induction were collected. The induced bacterial cells were sonicated (power 200 w, sonication for 2 s, intermittent for 4 s, about 15 times in total), and centrifuged at 12000 r / min for 15 min to collect the supernatant; the precipitate was resuspended with 50 μL of LE buffer and the inclusion bodies were dissolved at 4 °C overnight. The dissolved inclusion bodies were centrifuged at 12000 r / min for 20 min to collect the supernatant, and the expression was detected by SDS-PAGE.

[0108] The results are as Figure 4 shown. The results showed that under these conditions, the p30-54-72 recombinant protein was successfully expressed.

[0109] Finally, the bacterial cells were transferred into 400 ml of medium with K + resistance at a ratio of 1:100 for large-scale shaking culture of the bacterial cells.

[0110] 3. Purification of p30-54-72 recombinant protein

[0111] The induced expression p30-54-72 recombinant protein was purified using the nickel column purification kit from Nanjing Genscript Biotech Co., Ltd. The purified protein was verified by SDS-PAGE, and the results are as Figure 5 shown.

[0112] The results showed that: highly purified p30-54-72 recombinant protein was obtained. The amino acid sequence of the p30-54-72 recombinant protein is shown in SEQ ID NO.1.

[0113] Example 4: Optimization of ELISA Conditions

[0114] Optimize each step in the ELISA process, specifically as follows:

[0115] A. Antigen coating amount: Using 50 - 1000 ng of purified p30 - 54 - 72 recombinant protein as the coating antigen, it was found that the P / N value was the highest when 500 ng was coated;

[0116] B. Coating conditions: Detecting with different coating buffers and reaction times, it was found that when the coating buffer was phosphate buffer with pH = 7.2, containing 0.356 g of NaH2PO4, 2.772 g of Na2HPO4·12H2O, and 8.5 g of NaCl in 1 L of solution, and coating overnight at 4℃, the P / N value was the highest;

[0117] C. Blocking conditions: Blocking with a blocking solution of PBS'T buffer with pH 7.2 containing 2.5% skim milk powder, and incubating at 37℃ for 1 h resulted in the highest P / N value;

[0118] D. Serum sample dilution: Diluting with PBS with pH 7.2 at a ratio of 1:40, and adding 100 μL resulted in the highest P / N value;

[0119] E. Enzyme - labeled antibody: The antibody of horseradish peroxidase - labeled goat anti - pig was diluted 1:5000 with PBS'T buffer with pH 7.2 containing 2.5% skim milk powder, 100 μL, and incubating at 37℃ for 60 min resulted in the highest P / N value;

[0120] F. Washing conditions: Using phosphate buffer (PBS'T) with pH 7.2 containing 2.5% (v / v) Tween - 20, discarding after gently shaking for 1 min, and repeating 5 times resulted in the highest P / N value;

[0121] G. Other conditions: Chromogenic reaction: Adding 100 μL of substrate TMB to each well and developing color in the dark at 37℃ for 15 min; Termination: Adding 50 μL of 3 mol / L sulfuric acid to each well.

[0122] H. Terminate the reaction; Reading: Reading the OD value with an enzyme - linked immunosorbent assay reader 450nm Reading.

[0123] The optimized ELISA conditions are as follows:

[0124] (1) Coating: Using p30 - 54 - 72 recombinant protein as the coating antigen, the coating amount of p30 - 54 - 72 recombinant protein is 500 ng / well; Coating overnight at 4℃ in the coating buffer; The coating buffer contains 0.356 g / L NaH2PO4, 2.772 g / L Na2HPO4·12H2O, 8.5 g / L NaCl, and pH = 7.2;

[0125] (2) Washing: Wash with phosphate buffer (PBST) with a pH of 7.2 containing 0.5% (v / v) Tween-20. After gently shaking for 1 min, discard the liquid and repeat 5 times.

[0126] (3) Blocking: Block with a blocking solution containing PBST buffer with a pH of 7.2 and 2.5% non-fat milk powder, and incubate at 37 °C for 1 h.

[0127] (4) Washing: Wash with phosphate buffer (PBST) with a pH of 7.2 containing 0.5% (v / v) Tween-20. After gently shaking for 1 min, discard the liquid and repeat 5 times.

[0128] (5) Serum reaction conditions: Add the diluted mixed solution of the test serum to each well, incubate at 37 °C for 1 h, then drain off the liquid and wash with PBST.

[0129] (6) Adding enzyme-labeled antibody: Dilute the goat anti-pig antibody labeled with horseradish peroxidase with PBST containing 2.5% non-fat milk powder at a ratio of 1:5000, add 100 μL to each well, incubate at 37 °C for 1 h, then drain off the liquid and wash with PBST.

[0130] (7) Substrate color development: Add 100 μL of TMB substrate color development solution to each well and develop color in the dark at room temperature for 15 min.

[0131] (8) Terminating the reaction: Add 50 μL of termination solution to each well to terminate the color development reaction; read the data at an absorbance of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0132] Determination of the critical value

[0133] 82 positive serum samples and 38 negative serum samples of African swine fever virus (ASFV) antibody in pigs stored in the laboratory were detected under the optimized ELISA conditions described above. The test results were analyzed using TG-ROC software, and the cut-off value with p30-54-72 recombinant protein as the coating antigen was 0.371.

[0134] Example 5: Application of establishing an ELISA method

[0135] Take 100 pig sera, among which 50 are clinically determined to be ASFV positive and 50 are clinically determined to be ASFV negative. Place the pig sera in centrifuge tubes, centrifuge at 6000 rpm for 5 min to remove red blood cells and impurities, collect the supernatant as the test serum.

[0136] Using p30-54-72 recombinant protein as the coating antigen, detect the test serum under the optimized ELISA conditions in Example 4.

[0137] Meanwhile, the proteins expressed by concatenating two dominant epitope regions (78 - 132aa and 134 - 174aa) of p30, the protein expressed by the dominant epitope region 54 - 124aa of p54, and the protein expressed by concatenating three dominant epitope regions (12 - 89aa, 139 - 324aa, and 445 - 524aa) of p72 were used as coating antigens, and the serum to be tested was detected according to the optimized ELISA conditions in Example 4.

[0138] The results are shown in Table 1:

[0139]

[0140]

[0141] Note: Positive detection rate = (Number of positive detection results / Number of 50 positive samples) × 100%.

[0142] The results show that using the p30 - 54 - 72 recombinant protein as the coating antigen can make up for the deficiency of too low positive numbers in detection when using a single protein as the coating, and it can be used for the routine clinical detection of African swine fever virus.

[0143] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A p30-54-72 recombinant protein, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. A gene encoding the p30-54-72 recombinant protein according to claim 1, the nucleotide sequence of which is shown in SEQ ID NO.

2.

3. A recombinant expression vector or genetically engineered bacterium comprising the gene according to claim 2.

4. Use of the p30-54-72 recombinant protein according to claim 1 in the preparation of a kit for detecting African swine fever virus antibodies.

5. The application according to claim 4, wherein The kit is an ELISA detection kit.

6. An ELISA kit for detecting African swine fever virus antibodies, characterized in that, The ELISA kit uses the p30-54-72 recombinant protein as the coating antigen; the amino acid sequence of the p30-54-72 recombinant protein is shown in SEQ ID NO.

1.

7. The ELISA kit according to claim 6, characterized in that, In the ELISA kit, the coating amount of the p30-54-72 recombinant protein is 500 ng / well.

8. The ELISA kit according to claim 7, wherein The ELISA kit further comprises: enzyme-labeled antibody, sample diluent, washing solution, negative control serum, positive control serum, TMB substrate chromogenic solution and stop solution.

9. The ELISA kit according to claim 8, wherein The enzyme-labeled antibody is a horseradish peroxidase-labeled goat anti-pig antibody.

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