CN110698543A

CN110698543A - Double-antigen indirect ELISA kit for African swine fever virus antibody detection

Info

Publication number: CN110698543A
Application number: CN201910993746.XA
Authority: CN
Inventors: 徐高原; 张华伟; 周明光; 曾小燕; 郝根喜; 安春敬; 朱娴静; 孙芳
Original assignee: WUHAN KEQIAN BIOLOGICAL Co Ltd
Current assignee: WUHAN KEQIAN BIOLOGICAL Co Ltd
Priority date: 2019-10-18
Filing date: 2019-10-18
Publication date: 2020-01-17

Abstract

The invention relates to the field of veterinary biotechnology, in particular to a double-antigen indirect ELISA kit for antibody detection of African swine fever virus. The double-antigen indirect ELISA kit comprises African swine fever virus P30 protein and truncated P72 protein which are obtained by expression of an escherichia coli prokaryotic expression system. The kit can effectively enlarge the time range of antibody detection of the African swine fever virus, effectively improves the soluble expression quantity of the protein by truncation of the P72 protein, codon optimization of the P72 protein and the P30 protein, fusion protein labeling and the like, furthest retains the immunogen determinant, greatly improves the specificity and sensitivity of the ELISA kit, effectively reduces the preparation cost of the kit, and plays an important role in early diagnosis, specificity detection and epidemiological investigation of the African swine fever virus.

Description

Double-antigen indirect ELISA kit for African swine fever virus antibody detection

Technical Field

The invention relates to the field of veterinary biotechnology, in particular to an antigen composition of African swine fever virus, a preparation method thereof and a double-antigen indirect ELISA kit for detecting antibody of the African swine fever virus.

Background

African Swine Fever (ASF) is an acute, febrile, highly contagious disease of swine caused by African Swine Fever Virus (ASFV). African swine fever is characterized by short course of disease, high mortality and is classified as a type A epidemic disease by the world animal Organization (OIE).

The African swine fever virus is an important member of African swine fever virus of African swine fever family, has some characteristics similar to those of iridoviridae and poxviridae, is a large enveloped double-stranded DNA virus with a plus 20-face body and a diameter of about 175-215 nm, and consists of an outer envelope, a virus capsid, an inner envelope, a nucleocapsid and virus nucleic acid. The African swine fever virus genome is single molecular linear double-stranded DNA with covalently closed ends, the total length of the virus genome is 170kb-190kb, the center has a conserved region of about 125kb, the two ends are variable regions containing inverted terminal repetitive sequences, and the increase or deletion of the repetitive sequences is the main reason for the difference of the genome lengths of different isolates.

The African swine fever virus gene can code more than 200 proteins, wherein 54 structural proteins are provided. P30 is one of the major structural proteins and strong immunogenic proteins of African swine fever virus, encoded by CP204L gene, and has a molecular weight of about 36 kD. P30 has strong immunogenic determinants, can induce the body to produce neutralizing antibodies, is usually used as a diagnostic antigen, is an early protein of virus, can be detected in cytoplasm 4h after infection, is usually used for early detection of immune response after infection, and can detect ASFV specific antibodies one week in advance by an ELISA method based on P30. In the serological detection technology, ASFV infected pigs can generate specific antibodies 7-10 days after infection and can maintain for a long time, so indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and other methods can be used for detection. Among them, ELISA is an international trade test method stipulated by the world animal health Organization (OIE). The ELISA detection method based on P30 is more suitable for early detection after virus infection, so the development of a detection method which is suitable for detection in a larger time range and has higher specificity and sensitivity is still needed.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide an antigen composition of the African swine fever virus, a preparation method thereof and a double-antigen indirect ELISA kit for detecting the antibody of the African swine fever virus.

In order to achieve the purpose, the technical scheme of the invention is as follows:

firstly, the invention discovers that the combination of the P72 protein and the P30 protein as the detection antigen can effectively expand the infection time range applicable to detection and has higher specificity by researching the sequence and the function of the structural protein of the African swine fever virus and screening and combining the antigenicity of different structural proteins. However, the full-length P72 protein has a problem that it is expressed in a low amount and cannot be expressed in a soluble form. The invention discovers that the soluble expression quantity of the P72 protein can be improved by adopting the P72 protein truncated at the N terminal.

In a first aspect, the present invention provides an antigenic composition comprising the P72 protein and the P30 protein of african swine fever virus; the P72 protein is an N-terminal truncated protein.

Specifically, the N-terminal truncated P72 protein removes the 100 th and 410 th amino acids from the N-terminal of the full-length P72 protein. The N-terminal truncated protein can well maintain the immunogenicity of the P72 protein, and can effectively promote the mass expression and soluble expression of the P72 protein.

Preferably, the P72 protein has any one of the following amino acid sequences:

(1) an amino acid sequence shown as SEQ ID NO. 1;

(2) the amino acid sequence of the protein with the same function is obtained by deleting, replacing or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1.

The P30 protein has any one of the following amino acid sequences:

(1) an amino acid sequence shown as SEQ ID NO. 2;

(2) the amino acid sequence of the protein with the same function is obtained by deleting, replacing or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2.

Preferably, in the antigen composition, the mass ratio of the P72 protein to the P30 protein is (1-3): (1-3). The components are compounded in the proportion range, so that the specificity and the sensitivity of the African swine fever virus antibody detection can be better ensured.

The P30 protein also has the problem of low soluble expression amount, and in order to further improve the soluble expression amount, purity and yield of each protein in the antigen composition, further improve the specificity and sensitivity of detection and reduce the preparation cost of the antigen composition, the antigen composition preferably utilizes a prokaryotic expression system to respectively express coding genes of the P72 protein and the P30 protein of the African swine fever virus which are optimized by codons.

Preferably, the prokaryotic expression system is an escherichia coli expression system; the nucleotide sequences of the coding genes of the P72 protein and the P30 protein of the African swine fever virus which is optimized by the codon are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.

The codon-optimized coding sequence is obtained by a large amount of artificial optimization and screening, and can realize high-level and high-purity expression of the P72 protein and the P30 protein while ensuring the natural structures and higher immunogenicity of the P72 protein and the P30 protein to the maximum extent.

More preferably, the coding genes of the codon-optimized African swine fever virus P72 protein and P30 protein are connected with the SUMO tag protein coding gene at the 5 'end or the 3' end.

The coding gene sequence of the SUMO tag protein is shown in SEQ ID NO. 7.

By connecting the SUMO tag protein, high-level expression of soluble P72 protein and P30 protein can be better ensured, the purification process is simplified, and the immunogenicity of the P72 protein and the P30 protein is effectively ensured.

The invention also provides a preparation method of the African swine fever virus antigen composition, which comprises the following steps: and respectively expressing the coding genes of the P72 protein and the P30 protein of the African swine fever virus which are optimized by codons by utilizing a prokaryotic expression system.

Preferably, the prokaryotic expression system is an escherichia coli expression system; the nucleotide sequences of the coding genes of the codon optimized P72 protein and the P30 protein are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.

More preferably, the coding genes of the codon-optimized African swine fever virus P72 protein and P30 protein are connected with a tag protein coding gene at the 5 'end or the 3' end.

The tag protein is preferably SUMO tag protein.

As a preferred embodiment of the invention, the coding genes of the P72 protein and the P30 protein of the African swine fever virus which is optimized by codons are expressed by using an Escherichia coli BL21-pET42b expression system.

The invention provides recombinant escherichia coli, which comprises a codon-optimized coding gene of P72 protein and/or a codon-optimized coding gene of P30 protein; the nucleotide sequence of the coding gene of the codon optimized P72 protein is shown as SEQ ID NO. 3; the nucleotide sequence of the coding gene of the codon optimized P30 protein is shown as SEQ ID NO. 4.

Preferably, the present invention provides escherichia coli BL21(DE3) containing pET42b vector carrying the gene encoding the codon-optimized P72 protein; also provided is E.coli BL21(DE3) containing pET42b vector carrying the gene encoding the codon-optimized P30 protein.

Furthermore, the invention provides an application of the antigen composition or the antigen composition prepared by the preparation method or the recombinant escherichia coli in preparation of a reagent or a kit for detecting antibodies of African swine fever viruses or African swine fever viruses.

The invention also provides an application of the antigen composition or the antigen composition prepared by the preparation method or the recombinant escherichia coli in preparation of a positive standard substance for detecting African swine fever viruses.

The invention also provides an application of the antigen composition or the antigen composition prepared by the preparation method or the recombinant escherichia coli in preparation of an African swine fever virus vaccine.

Further, the invention provides a double-antigen indirect ELISA kit for detecting antibody of African swine fever virus, which comprises the antigen composition, or comprises the antigen composition prepared by the preparation method, or comprises P30 protein and P72 protein expressed by the recombinant Escherichia coli.

Preferably, in the double-antigen indirect ELISA kit, the P30 protein and the P72 protein are mixed in a mass ratio of 1: 1.

Preferably, the double-antigen indirect ELISA kit further comprises one or more selected from a 96-well ELISA plate, an enzyme-labeled secondary antibody, a standard positive control, a standard negative control, a concentrated washing solution, a diluent, a TMB substrate and a stop solution.

Preferably, the standard positive control comprises serum obtained by mixing the serum obtained by immunizing a pig with the P72 protein and the P30 protein, respectively, in equal volumes. The standard negative control comprises serum from a non-immunized healthy pig.

When the double-antigen indirect ELISA kit for detecting the antibody of the African swine fever virus is used for detecting the African swine fever, the coating concentration of the P72 protein and the P30 protein is 0.5-2 mu g/ml.

The invention has the beneficial effects that:

1. the antigen composition consisting of the African swine fever virus P30 protein and the P72 protein and the double-antigen indirect ELISA kit provided by the invention can effectively expand the time range of antibody detection of the African swine fever virus, simultaneously improve the specificity and sensitivity of detection, and play an important role in early diagnosis, specificity detection and epidemiological investigation of the African swine fever virus.

2. According to the invention, through the truncation of the P72 protein and the codon optimization of the P72 protein and the P30 protein, the expression amount of the protein is effectively improved, the SUMO label is fused, the soluble expression of the protein is improved, the structural states of the obtained P72 protein and the P30 protein are completely consistent with the natural structural state, the immunogen determinant is retained to the greatest extent, and compared with the protein expressed in the form of inclusion bodies, the protein has better reactogenicity and stronger affinity, is more easily combined with an antibody, and the specificity and the sensitivity of an ELISA kit are greatly improved.

3. The escherichia coli prokaryotic expression system used by the invention is efficient and safe, and is harmless to people and livestock, and the P72 protein and the P30 protein can be prepared by simple expression and purification processes, so that the preparation cost of the kit is effectively reduced.

Drawings

FIG. 1 shows the SalI/XhoI double restriction enzyme identification results of recombinant plasmids pET42b-SUMO-P30 and pET42b-SUMO-P72 in example 1 of the present invention, wherein A is recombinant plasmid pET42 b-SUMO-P30; b is recombinant plasmid pET 42B-SUMO-P72.

FIG. 2 is a SDS-PAGE result of the recombinant proteins P30 and P72 purified in example 1 of the present invention, wherein A is recombinant protein P30, lane 1 is a control using a conventional codon optimization method, lane 2 is specific codon-optimized P30 protein, and M is protein marker; b is recombinant protein P72, lane 1 is a control using conventional codon optimization, lane 2 is P72 protein optimized by specific codons, and M is protein marker.

FIG. 3 shows the Western Blot detection results of recombinant proteins P30 and P72 purified in example 1 of the present invention, wherein A is recombinant protein P30, lane 1 is a control employing conventional codon optimization, lane 2 is specific codon-optimized P30 protein, and M is protein marker; b is recombinant protein P72, lane 1 is a control using conventional codon optimization, lane 2 is P72 protein optimized by specific codons, and M is protein marker.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of African Swine fever Virus P72 protein and P30 protein antigens

1. Construction of vectors pET42b-SUMO-P30 and pET42b-SUMO-P72

Referring to the coding gene sequences of African swine fever virus (GenBank No. KM111294.1) P30 and P72, the amino acid sequence of the N-terminal truncated protein (hereinafter referred to as P72 protein) of the P72 protein is shown in SEQ ID NO. 1; the amino acid sequence of the P30 protein is shown in SEQ ID NO. 2. Carrying out artificial codon optimization according to the codon preference of an escherichia coli prokaryotic expression system, designing a plurality of codon optimization sequences, and determining the codon optimization sequence with the optimal expression effect through screening, wherein the coding gene sequence of the P72 protein subjected to codon optimization is shown as SEQ ID No.3, and the coding gene sequence of the P30 protein subjected to codon optimization is shown as SEQ ID No. 4. Artificially synthesizing a P72 protein coding gene (930bp) shown in SEQ ID NO.3 and a P30 protein coding gene (579bp) fragment shown in SEQ ID NO. 4. The above-mentioned fragments were artificially synthesized and placed in a plasmid vector pUC-18 (Invitrogen), which was obtained as pUC-P30 and pUC-P72.

The conventional recombinant plasmid construction process and method are adopted to construct recombinant vectors pET42b-SUMO-P30 and pET42b-SUMO-P72, and the specific steps are as follows:

(1) construction of pET42b-SUMO vector:

① A DNA fragment of SUMO tag (SUMO tag sequence is shown in SEQ ID NO. 7) with NdeI and XhoI cutting site sequences at both ends is artificially synthesized and connected to a pUC18 plasmid vector to be named as pUC-SUMO.

② the pET42b and pUC-SUMO are subjected to NdeI and XhoI enzyme cutting simultaneously, the target fragments are recovered by glue, and then are connected, DH5 alpha is transformed, and positive clones are screened, so that pET42b-SUMO recombinant plasmid vectors are obtained.

(2) Enzyme digestion: carrying out double enzyme digestion on pUC-P30 and pUC-P72 vectors by using SalI and XhoI enzymes (purchased from TAKARA) to obtain the artificially synthesized P72 protein coding gene fragment shown as SEQ ID NO.5 and P30 protein coding gene fragment shown as SEQ ID NO. 6; meanwhile, the vector pET42b-SUMO was digested with the same endonuclease. The digestion reaction system and reaction conditions are shown in table 1:

TABLE 1 double digestion reaction System and reaction conditions

After the digestion, the fragments were separated by agarose gel electrophoresis and recovered.

(2) Connecting: the pET42b-SUMO vector was ligated to the P72 protein-encoding gene and the P30 protein-encoding gene, and the reaction system and reaction conditions for the ligation were as shown in Table 2.

TABLE 2 connected reaction systems and reaction conditions

(3) And (3) transformation: the ligation product was transformed into E.coli competent DH5 alpha.

(4) Extracting recombinant plasmids pET42b-SUMO-P30 and pET42 b-SUMO-P72: selecting a monoclonal colony from the plate, extracting plasmids by using a plasmid extraction kit, carrying out enzyme digestion identification on SalI and XhoI, wherein the enzyme digestion identification result is shown in figure 1.

2. Expression and purification of African swine fever virus P30 and P72 proteins

(1) Induced expression of recombinant protein: the recombinant plasmids pET42b-SUMO-P30 and pET42b-SUMO-P72 which are verified to be correct by enzyme digestion are respectively transformed into a host bacterium BL21(DE3), and recombinant Escherichia coli BL21(DE3) carrying pET42b-SUMO-P30 and pET42b-SUMO-P72 respectively is obtained. Picking single clone to containCultured overnight in 50. mu.g/mL LB medium at a rate of 1: 100 volume ratio, and culturing at 37 deg.C to OD₆₀₀About 0.6, IPTG was added to a final concentration of 1mM and induced at 37 ℃ for 4 h.

(2) And (3) purifying the recombinant protein: and (4) centrifuging the bacterial liquid after induction culture at 8000rpm for 5min, collecting the thallus, and resuspending by PBS. The thalli is crushed by a pressure crusher, and the bacterial liquid is in a clear state after crushing, which indicates that the protein is soluble expression. The mixture was centrifuged at 12000rpm for 30min at 4 ℃ to collect the supernatant. The recombinant proteins P30 and P72 were purified according to the Ni-NTA agarose column (Qiagen, Cat No:30210) instructions.

(3) Detection of recombinant protein: the purified recombinant proteins P30 and P72 are taken for SDS-PAGE detection and Western Blot detection, single target bands are obtained, and the antigen is good. The SDS-PAGE detection and Western Blot detection results of the purified recombinant proteins P30 and P72 after codon optimization are respectively shown in fig. 2 and fig. 3 (the coding gene obtained by adopting a conventional codon optimization mode is expressed, and the purified recombinant proteins P30 and P72 are used as a reference, and the coding gene sequences of P30 and P72 obtained by conventional codon optimization are respectively shown in SEQ ID NO.5 and SEQ ID NO. 6), and the results show that compared with the coding gene obtained by adopting the conventional codon optimization mode, the expression quantity of P30 and P72 is remarkably improved after the specific codon optimization of the invention, and soluble expression can be realized.

Example 2P 30/P72 Dual antigen Indirect ELISA kit for African swine fever virus antibody detection

The embodiment provides a P30/P72 double-antigen indirect ELISA kit for detecting African swine fever virus antibodies, and the detection principle is as follows: the detection is carried out by indirect ELISA, and the purified recombinant proteins P30 and P72 prepared in example 1 are mixed according to the mass ratio of 1:1 and mixing uniformly. The mixed solution of the P30 and the P72 proteins is coated in the micropores of an enzyme label plate, then the enzyme label plate is blocked by PBST containing 1% BSA, and a sample to be detected and a standard positive control and a standard negative control are respectively added into the enzyme label plate. The African swine fever virus antibodies in the samples and the standard products can react with the antigen coated by the ELISA plate; adding a horseradish peroxidase-labeled secondary antibody, namely goat anti-pig IgG (Biodragon, Cat: BF03030) antibody, and reacting the enzyme-labeled secondary antibody with the goat anti-pig IgG antibodyAnd (4) combining the antigen-antibody complex after the reaction in the previous step is finished. Then adding horseradish peroxidase substrate TMB for color development, adding stop solution to stop reaction, and measuring absorbance value (OD) of each well by an enzyme-labeling instrument at a wavelength of 450nm₄₅₀) Size of (1) (shade of color after color development reaction of stop solution), OD₄₅₀Is in direct proportion to the content of the African swine fever virus antibody in the sample to be detected.

1. Preparation of Standard Positive serum

The concentrations of the purified recombinant proteins P30 and P72 prepared in example 1 were adjusted to 1mg/ml with 0.01M PBS (pH 7.6), and each was mixed with an equal volume of Freund's complete adjuvant (Sigma Co.), and sufficiently emulsified (power 30W, 5min) with an ultrasonic instrument (Bandelin Co.) to obtain P30 antigen and P72 antigen. P30 antigen and P72 antigen were injected intramuscularly into 2 healthy 2-month-old piglets, 2ml (1mg protein)/head; after 10 days of the first immunization, the immunization was boosted 1 time with the same dose of antigen mixed with Freund's incomplete adjuvant; after 10 days, the antigen with the same dose is used for strengthening immunization for 1 time; on day 7 after the third immunization, serum was aseptically collected from swine blood; according to a conventional indirect ELISA method, 5 mu g/ml of recombinant proteins P30 and P72 are respectively used for coating an ELISA plate and react with recombinant protein immune pig serum which is continuously diluted by 10 times, and the ELISA antibody titer of the two recombinant protein immune pig sera is measured to be more than 10⁵. Mixing the two recombinant protein immune pig sera in equal volume, adding thimerosal with final concentration of 0.01% for antisepsis to obtain standard positive sera, subpackaging, and storing at-20 deg.C.

2. Preparation of Standard negative serum

Selecting fattening pigs with good body conditions, normal body temperature, appetite and excrement and urine, and qualified through inspection as pigs for blood collection. After sterilization, jugular vein and artery blood sampling, serum separation, adding thimerosal with the final concentration of 0.01 percent for antisepsis to obtain standard negative serum, subpackaging and storing at-20 ℃.

3. Preparation method of antigen-coated ELISA plate

An antigen mixture obtained by mixing the purified recombinant P30 and P72 proteins prepared in example 1 at a mass ratio of 1:1 was added to a microplate at 100. mu.l/well using a carbonate buffer solution of 0.05M at pH 9.6 as a coating buffer. Coating at 4 deg.C overnight, discarding the coating solution the next day, adding blocking solution containing 1% BSA at 200 μ l/well, standing at 37 deg.C for 2 hr, washing and spin-drying. Drying at room temperature, packaging, adding desiccant, and vacuum preserving.

4. Preparation of other working Agents

(1) Wash (PBST pH 7.4): KH (Perkin Elmer)₂PO₄0.2g，Na₂HPO₄-12H₂O2.9 g, NaCl 8.0g, KCl0.2g, Tween-20 (working concentration 0.05%) 0.5ml, and distilled water to 1L. Concentrate 25-fold as stock solution (concentrate wash).

(2) Sealing liquid: bovine Serum Albumin (BSA) 0.1g, and washing buffer (PBS) to 100 ml.

(3) Diluting liquid: the same as the sealing liquid.

(4) Substrate buffer (citric acid phosphate pH 5.0): 0.2M Na₂HPO₄25.7ml, 24.3ml of 0.1M citric acid, and distilled water to 100 ml.

(5) TMB (tetramethylbenzidine) use solution: 0.5ml of TMB (10mg/5ml of absolute ethanol), 10ml of substrate buffer, 0.75% H₂O₂32μl。

(6) Stopping liquid: 2M H₂SO₄:。

5. Construction of double-antigen indirect ELISA kit for African swine fever virus antibody detection

The double-antigen indirect ELISA kit for detecting the African swine fever virus antibody comprises the following components:

(1) the 96-hole enzyme label plate is coated by antigen mixed solution obtained by mixing recombinant proteins P30 and P72 in a mass ratio of 1: 1;

(2) horse radish peroxidase-labeled goat anti-porcine IgG polyclonal antibody (Biodragon, Cat: BF 03030);

(3) standard positive sera;

(4) a standard negative serum;

(5) concentrating the washing solution;

(6) diluting the solution;

(7) a TMB substrate;

(8) a stop solution;

(9) and (5) product instructions.

Example 3 determination of the detection method for African Swine fever Virus antibodies

In this example, the purified ASFV recombinant proteins P30 and P72 prepared in example 1 and the P30/P72 double antigen indirect ELISA kit for detecting African swine fever virus antibody of example 2 were used to detect African swine fever virus antibody.

1. Determination of concentration of coating antigen and enzyme-labeled antibody

(1) Monoclonal antigen ELISA: and (3) respectively taking ASFV recombinant proteins P30 and P72 which are diluted in a gradient manner as envelope antigens, and carrying out standard positive serum and standard negative serum according to the ratio of 1: 100,1: 200,1: 400,1: 800,1: 1600,1: 3200,1: 6400,1: 12800 diluting, using horse radish peroxidase labeled goat anti-pig IgG as second antibody, determining OD₄₅₀. The results are shown in tables 3 and 4, and show that the optimal dilution of the ASFV recombinant proteins P30 and P72 is 1: 800, i.e. the optimal coating concentration is 1. mu.g/ml, and the optimal dilution of standard positive serum and standard negative serum is 1: 800.

TABLE 3 determination of the amount of antigen P30 coating and the dilution of standard negative and positive sera

TABLE 4 determination of the amount of antigen P72 coating and the dilution of standard negative and positive sera

(2) Establishment of MA-ELISA: the recombinant proteins P30 and P72 are mixed in a ratio of 1:1, mixing with a mixture of 1: the standard positive serum and the standard negative serum are diluted by 800 percent to react, and then the reaction product reacts with the enzyme-labeled secondary antibody diluted by different times to obtain a positive hole OD₄₅₀The net/negative well OD450 net was used as a criterion (the antigen coating concentration corresponding to the maximum value was the optimum concentration), and the results are shown in table 5, where the optimum dilution of the mixed antigen was measured as 1: 800, i.e. the coating concentration is 2. mu.g/ml. The enzyme-labeled antibody is optimally used at a concentration of 1: 6400 for convenient use, follow-up experimentThe concentration of the secondary enzyme labeled with the secondary enzyme is 1: 10000.

TABLE 5 determination of coating amount of mixed solution of antigens P30 and P72 and dilution of enzyme-labeled secondary antibody

(3) ELISA quantification of standard positive, negative sera: performing 10 times of repeated detection on the prepared standard positive and negative sera respectively according to ELISA detection program, and using positive control serum OD₄₅₀The mean value of the net values-2 XSD (standard deviation) is a calculation formula, and the OD of the positive control serum is calculated₄₅₀The lower limit of the net value is 1.4, and the OD of the negative control serum is used₄₅₀The mean value of the net value +2 XSD (standard deviation) was used as a calculation formula to calculate the OD of the negative control serum₄₅₀The upper limit of the net value was 0.1, thus determining the OD when the standard positive serum was used₄₅₀Net > 1.4, OD of standard negative serum₄₅₀When the net value is less than or equal to 0.1, the MA-ELISA system is effective.

2. Detection procedure for African swine fever virus antibody

(1) The microplate was removed from the kit and allowed to equilibrate to room temperature (16-26 deg.C) to avoid direct sunlight.

(2) Diluting the serum to be detected, the standard positive serum and the standard negative serum according to the proportion of 1: diluting at a ratio of 200, adding 100 μ l/well of enzyme label plate (detection loading mode is shown in Table 6), incubating at 37 deg.C for 1h, and washing with washing solution for 3-5 times.

TABLE 6 test sample application

Note: p: positive control serum, N: negative control serum, S1-S46: and (4) detecting the serum sample.

(3) Add 1: 10000 mu l of enzyme-labeled goat anti-pig IgG antibody, incubating for 30min at 37 ℃, and washing for 3-5 times by using a washing solution.

(4) 100ul of TMB substrate solution is added into each well, and the reaction is carried out for 10min at room temperature in a dark place.

(5) Add 100. mu.l of 2M H per well₂SO₄The reaction is stopped, and the absorbance value of 450nm is detected by an enzyme-labeling instrument.

(6) And (3) analyzing a detection result: and (4) using multiple wells for detection, and taking an average value.

And (3) judging the effectiveness: OD of standard negative serum₄₅₀Average value less than or equal to 0.1, OD of standard positive serum₄₅₀Mean value greater than 1.4, and OD of standard positive serum and standard negative serum₄₅₀The difference in values is greater than 0.4, the test is considered valid;

and (5) judging a result: when the S/P value of the sample is more than or equal to 0.5, the sample is judged to be positive, and the sample contains an African swine fever virus antibody; when the S/P value of the sample is less than 0.3, the sample is judged to be negative, and the sample does not contain the African swine fever virus antibody;

S/P value ═ sample OD₄₅₀value-OD of Standard negative serum₄₅₀Value) ÷ (standard positive serum OD)₄₅₀mean-OD of Standard negative serum₄₅₀Average value).

Example 4 sensitivity assay for African Swine fever Virus antibody detection

The standard positive serum prepared in example 2 was diluted in multiple proportions, and the standard negative serum was set as a control. Meanwhile, parallel detection is carried out by using the P30/P72 double-antigen Indirect ELISA kit (DA-ELISA) for detecting African Swine Fever virus antibodies provided by the embodiment 2 of the invention, the ELISA kit (INGEZIM PPA COMPAC) of Ingenasa Spanish and the ELISA kit (ID Screen African Swine river Indirect ELISA kit) of French TD-Vet, and the detection results are shown in Table 7.

TABLE 7 DA-ELISA sensitivity analysis

Example 5 specificity assay for African Swine fever Virus antibody detection

The P30/P72 antibody standard positive serum prepared in the embodiment 2, the porcine circovirus antibody standard positive serum, the porcine pseudorabies virus antibody standard positive serum, the porcine reproductive and respiratory syndrome virus antibody standard positive serum, the porcine encephalitis B virus antibody standard positive serum and the porcine influenza virus (H1 subtype) antibody standard positive serum are respectively used as detection objects, and the P30/P72 double-antigen indirect ELISA kit (DA-ELISA) for detecting African swine fever virus antibodies, the porcine pseudorabies virus ELISA antibody detection kit, the porcine circovirus ELISA antibody detection kit, the porcine reproductive and respiratory syndrome virus ELISA antibody detection kit, the porcine encephalitis B virus ELISA antibody detection kit and the porcine influenza virus (H1 subtype) ELISA antibody detection kit provided in the embodiment 2 of the invention are simultaneously used for parallel detection of various standard positive sera, the detection results are shown in Table 8, and the results show that the double-antigen indirect ELISA kit (DA-ELISA) for detecting antibody of African swine fever virus provided in example 2 can only detect standard positive serum of P30/P72 antibody, and has good specificity.

TABLE 8 DA-ELISA specificity analysis

Note: 1: standard positive serum for P30/P72 antibody; 2: standard positive serum of porcine circovirus antibody; 3: standard positive serum of porcine pseudorabies virus antibody; 4: standard positive serum of porcine reproductive and respiratory syndrome virus antibody; 5: standard positive serum of swine Japanese encephalitis virus antibody; 6: standard positive serum for swine influenza virus (subtype H1) antibodies. A: P30/P72 double antigen indirect ELISA kit (DA-ELISA); b: a porcine circovirus ELISA antibody detection kit; c: a porcine pseudorabies virus ELISA antibody detection kit; d: porcine reproductive and respiratory syndrome virus ELISA antibody detection kit; e: a pig encephalitis B virus ELISA antibody detection kit; f: swine influenza virus (subtype H1) ELISA antibody detection kit; +: the detection result is positive; and (2) preparing: the detection result is negative.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Pro Leu Glu Gly Ala Val Tyr Thr Leu Val Asp Pro Phe Gly Arg Pro

50 55 60

Ile Val Pro Gly Thr Lys Asn Ala Tyr Arg Asn Leu Val Tyr Tyr Cys

65 70 75 80

Glu Tyr Pro Gly Glu Arg Leu Tyr Glu Asn Val Arg Phe Asp Val Asn

85 90 95

Gly Asn Ser Leu Asp Glu Tyr Ser Ser Asp Val Thr Thr Leu Val Arg

100 105 110

Lys Phe Cys Ile Pro Gly Asp Lys Met Thr Gly Tyr Lys His Leu Val

115 120 125

Gly Gln Glu Val Ser Val Glu Gly Thr Ser Gly Pro Leu Leu Cys Asn

130 135 140

Ile His Asp Leu His Lys Pro His Gln Ser Lys Pro Ile Leu Thr Asp

145 150 155 160

Glu Asn Asp Thr Gln Arg Thr Cys Ser His Thr Asn Pro Lys Phe Leu

165 170 175

Ser Gln His Phe Pro Glu Asn Ser His Asn Ile Gln Thr Ala Gly Lys

180 185 190

Gln Asp Ile Thr Pro Ile Thr Asp Ala Thr Tyr Leu Asp Ile Arg Arg

195 200205

Asn Val His Tyr Ser Cys Asn Gly Pro Gln Thr Pro Lys Tyr Tyr Gln

210 215 220

Pro Pro Leu Ala Leu Trp Ile Lys Leu Arg Phe Trp Phe Asn Glu Asn

225 230 235 240

Val Asn Leu Ala Ile Pro Ser Val Ser Ile Pro Phe Gly Glu Arg Phe

245 250 255

Ile Thr Ile Lys Leu Ala Ser Gln Lys Asp Leu Val Asn Glu Phe Pro

260 265 270

Gly Leu Phe Val Arg Gln Ser Arg Phe Ile Ala Gly Arg Pro Ser Arg

275 280 285

Arg Asn Ile Arg Phe Lys Pro Trp Phe Ile Pro Gly Val Ile Asn Glu

290 295 300

Ile Ser Leu Thr Asn Asn

305 310

<210>2

<211>193

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Asp Phe Ile Leu Asn Ile Ser Met Lys Met Glu Val Ile Phe Lys Thr

1 5 10 15

Asp Leu Arg Ser Ser Ser Gln Val Val Phe His Ala Gly Ser Leu Tyr

2025 30

Asn Trp Phe Ser Val Glu Ile Ile Asn Ser Gly Arg Ile Val Thr Thr

35 40 45

Ala Ile Lys Thr Leu Leu Ser Thr Val Lys Tyr Asp Ile Val Lys Ser

50 55 60

Ala Arg Ile Tyr Ala Gly Gln Gly Tyr Thr Glu His Gln Ala Gln Glu

65 70 75 80

Glu Trp Asn Met Ile Leu His Val Leu Phe Glu Glu Glu Thr Glu Ser

85 90 95

Ser Ala Ser Ser Glu Asn Ile His Glu Lys Asn Asp Asn Glu Thr Asn

100 105 110

Glu Cys Thr Ser Ser Phe Glu Thr Leu Phe Glu Gln Glu Pro Ser Ser

115 120 125

Glu Val Pro Lys Asp Ser Lys Leu Tyr Met Leu Ala Gln Lys Thr Val

130 135 140

Gln His Ile Glu Gln Tyr Gly Lys Ala Pro Asp Phe Asn Lys Val Ile

145 150 155 160

Arg Ala His Asn Phe Ile Gln Thr Ile Tyr Gly Thr Pro Leu Lys Glu

165 170 175

Glu Glu Lys Glu Val Val Arg Leu Met Val Ile Lys Leu Leu Lys Lys

180185 190

Lys

<210>3

<211>930

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

ttccacgata tggttggtca ccacatcctg ggtgcgtgcc acagctcttg gcaggatgcg 60

ccgatccagg gcaccagcca gatgggcgcg cacggtcagc tgcagacctt cccgcgtaac 120

ggttacgatt gggataacca gactccgctg gaaggcgcag tttacaccct ggttgatccg 180

ttcggtcgtc cgatcgttcc gggtactaaa aacgcgtacc gtaacctggt ttactattgc 240

gaatatccgg gtgaacgtct gtacgaaaac gttcgtttcg atgttaacgg caactctctg 300

gatgaataca gctctgatgt taccaccctg gtgcgtaaat tctgcatccc aggcgataaa 360

atgaccggct acaaacacct ggttggtcag gaagttagcg ttgaaggcac cagcggtccg 420

ctgctgtgca acatccacga tctgcacaaa ccgcaccagt ctaaaccgat cctgaccgat 480

gaaaacgata cccagcgtac ctgctctcac accaacccga aattcctgag ccagcacttc 540

ccggaaaact ctcacaacat ccagaccgcg ggtaaacagg atatcacccc gatcaccgat 600

gctacctatc tggatatccg tcgtaacgtt cactacagct gcaacggtcc gcagactccg 660

aaatactacc agccgccgct ggcgctgtgg atcaaactgc gtttctggtt caacgaaaac 720

gttaacctgg ctatcccgag cgttagcatc ccgttcggcg aacgtttcat caccatcaaa 780

ctggctagcc agaaagatct ggttaacgaa tttccgggcc tgttcgttcg tcagtctcgt 840

ttcatcgcgg gccgtccgag ccgtcgtaac atccgtttca aaccgtggtt cattccgggt 900

gttatcaacg aaatcagcct gaccaacaac 930

<210>4

<211>579

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gacttcatcc tgaacatcag catgaaaatg gaagttatct tcaaaaccga tctgcgtagc 60

agcagccagg ttgttttcca cgcgggtagc ctgtacaact ggttcagcgt tgaaatcatc 120

aacagcggcc gtatcgtgac caccgcgatc aaaaccctgc tgagcaccgt taaatacgat 180

atcgttaaaa gcgcgcgtat ctacgcgggc cagggctaca ccgaacacca ggcgcaggaa 240

gaatggaaca tgatcctgca cgttctgttc gaagaagaaa ccgaaagcag cgcgagcagc 300

gaaaacatcc acgaaaagaa cgataacgaa accaacgaat gcaccagcag cttcgaaacc 360

ctgttcgaac aggaaccgag cagcgaagtt ccgaaagata gcaaactgta catgctggcg 420

cagaaaaccg ttcagcacat cgaacagtac ggcaaagcgc cggatttcaa caaagttatc 480

cgtgcgcaca acttcatcca gaccatctac ggcaccccgc tgaaagaaga agaaaaagaa 540

gttgttcgtc tgatggttat caaactgctg aaaaagaaa 579

<210>5

<211>930

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

ttccacgata tggttggtca ccacatcctg ggtgcgtgcc acagctcttg gcaggatgcg 60

ccgatccagg gcaccagcca gatgggcaaa cacggtcagc tgcagacctt cccgcgtaac120

ggttacgatt gggataacca gactccgctg gaaggcgcag tttaccgcct ggttgatccg 180

aacggtcgtc cgatcgttcc gggtactaaa aacgcgtacc gtaacctggt ttactattgc 240

gccggtccgg gtgaacgtct gtacgaaaac gttcgtttcg atgttaacgg caactctctg 300

gatgaataca gctctgatgt taccaccctg gtgcgtaaat tctgcatccc aggcgataaa 360

atgaccggct acaaacacct ggttggtcag gaagttagcg ttgaaggcac cagcggtccg 420

ctgctgtgca acatccacga tctgcacaaa ccgcaccagt ctaaaccgat cctgaccgat 480

gaaaacgata cccagcgtac ctgctctcac accaacccga aattcctgag ccagcacttc 540

ccggaaaact ctcacaacat ccagaccgcg ggtaaacagg atatcacccc gatcaccgat 600

gctacctatc tggatatccg tcgtaacgtt cactacagct gcaacggtcc gcagactccg 660

aaatactacc agccgccgct ggcgctgtgg atcaaactgc gtttctggtt caacgaaaac 720

gttaacctgg ctatcccgag cgttagcatc ccgttcccgg aacgtttcat caccatcaaa 780

ctggctagcc agaaagatct ggttaacgaa tttccgggcc tgttcgttcg tcagtctcgt 840

ttcatcgcgg gccgtccgag ccgtcgtaac atccgtttca aaccgtggtt cattccgggt 900

gttatcaacg aaatcagcct gaccaacaac 930

<210>6

<211>579

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

gacttcatcc tgaacatcag catgaaaatg ccagttatct tcaaaaccga tctgcgtagc 60

agcagccagg ttgttttcca cgcgggtagc ctgtacaact gattcagcgt tgaaatcccc 120

aacagcggcc gtatcgtgac caccgcgatc aaaaccctgc tgagcaccgt taaataccat 180

atcgttaaaa gcgcgcgtat ctacgcgggc cagggctaca ccgcgcacca ggcgcaggaa 240

gaatggaaca tgatcctgca cgttctgttc gaagaagaaa ccgaaagcag cgcgagcagc 300

gaaaacatcc acgaaaagtt cgataacgaa accaacgaat gcaccagcag cttcgaaacc 360

ctgttcgaac aggaaccgag cagcgaagtt ccgaaagata gcaaactgta catgctggcg 420

cagaaaaccg ttcagcacat cgaacagtac ggcaaagcgc cggatttcaa caaagttatc 480

cgtgcgcaca acttcatcca gaccatctac ggcaccccgc tgaaagaaga agaaaaagaa 540

gttgttcgtc tgatggttat caaactgctg aaaaagaaa 579

<210>7

<211>294

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60

gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120

aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180

gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240

ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggt 294

Claims

1. An antigenic composition comprising the P72 protein and the P30 protein of african swine fever virus; the P72 protein is an N-terminal truncated protein.

2. The antigenic composition of claim 1, wherein said P72 protein has the amino acid sequence of any one of seq id no:

(1) an amino acid sequence shown as SEQ ID NO. 1;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the amino acid sequence shown as SEQ ID NO. 1;

and/or the presence of a gas in the gas,

the P30 protein has any one of the following amino acid sequences:

(1) an amino acid sequence shown as SEQ ID NO. 2;

3. The antigenic composition of claim 1 or 2, wherein said P72 protein and said P30 protein are present in a mass ratio of (1-3): (1-3).

4. A preparation method of an African swine fever virus antigen composition is characterized by comprising the following steps: respectively expressing coding genes of the P72 protein and the P30 protein of the African swine fever virus which are optimized by a codon by utilizing a prokaryotic expression system;

5. The method according to claim 4, wherein the genes encoding the codon-optimized African swine fever virus P72 protein and P30 protein are linked to a tag protein-encoding gene at the 5 'end or 3' end; preferably, the tag protein coding gene is a SUMO tag protein coding gene.

6. A recombinant Escherichia coli, which comprises a coding gene of a codon-optimized African swine fever virus P72 protein and/or a coding gene of a codon-optimized African swine fever virus P30 protein; the nucleotide sequence of the coding gene of the African swine fever virus P72 protein optimized by the codon is shown as SEQ ID NO. 3; the nucleotide sequence of the coding gene of the African swine fever virus P30 protein optimized by the codon is shown as SEQ ID NO. 4.

7. Use of the antigen composition according to any one of claims 1 to 3 or the antigen composition prepared by the preparation method according to claim 4 or 5 or the recombinant escherichia coli according to claim 6 for preparing a reagent or a kit for detecting antibodies against african swine fever virus or african swine fever virus.

8. Use of the antigen composition according to any one of claims 1 to 3 or the antigen composition prepared by the preparation method according to claim 4 or 5 or the recombinant escherichia coli according to claim 6 for preparing a positive standard for detecting african swine fever virus.

9. Use of the antigenic composition according to any one of claims 1 to 3 or the antigenic composition produced by the production process according to claim 4 or 5 or the recombinant E.coli according to claim 6 for the production of an African swine fever virus vaccine.

10. A double-antigen indirect ELISA kit for antibody detection of African swine fever virus, comprising the antigen composition of any one of claims 1-3, or the antigen composition prepared by the preparation method of claim 4 or 5, or the African swine fever virus P30 protein and P72 protein expressed by the recombinant Escherichia coli of claim 6.