CN118221784A - African swine fever virus p72 protein antigen epitope peptide, monoclonal antibody and application thereof - Google Patents

Abstract

The invention discloses an African swine fever virus p72 protein epitope peptide, which is 102-114 peptide segments or/and 239-248 peptide segments of the African swine fever virus p72 protein, and the amino acid sequence of the epitope peptide is ¹⁰²FHDMVGHHILGACH¹¹⁴ or/and ²³⁹GPLLCNIHDL²⁴⁸. The antigen epitope peptide and the monoclonal antibody provided by the invention are used for detecting African swine fever virus, have the characteristics of good specificity and high accuracy, and provide a new tool for immunological detection of African swine fever virus p72 protein.

Description

African swine fever virus p72 protein antigen epitope peptide, monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of medicine and biology, and particularly relates to an African swine fever virus p72 protein antigen epitope peptide, a monoclonal antibody thereof and application thereof.

Background

Monoclonal antibody technology is an important technology in modern life science research, and plays an irreplaceable important role in research on the structure and function of proteins and in immunodiagnosis of human beings and animals. Monoclonal antibodies have numerous applications in disease prevention, clinical treatment, disease diagnosis and detection, and are also widely used as research tools in the field of biological research. African swine fever (AFRICAN SWINE FEVER, ASF) is an acute, virulent, hemorrhagic, highly contagious animal infectious disease caused by infection of pigs with African swine fever virus (AFRICAN SWINE FEVER virus, ASFV), and has the characteristics of short course, strong infection capability, wide infection range, high morbidity and mortality, and the like, and is a huge hazard to the pig industry worldwide. The primary target cells for viral infection are mononuclear-macrophages, whose regulation of macrophage function is critical for pathogenic and immune evasion mechanisms. ASFV is the only known arbo DNA virus currently available for transmission by ticks biting ASFV and by pigs exposed to ASFV infection and products thereof. ASF rapidly spreads to most provinces and regions of China after the first occurrence of Shenyang in China in 8 months of 2018, which causes serious economic loss to pig industry in China. Until now, there is no effective vaccine and specific drug against ASF, and prevention and control mainly depends on rapid detection and early diagnosis.

ASFV is a linear double-stranded DNA virus of regular icosahedron, the whole length of the viral genome is 170-194kbp, encoding 151-167 open reading frames, and the particle diameter is 175-215nm. The structure is more complicated, and the five layers from inside to outside are formed: the nucleoids, core shells, inner membranes, capsids and outer membranes have corresponding structural proteins in each layer, and play an important role in virus particle adsorption, entry and replication. Although the function of some viral proteins has been widely studied, there are many viral proteins whose functions are unknown. In recent years, as many proteins of ASFV are being studied, the importance of p72 protein has been developed. Accordingly, intensive studies on p72 protein are necessary.

Wherein the p72 protein is an important antigen protein encoded by the B646L gene and having a size of 73.2kD, and is also a main structural protein of ASFV. The p72 protein, also known as p73 protein, accounts for 31% -33% of the viral particles. p72 forms homotrimers, each monomer adopts a double jelly roll structure consisting of pseudo-hexamer capsules. The protein is mainly involved in viral capsid assembly and plays an important role in virus adsorption and invasion of susceptible cells. Late viral promoters synthesize late proteins, B646L genes are expressed later in the virus infection. The newly synthesized p72 is homogeneously distributed between the soluble cytoplasm and the membrane bound to the Endoplasmic Reticulum (ER) and assembles on the ER membrane to form a large capsid or matrix precursor. The p72 protein has conformational dependence in the ASFV invasion process and its expression is an indicator of viral replication. The p72 antibody can inhibit binding of ASFV to macrophages, but in antibody-mediated immunoprotection, the p72 antibody cannot play a decisive role.

P72 is highly conserved among ASFV strains isolated from different parts of the world. This demonstrates the stability of p72 antigenicity and lays a theoretical foundation for serological testing of ASFV. At present, based on p72, a double-antibody sandwich ELISA detection method, a qPCR detection method or an indirect ELISA detection and other diagnostic methods are established.

As a preferred serological detection method of ASFV recommended by world health organization (WOAH), the enzyme-linked immunosorbent assay (ELISA) has the characteristics of simple operation, high detection speed, good sensitivity and specificity, and the like, can be used for mass detection in pig farms, and is one of ASFV detection methods with the widest application range at present. The ELISA detection method of ASFV is mostly an indirect ELISA established based on ASFV structural proteins, and has good sensitivity and specificity. Among a plurality of structural proteins of ASFV, p72, p54 and p30 proteins are the most widely used detection target proteins at present due to good immunogenicity.

In summary, p72 protein is widely used as an antigen target in the development of diagnostic methods, and its monoclonal antibody plays an important role in the diagnostic prevention of ASFV and the functional research of p72 protein. Thus, there is a need for specific monoclonal antibodies to identify monoclonal antibodies that recognize novel epitopes.

Disclosure of Invention

Aiming at the technical problems to be solved, the invention provides an African swine fever virus p72 protein epitope peptide, a monoclonal antibody and application thereof, and overcomes the defects of the prior art.

The invention aims to provide an African swine fever virus p72 protein epitope peptide, wherein the epitope peptide is 102-114 peptide fragments or/and 239-248 peptide fragments of the African swine fever virus p72 protein, and the amino acid sequence of the epitope peptide is ¹⁰²FHDMVGHHILGACH¹¹⁴ or/and ²³⁹GPLLCNIHDL²⁴⁸.

The second purpose of the invention is to provide the application of the antigen epitope peptide in detecting the specific antibody of the African swine fever virus.

The third object of the present invention is to provide a monoclonal antibody of the p72 protein of African swine fever virus, which is specific to the epitope peptide.

A preparation method of a monoclonal antibody for resisting African swine fever virus p72 protein comprises the following steps:

1) PCR amplification is carried out by taking pCMV-myc-p72 as a template to obtain the African swine fever virus p72 gene;

2) Connecting the African virus p72 gene with a target expression vector pCold-I to obtain a recombinant prokaryotic expression plasmid pCold-I-p72;

3) Converting the recombinant prokaryotic expression plasmid pCold-I-p72 into DE3/BL21 competent escherichia coli (E.coli), and carrying out induced expression to obtain p72 protein;

4) Immunizing a mouse by using p72 protein, and fusing spleen cells of the immunized mouse with myeloma cells SP2/0 to obtain five hybridoma cells;

5) The mouse is injected into the abdominal cavity to inject mouse hybridoma cells, ascites is collected, supernatant is collected by centrifugation, and five African swine fever virus p72 protein monoclonal antibodies are obtained.

Five monoclonal antibodies are prepared by prokaryotic expressed ASFV structural protein p72, and two new antigen epitopes '¹⁰²FHDMVGHHILGACH¹¹⁴' and '²³⁹GPLLCNIHDL²⁴⁸' are identified. Five monoclonal antibodies specific for the above epitopes are used simultaneously for a variety of immunological assays including, but not limited to ELISA, WB, IFA. And then, based on the two identified antigen epitopes and one monoclonal antibody 2F3 clone, respectively exploring and optimizing an indirect ELISA method for detecting ASFV antibodies and a double-sandwich ELISA method for detecting ASFV antigens. According to the invention, 4 different combinations of 2 monoclonal antibodies, namely 2F3 and 1D7, which respectively recognize two epitopes, are tested, and one monoclonal antibody 2F3 is found to be the strongest signal of a capture antibody and a detection antibody after optimization, so that the effect is the best.

The fourth object of the invention is to provide the application of the african swine fever virus p72 protein monoclonal antibody in immunological detection.

Such applications include ELISA assays, WB assays, IFA assays.

In order to supplement an ASF detection and diagnosis method, the invention uses a cell fusion hybridoma technology to prepare an ASFV structural protein p72 monoclonal antibody, and establishes an indirect ELISA and double-sandwich ELISA detection method based on the prepared monoclonal antibody and antigen epitope peptide thereof.

Finally, the invention also provides application of the antigen epitope peptide and the monoclonal antibody in diagnosis or detection of African swine fever virus.

Applications described above include any one or more of the following:

(1) The antigen epitope peptide and the monoclonal antibody are applied to the preparation of a kit for diagnosing or detecting African swine fever virus;

(2) The antigen epitope peptide and the monoclonal antibody are applied to the preparation of medicines for diagnosing or detecting African swine fever virus infection;

(3) The application of the antigen epitope peptide and the monoclonal antibody in preparing a reagent for diagnosing or detecting African swine fever virus infection.

The invention establishes a method for detecting the African swine fever virus antibody by indirect ELISA based on antigen epitope peptide identified by the African swine fever virus monoclonal antibody.

The invention provides an indirect ELISA detection method for African swine fever virus antibodies based on the epitope peptide.

The indirect ELISA detection method comprises the following steps:

1) African swine fever virus p72 protein epitope peptide ¹⁰²FHDMVGHHILGACH¹¹⁴ and/or ²³⁹GPLLCNIHDL²⁴⁸ are/is coated, the coating concentration of the epitope peptide is 0.625 ng/. Mu.L, 100. Mu.L of each ELISA hole is added, incubated overnight at 4 ℃, and washed after blocking for 2 hours by 5% BSA;

2) Serum to be tested was added and 100 μl of PBS buffer was added to each well to dilute 1:5 times of African swine fever positive recovery pig serum is incubated for 2 hours;

3) The second enzyme-labeled antibody was added, and 100. Mu.L of the solution diluted 1 with PBS buffer was added to each well: 10000 times of pig-derived enzyme-labeled antibody is incubated for 1h;

4) Adding a color development liquid, developing for 10min, and adding a stopping liquid to stop the reaction;

5) And (3) measuring the OD ₄₅₀ value by adopting an enzyme-labeled instrument, wherein the P/N is more than or equal to 2.0 and positive.

The invention also provides a double-sandwich ELISA detection method for the African swine fever virus antigen based on the monoclonal antibody.

The double-sandwich ELISA detection method comprises the following steps:

(1) Coating an ELISA plate by using the African swine fever virus p72 protein monoclonal antibodies 2F3 and 1D7 as capture antibodies, wherein the coating concentration is 1 mug/ml, incubating overnight at 4 ℃, sealing and washing;

(2) The biotin-labeled monoclonal antibodies 2F3 and 1D7 were diluted at a concentration of 1. Mu.g/mL and incubated as detection antibodies;

(3) Adding a color development liquid for developing color, and adding a stopping liquid for stopping the reaction;

(4) And (3) measuring the OD ₄₅₀ value by adopting an enzyme-labeled instrument, and selecting the pairing of the capture antibody with the maximum P/N value and the detection antibody.

The specific monoclonal antibody of the structural protein p72 of African Swine Fever Virus (ASFV) is a specific and useful tool for ASFV research, has been successfully applied to various immunological tests, including enzyme-linked immunosorbent assay (ELISA), western Blot (WB) and Immunofluorescence (IFA) tests, and can also be applied to ASFV function research. Meanwhile, the invention establishes an indirect ELISA method for detecting ASFV antibody based on monoclonal antibody recognition epitope peptide and a double-sandwich ELISA method for detecting ASFV antigen based on monoclonal antibody, and provides a new protein reference for ASF detection. The invention utilizes a prokaryotic expression system to obtain recombinant ASFV p72 protein to prepare a specific monoclonal antibody thereof, identifies a novel antigen epitope recognized by the monoclonal antibody, establishes and optimizes an ASFV serological antibody and an antigen detection method, and provides an effective tool for subsequent ASF early diagnosis and prevention and control.

Drawings

FIG. 1 shows the SDS-PAGE identification of purified p72 protein and Western Blotting verification result of recombinant protein in the present invention.

FIG. 2 shows the result of Western blotting identification of ASFV p72 protein 5 monoclonal antibody in the present invention.

FIG. 3 shows the result of IFA detection of ASFV p72 protein 5 monoclonal antibody of the present invention.

FIG. 4 is a schematic diagram of the identification of an epitope recognized by ASFV p72 monoclonal antibody in the present invention.

FIG. 5 shows the identification of the epitope recognized by the p72 monoclonal antibody by Western blotting experiments in the present invention.

FIG. 6 shows the SDS-PAGE identification of purified monoclonal antibodies of the invention.

Detailed Description

The following describes the technical scheme of the present invention in further detail by combining examples: the present embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are provided, but the protection rights of the present invention are not limited to the following embodiments.

The materials and reagents mentioned in the invention are all available from domestic and foreign sources or are paid out by the public, and will not be described here.

Example 1

(1) ASFV p72 gene clone expression, protein purification and monoclonal antibody preparation

Genomic DNA was extracted from ASFV positive samples, and p72/B646L gene was PCR amplified, cloned into KpnI and SalI sites of pCMV-myc vector to obtain pCMV-myc-p72 recombinant plasmid, which was stored in the university of Yangzhou veterinary medicine laboratory. The stored recombinant plasmid pCMV-myc-p72 is used as a template, cloning and detection PCR primers are designed (see Table 18), and ASFV p72 gene is connected with a target expression vector pCold-I to obtain a recombinant prokaryotic expression plasmid pCold-I-p72. The recombinant prokaryotic expression plasmid is transformed into DE3/BL21 competent escherichia coli (E.coli), and the optimal induction condition of prokaryotic protein expression is as follows: the IPTG inducer was cultured at a concentration of 1mM at 25℃for 6 hours. The protein was found to be expressed mainly in inclusion bodies by supernatant and precipitation analysis of the cell lysate. After the thalli are fully crushed by ultrasonic, collecting the precipitate by high-speed centrifugation, washing, then re-suspending the precipitate by PBS solution containing 8M urea, crushing overnight suspension by an ultrasonic crusher for 30min at 4 ℃, and putting the suspension into a dialysis bag for gradient dialysis. After the dialysis is completed, the dialysis bag is taken out, the PEG20000 concentrate is spread, the liquid in the bag is taken out after the concentration, 30 mu L of sample is taken out for SDS-PAGE coomassie brilliant blue staining analysis, and the size and concentration of the purified protein are determined. SDS-PAGE detection is carried out on the purified protein, the result is shown in figure 1, the p72 protein band after purification is single, the protein concentration is 1 mug/uL, and the requirement of later immunization is met; western Blotting test is carried out on the purified protein sample, and the labeled recombinant protein p72 can be specifically identified by His antibody, and the band size is 72kD.

BALB/c mice 6-8 weeks old were immunized subcutaneously with purified p72 protein, with a second time interval of 14 days from the first immunization and a third time interval of 7 days from the second immunization. The mice with highest ELISA titers of antibodies in serum are subjected to intraperitoneal injection for boosting, and serum is collected and separated 5 days after three immunizations. The spleen cells of the mice and the myeloma SP2/0 cells of the mice are fused within 72 hours after the immunization (additional information after specific operation steps) to obtain hybridoma cell clones. Wherein, 5 ELISA positive cell clones exist, and further Western-blot detection finds that the supernatant of 5 hybridoma cell clones can generate specific reaction with p72 protein in eukaryotes and viruses. .

BALB/c mice of 8-12 weeks old were selected and injected intraperitoneally with 0.2-0.3 mL/mouse sterile liquid paraffin. Mice were intraperitoneally injected with hybridoma cells (1-3X 10 ⁶ cells/0.3 mL/mouse) 3 days later and the degree of abdominal distension was observed 5 days later to determine the ascites production. If the abdomen of the mouse is touched by hands and has obvious swelling feel and the abdomen of the mouse is in a tight state, the ascites can be collected by the 16-gauge needle. Continuously collecting 2-3 times, centrifuging the collected ascites at 2000rpm for 5min, removing the uppermost adipose tissue and erythrocytes mixed into the ascites, and collecting the supernatant. And (5) after sub-packaging, freezing and storing in a refrigerator at the temperature of minus 80 ℃. Thus, 5 ASFV p72 monoclonal antibodies 1B7, 1D7, 2F3, 5D4 were obtained.

Table 18ASFV p72 amplification primer sequence and truncated protein gene PCR amplification primer sequence

(2) ASFV p72 protein monoclonal antibody identification

(A) Monoclonal antibody Western blotting identification

293T cells were plated on 12-well cell culture plates (2X 10 ⁵ cells/well). When the cell density reaches 70% -80%, the Lip2000 is used to transfect pCMV-myc-p72 plasmid and empty plasmid pCMV-myc each 1 mug/hole. Cells were collected 24h after transfection. Meanwhile, primary Porcine Alveolar Macrophages (PAM) were plated in 12-well cell culture plates (2×10 ⁵ cells/well), and the cells were infected with ASFV virus stored in the present laboratory, and collected after 72 hours. Western blotting detection is carried out by using the prepared ascites monoclonal antibody as a primary antibody of the cell lysis protein, and the specific reaction of the monoclonal antibody on the p72 protein is identified (see figure 2). As shown in FIG. 2, 5 strains of p72 monoclonal antibody were prepared as ascites fluid according to 1: and (3) diluting with 1000, and respectively verifying the reactivity of 5 monoclonal antibodies with a protein sample of 293T cells transfected with pCMV-myc-p72 and a protein sample of PAM infected by ASFV through WB, wherein Western Blot analysis shows that the 5 monoclonal antibodies can specifically recognize exogenous and endogenous p72.

(B) Monoclonal antibody IFA identification

293T cells were plated on 12-well cell culture plates (2X 10 ⁵ cells/well). When the cell density reaches 70% -80%, the Lip2000 is used to transfect pCMV-myc-p72 plasmid and empty plasmid pCMV-myc each 1 mug/hole. The IFA experiments were performed 24h after transfection. Meanwhile, primary PAM cells were plated in 12-well cell culture plates (2×10 ⁵ cells/well), and the cells were infected with ASFV virus stored in this laboratory, and IFA experiments were performed after 72 hours. IFA reactivity of the monoclonal antibodies to the p72 protein was identified (see fig. 3). In FIG. 3, 5 strains of p72 monoclonal antibody were prepared as ascites fluid according to 1:200 dilution, carrying out immunofluorescence detection on 293T cells transfected with pCMV-myc-p72 recombinant genes and PAM infected by ASFV, and the experimental results show that 5 p72 monoclonal antibodies can specifically react with exogenous and endogenous p 72.

(C) Monoclonal antibody subclass identification

The obtained five monoclonal antibodies were subjected to antibody subclass identification using a monoclonal antibody subclass identification kit of the company salvinsis. The specific method is to see additional information. After the completion, the result can be directly observed with naked eyes, and the enzyme-labeled secondary antibodies corresponding to the developed dark blue holes are the subclasses of the monoclonal antibodies to be detected (see table 1).

TABLE 1 identification of ASFV p72 monoclonal antibody subclasses

(D) Indirect ELISA determination of antibody titers

The titers of the five monoclonal antibodies were determined by indirect ELISA. ELISA plates were coated with 100. Mu.L of p72 recombinant purified protein per well (1 ng/mL concentration of antigen diluted) as detection antigen, and the monoclonal antibody ascites (1:800, 1:1600, 1:3200, 1:64 000, 1:12800, 1:25600, 1:51200, 1:102400) were measured at different dilutions, 2 replicate wells were set per dilution, non-immune mouse serum was used as negative control, immune p72 protein mouse serum was used as positive control, OD450nm values were measured with an enzyme-labeled instrument, and five monoclonal antibody titers were determined (see Table 2).

TABLE 2 determination of titers of ASFV p72 monoclonal antibody ascites

(E) ASFV p72 monoclonal antibody epitope identification

The minimal epitope recognized by the monoclonal antibody was identified by Western blotting. The p72 protein is firstly divided into three fragments (aa 1-215, aa 216-430 and aa 431-646 respectively), the coding sequence of the p72 protein fragment is connected to a eukaryotic expression vector pEGFP-N1, and then the p72 truncated recombinant pEGFP-N1 expression fragment is constructed by shortening from the N segment and the C end according to the condition of ascites monoclonal antibody WB reaction (see figure 4 for the truncation condition). 4 shows the length and amino acid position of truncated recombinant p72 proteins, a series of N-terminally truncated p72 was constructed on pEGFP-N1, C-terminally truncated p72 was constructed on pEGFP-N1, and successfully expressed in 293T cells. Blue indicates that the antibody reacted with the monoclonal antibody, and black indicates that the antibody did not react with the monoclonal antibody.

After verification by sequencing, plasmids were extracted, 293T cells were transfected separately, and after 24h cells were collected and lysates were added. Western blotting was used to verify the fragment of the truncated protein that was recognized by the monoclonal antibody. Based on the results, the p72 protein fragment was further truncated until it was unrecognizable by the p72 mab. The smallest fragment that can be recognized by the mab is the epitope peptide of the p72 mab (see figure 5). In FIG. 5, the Western blot analysis of the reactivity between the truncated p72 protein fragment and two monoclonal antibodies, aa is an abbreviation for amino acids, and the above experimental results show that the epitope recognized by the 1B7, 2F3, 5D4 monoclonal antibodies is ¹⁰²FHDMVGHHILGACH¹¹⁴, as shown in SEQ ID NO.1, and the epitope recognized by the 1D7 monoclonal antibody is ²³⁹GPLLCNIHDL²⁴⁸, as shown in SEQ ID NO. 2.

(F) Ascites purification and labelling of ASFV p72 protein monoclonal antibody

And selecting two monoclonal antibodies of 2F3 and 1D7 according to the antigen epitope and the ascites titer for purification. The prepared monoclonal antibody ascites was purified according to the following procedure. Before loading, the monoclonal antibody ascites (1 mL of monoclonal antibody ascites at a time) is diluted by PBS according to the ratio of 1:3, filtered by a 0.45um filter membrane, then packed into a column (1 mL), the bottom cap of the purification column is taken out, and 200 mu L of Protein A/G PLUS-Agarose is taken and added into the purification column. Washing with 10mL PBS and balancing the pre-packed column, controlling the flow rate at 1mL/min, covering the bottom cap, adding the filtered ascites into the purification column, and rotating at 4deg.C for 2-4h. After sufficient binding of the antibodies, the pre-loaded column was washed with 20 volumes of PBS to remove non-specifically adsorbed heteroproteins, with a flow rate of 1mL/min being recommended. Finally, 100L of 1M Tris-HCl (pH 8.5) was previously added to a 1.5mL centrifuge tube, and the bound antibody was eluted with 5mL 100mM glycine (pH 3.0), and the eluate was collected in 5 tubes, 1mL per tube. And then dialyzing and concentrating the purified antibody, taking 9 mu L of the purified target antibody, adding 3 mu L of 4×loading, shaking and mixing uniformly, and carrying out denaturation at 100 ℃ for 5-10min to prepare a protein sample. After SDS-PAGE, coomassie brilliant blue staining was performed to identify the purity of the antibody (see FIG. 6), and SDS-PAGE was performed to verify that the purified 2 monoclonal antibodies (lanes 3 and 4) had distinct bands at 55kDa and 25kDa, respectively, and that the heavy chain and the light chain of the antibody were identical in molecular weight. The concentration of the purified monoclonal antibody after concentration was determined by BCA method, and the purified monoclonal antibody was stored at-80℃for later use after being split into 1.5mL EP tubes and labeled with the name and concentration. Part of the ascites was then sent to the company for HRP labeling.

(3) Establishment, optimization and application of ASFV p72 epitope peptide-based indirect ELISA detection method

(A) Selection of optimal epitope peptides

Two synthesized epitope peptides "¹⁰²FHDMVGHHILGACH¹¹⁴" and "²³⁹GPLLCNIHDL²⁴⁸" were coated individually or in combination, and incubated overnight at 4℃at a concentration of 1 ng/. Mu.L. The inactivated ASF positive recovered pig serum is diluted 1:10 to be used as a primary antibody, and a pig-derived enzyme-labeled antibody is used as an ELISA secondary antibody. The subsequent ELISA operation is carried out with additional information, finally, the OD ₄₅₀ value is read by an enzyme-labeled instrument, the P/N value is calculated, the corresponding epitope peptide mixed mode with large P/N value is selected as the optimal epitope peptide (see table 3), epitope peptide 1 '¹⁰²FHDMVGHHILGACH¹¹⁴' is the optimal epitope peptide, the mixing of epitope peptide 1 and epitope peptide 2 is carried out again, and epitope peptide 2 '²³⁹GPLLCNIHDL²⁴⁸' is carried out again.

TABLE 3 Indirect ELISA detection of P/N values in selection experiments for optimal epitope peptides

(B) Optimization of optimal antigen peptide coating concentration and optimal serum dilution

The antigen epitope peptide selected in the experimental results is used for coating a 96-well ELISA plate, the concentration of each row is respectively 10, 5, 2.5, 1.25, 0.625 and 0.3125 ng/mu L from top to bottom, 100 mu L is added to each well, and the mixture is incubated at 4 ℃ overnight. The blocking and washing procedure was identical to the second chapter ELISA procedure. The inactivated African swine fever positive recovered pig serum is used as a primary antibody, the dilution ratio of positive pig serum from left to right in each column is 1:5, 1:10, 1:50, 1:100, 1:200, 1:400 and 1:800, meanwhile, negative pig serum is used as a negative control, and the last column is added with antibody diluent to be used as a blank control. The secondary antibody uses a swine enzyme-labeled antibody, and the subsequent color development operation is the same as above. Finally, 2M sulfuric acid is added to stop the reaction, an enzyme-labeled instrument is used for reading the OD ₄₅₀ value, and the antigen peptide coating concentration and the serum dilution with the large P/N value are selected as the optimal ELISA conditions by calculating the P/N value (see table 4).

TABLE 4 indirect ELISA detection of P/N values in an optimization experiment for antigen coating concentration and serum dilution

(C) Optimization of optimal coating conditions and optimal blocking conditions for antigens

And further optimizing subsequent experimental conditions according to the optimal epitope peptide, the optimal antigen coating concentration and the optimal serum dilution ratio selected from the experimental results. The experiment was performed at 4℃overnight, 37℃for 1h, and 37℃for 2h. In selection of blocking method, five conditions of unblocking, 2% BSA, 5% BSA, 2% skimmed milk powder, and 5% skimmed milk powder were set for experiments. Other conditions are the same as those described above. Finally, the OD ₄₅₀ value is read by an enzyme label instrument, and the corresponding condition with large P/N value is selected as the optimal condition by calculating the P/N value (see table 5).

TABLE 5 indirect ELISA detection of P/N values in epitope peptide coating conditions and blocking condition optimization experiments

(D) Optimization of optimal primary antibody and secondary antibody incubation time and optimal color development time

And (5) continuously optimizing the experimental conditions on the basis of the experimental results. The incubation time of the primary antibody pig serum is set to be 1h and 2h, the incubation time of the enzyme-labeled secondary antibody is also set to be 1h and 2h, the color development time is set to be 10min, 15min and 20min, and other conditions are the same as the operation. Finally, the OD ₄₅₀ value is read by an enzyme label instrument, and the corresponding condition with large P/N value is selected as the optimal condition by calculating the P/N value (see table 6).

TABLE 6 indirect ELISA detection of P/N values in primary and secondary antibodies and development time optimization experiments

(E) Determination of sample threshold

The optimal epitope peptide, the optimal coating concentration, the optimal sealing condition, the optimal primary antibody and secondary antibody incubation time and the optimal color development time are determined through the experiment. Experiments were then performed using the optimized ELISA optima described above, and the threshold values for the established ELISA method were determined. Short peptides were coated onto 96-well elisa plates at optimal concentration, 100 μl per well. 30 negative porcine serum was used as primary antibody, 100 μl was added per well, and the other procedures were the same as the optimal experimental conditions. Finally, the OD ₄₅₀ value is read by an enzyme label instrument and the average value is calculatedAnd error SD according to the statistical formula/>Obtain two critical values/>And/>(See Table 7).

TABLE 7 threshold results of indirect ELISA detection of epitope peptides

(F) Serum specificity and sensitivity detection

And (3) carrying out experiments according to the optimized conditions, and carrying out specificity and sensitivity detection on the established indirect ELISA detection method. And (3) sealing after coating the antigen peptide, performing chromogenic reaction after incubating the primary antibody and the secondary antibody, and finally stopping the reaction. Inactivated ASFV, PRRSV, PEDV, SIV positive and negative porcine serum were used as primary antibodies. The specificity of the indirect ELISA assay was determined by reading the OD ₄₅₀ values with an ELISA reader (see Table 8).

TABLE 8 detection of serum-specific OD450 values by indirect ELISA

(4) Establishment, optimization and application of ASFV p72 protein monoclonal antibody-based double-sandwich ELISA detection method

① Antibody pairing experiments

Two purified p72 monoclonal antibodies 2F3 and 1D7 are used as capture antibodies, and simultaneously the biotin-labeled antibodies are used as detection antibodies, and pairwise pairing experiments are carried out to screen optimal antibody pairs. Monoclonal antibodies 2F3, 1D7 were used as capture antibodies, diluted 1- μg/ml and coated with ELISA plates, each concentration repeated three times. The biotin-labeled monoclonal antibodies 2F3 and 1D7 are diluted according to 1 mug/ml, incubated as detection antibodies, additional information is seen in the subsequent ELISA operation, and finally the OD ₄₅₀ value is read by an enzyme-labeled instrument. The pairing of the capture antibody with the detection antibody with the greatest P/N value was selected as the optimal condition by calculating the P/N value (Table 9).

TABLE 9 determination of P/N values for double-sandwich ELISA assays in optimal pairing of Capture antibodies with detection antibodies

From this, monoclonal antibody clone 2F3 was found to have the best effect and was used as both capture and detection antibodies; after combination of two pairs, 2F3 was found to have the best effect as both capture antibody and detection antibody.

② Selection of optimal antibody concentration

Based on the best pairing result of the capture antibody and the detection antibody, the best coated antibody and the detection antibody concentration are searched by a chessboard titration method. The capture antibody was coated with a concentration gradient of 10,5,2.5,1.25,0.625. Mu.g/mL, the detection antibody was incubated with a concentration gradient of 2,1,0.5,0.25. Mu.g/mL, the remaining conditions were the same as described above, and duplicate wells were set. The OD450 value is read by an enzyme labeling instrument, and the corresponding condition with a large P/N value is selected as the optimal condition by calculating the P/N value (see Table 10).

TABLE 10 selection of optimal antibody concentration double sandwich ELISA detection P/N values in experiments

③ Determination of optimal coating conditions for Capture antibodies

The optimal pairing concentration result and the optimal antibody concentration of the capture antibody and the detection antibody are determined in the experiment, so that the subsequent experimental conditions are further optimized. The experiment was performed by setting three conditions of 4℃overnight, 37℃for 1h, and 37℃for 2h when the capture antibody was coated. Other conditions are the same as the above, finally, the OD450 value is read by using an enzyme-labeled instrument, and the corresponding condition with a large P/N value is selected as the optimal condition by calculating the P/N value (see Table 11).

Table 11 double-sandwich ELISA detection of P/N values in Capture antibody optimal coating Condition experiments

④ Determination of optimal incubation time for antigen

Based on the above working condition screening results, the optimal incubation time of the antigen was searched. The antigen incubation time was set to be 0.5h, 1h, 1.5h, 2h, and the other conditions were the same as described above. Finally, the OD450 value is read by an enzyme labeling instrument, and the corresponding condition with large P/N value is selected as the optimal incubation time of the antigen by calculating the P/N value (see Table 12).

Table 12 double sandwich ELISA assay P/N values in the best incubation time experiments for antigens

⑤ Determination of optimal incubation time for detection of antibodies

Based on the screening result of the working condition, the optimal incubation time of the detection antibody is searched, and the incubation time of the detection antibody is set to be four incubation times of 0.5h, 1h, 1.5h and 2h, and other conditions are the same as the operation. Finally, the OD450 value is read by an enzyme labeling instrument, and the corresponding condition with the large P/N value is selected as the optimal incubation time of the antibody by calculating the P/N value (see Table 13).

Table 13 double sandwich ELISA assay P/N values in the test for optimal incubation time of detection antibodies

⑥ Optimal closure condition determination

Based on the above screening results of working conditions, the best blocking conditions were found, and experiments were performed with the blocking conditions set to five conditions of unblocking, 2% bsa, 5% bsa, 2% skim milk powder, and 5% skim milk powder, and the other conditions were the same as those described above. Finally, the OD450 value is read by an enzyme labeling instrument, and the corresponding condition with large P/N value is selected as the optimal condition by calculating the P/N value (see table 14).

TABLE 14 double sandwich ELISA detection of P/N values in optimal blocking time experiments

⑦ Determination of optimal color development time

And (5) continuously optimizing the experimental conditions on the basis of the experimental results. The color development time of TMB color development liquid is set to be 10min, 15min and 20min, and other conditions are the same as the operation. Finally, the OD ₄₅₀ value is read by an enzyme label instrument, and the corresponding condition with large P/N value is selected as the optimal condition by calculating the P/N value (see table 15).

TABLE 15 double sandwich ELISA detection of P/N values in the best color development time experiment

⑧ Determination of sample threshold

Experiments were performed using the optimized ELISA optima described above, and the threshold values for the established ELISA method were determined. The capture antibodies were coated onto 96-well elisa plates at optimal concentrations, 100 μl per well. 30 negative porcine serum was used as primary antibody, 100 μl was added per well, and the other procedures were the same as the optimal experimental conditions. Finally, the OD ₄₅₀ value is read by an enzyme label instrument and the average value is calculatedAnd error SD according to the statistical formula/>Obtain two critical values/>And (3) with(See Table 16).

Table 16 threshold results for double sandwich ELISA assay

⑨ Antigen-specific detection

And (3) carrying out experiments according to the optimized conditions, and carrying out specific detection on the established double-sandwich ELISA detection method. Inactivated ASFV, PRRSV, PEDV, SIV positive and negative pig samples were used. The specificity of the two-sandwich ELISA assay was determined by reading the OD ₄₅₀ values with an ELISA reader (see Table 17).

Table 17 double sandwich ELISA for detecting OD450 values specific for ASFV positive samples

Additional information: cell fusion specific operation and monoclonal antibody subclass identification method

Cell fusion

① Mixing 1×10 ⁸ spleen cells with 1-2×10 ⁷ myeloma cell SP2/0 in 50mL fusion tube, adding serum-free DMEM medium to 30mL, and mixing thoroughly.

② Centrifugation at 1000rpm for 5-10min, and removal of supernatant (at this time, the bottom of the tube precipitated as lower red spleen cells and upper white SP2/0 cells, which were approximately equivalent in packed volume).

③ Holding the fusion tube bottom by palm rotation to make the cell mass in loose and homogeneous state.

④ Slowly dripping 1mL of PEG1500 at 37deg.C with a 1mL pipette, and rotating the centrifuge tube while adding to mix PEG and cells thoroughly.

⑤ 10-20ML of serum-free DMEM medium preheated to 37℃was added slowly and then quickly over 90s with a pipette, diluting and simultaneously terminating the PEG1500 effect. Standing at 37deg.C for 10min.

⑥ 1000Rpm, centrifuging for 5-10min, and discarding supernatant.

⑦ Adding 40mL HAT medium, gently stirring and mixing the precipitated cells.

⑧ Split charging into 96-well cell culture plate containing celiac feeder cells with a row gun, wherein each well is 100 μl; the plates were then incubated at 37℃in a 5% CO2 incubator.

⑨ 7-10D after fusion (hybridoma cell clones are very evident), 1/2 medium can be exchanged with fresh HAT medium.

⑩ Observing the growth condition of the hybridoma cells, and sucking out the supernatant for ELISA antibody detection when the area of the bottom of the hole exceeds 1/10 and the supernatant turns yellow.

Monoclonal antibody subclass identification method

① The kit was returned to room temperature, and the cleaning solution was prepared to a working concentration (1 part of concentrated cleaning solution plus 19 parts of pure water) with pure water.

② Taking out the ELISA plate, setting 8 holes for each monoclonal antibody sample to be detected, and setting 8 holes for positive control and positive control.

③ Firstly adding 50 mu L/hole of sample diluent into each sample detection hole, and then adding 50 mu L of cell culture supernatant into detection holes containing the sample diluent, wherein each sample is added with 8 holes. Positive control and negative control wells were added with universal positive control and negative control, respectively, 100 μl per well. And (5) attaching a sealing plate film and incubating for 30min at 37 ℃.

④ Discarding the liquid in the plate, washing for 5 times, and drying. 100 mu L of 8 enzyme-labeled secondary antibodies are respectively added into 8 sample detection holes, and the same enzyme-labeled secondary antibodies are also added into 8 holes of a general positive control. And (5) attaching a sealing plate film and incubating for 30min at 37 ℃.

⑤ Discarding the liquid in the plate, washing for 5 times, and drying. 50 mu L of each of the developing solution A and the developing solution B is added into each hole, and 1 new sealing plate film pasting plate is changed to develop color for 20min at the temperature of 37 ℃ in dark.

⑥ And after the detection, directly observing a result with naked eyes, wherein the enzyme-labeled secondary antibodies corresponding to the holes with dark blue color are the subclasses of the monoclonal antibodies to be detected.

Indirect ELISA procedure

① Using purified fusion protein as antigen, using coating liquid to make gradient dilution, and adding blank coating liquid into last row as negative control. 100. Mu.L of coated ELISA plates per well were placed in a refrigerator at 4℃overnight for 12h.

② Washing with PBST for 3 times, each for 5min, and drying on absorbent paper.

③ Blocking solution (PBS solution containing 1% BSA) was added at 200. Mu.L/well, incubated at 37℃for 2h, and washed as above.

④ Adding diluted serum: add 100 μl of 1: positive serum diluted 800, then 2-fold diluted positive serum was added from left to right per column at a final concentration of 1:819200. simultaneously, a row 1 is set: 800 diluted normal negative serum control wells, the last column is a blank control well of antibody dilution (PBS solution containing 0.2% BSA). Incubate at 37℃for 1.5h and wash as above.

⑤ Adding dilution ratio of 1:10000 HRP goat anti-mouse IgG as secondary antibody, 100. Mu.L/well, incubated at 37℃for 1h, washed as above.

⑥ Adding commercial substrate color development solution (50 μl/well), and reacting at 37deg.C for 10min.

⑦ The reaction was stopped by adding 2M H ₂SO₄ (50. Mu.L/well) and the OD ₄₅₀ value per well was determined.

Double-sandwich ELISA operation method

① Coating by diluting the capture antibody at known concentration with PBS, adding 100mL of the solution to each well, and coating the solution on a protein coating plate at 4 ℃ overnight.

② Washing with PBST for 3 times, each for 5min, and drying on absorbent paper.

③ Blocking solution (PBS solution containing 1% BSA) was added at 200. Mu.L/well, incubated at 37℃for 2h, and washed as above.

④ Incubating the sample, namely adding the sample to be detected into each antibody coated hole, taking a sample diluent as a blank control, incubating for 1.5h at 37 ℃ and washing the blank control, wherein the sample diluent is 100 mL/hole.

⑤ And incubating enzyme-labeled antibodies, namely adding 100mL biotin-labeled antibodies with the same concentration into each antibody coating hole, incubating for 30min at room temperature, and washing the same.

⑥ Adding commercial substrate color development solution (50 μl/well), and reacting at 37deg.C for 10min.

⑦ The reaction was stopped by adding 2M H2SO4 (50. Mu.L/well) and the OD ₄₅₀ per well was determined.

The foregoing is merely illustrative of the embodiments of the present invention, and the scope of the present invention is not limited thereto, and any person skilled in the art will appreciate that modifications and substitutions are within the scope of the present invention, and the scope of the present invention is defined by the appended claims.

Claims (10)

1. The african swine fever virus p72 protein epitope peptide is characterized in that the epitope peptide is 102-114 peptide fragments or/and 239-248 peptide fragments of the african swine fever virus p72 protein, and the amino acid sequence is ¹⁰²FHDMVGHHILGACH¹¹⁴ or/and ²³⁹GPLLCNIHDL²⁴⁸.

2. A monoclonal antibody to the p72 protein of african swine fever virus, wherein the monoclonal antibody is specific for the epitope peptide of claim 1.

3. The preparation method of the monoclonal antibody for resisting the African swine fever virus p72 protein is characterized by comprising the following steps:

1) PCR amplification is carried out by taking pCMV-myc-p72 as a template to obtain the African swine fever virus p72 gene;

2) Connecting the African virus p72 gene with a target expression vector pCold-I to obtain a recombinant prokaryotic expression plasmid pCold-I-p72;

3) Converting the recombinant prokaryotic expression plasmid pCold-I-p72 into DE3/BL21 competent escherichia coli, and carrying out induced expression to obtain p72 protein;

4) Immunizing a mouse by using p72 protein, and fusing spleen cells of the immunized mouse with myeloma cells SP2/0 to obtain five hybridoma cells;

5) The mouse is injected into the abdominal cavity to inject mouse hybridoma cells, ascites is collected, supernatant is collected by centrifugation, and five African swine fever virus p72 protein monoclonal antibodies are obtained.

4. The use of the african swine fever virus p72 protein monoclonal antibody according to claim 2 in immunological detection.

5. The use according to claim 4, comprising ELISA detection, WB detection, IFA detection.

6. Use of the epitope peptide of claim 1 and the monoclonal antibody of claim 3 for diagnosis or detection of african swine fever virus.

7. An indirect ELISA detection method of african swine fever virus antibodies based on the epitope peptide of claim 1.

8. The indirect ELISA detection method according to claim 7, characterized by comprising the following steps:

1) Coating an African swine fever virus p72 protein epitope peptide ¹⁰²FHDMVGHHILGACH¹¹⁴ or/and ²³⁹GPLLCNIHDL²⁴⁸, wherein the concentration of the epitope peptide coating is 0.625 ng/mL, adding 100 mL of each ELISA hole, incubating overnight at 4 ℃, blocking for 2 hours by adopting 5% BSA, and washing;

2) Serum to be tested was added, 100 mL dilution with PBS buffer was added per well 1:5 times of African swine fever positive recovery pig serum is incubated for 2 hours;

3) Adding enzyme-labeled secondary antibody, adding 100 mL to each well, diluting with PBS buffer solution for 1:10000 times of pig-derived enzyme-labeled antibody is incubated for 1h;

4) Adding a color development liquid, developing color 10 min, and adding a stopping liquid to stop the reaction;

5) And (3) measuring the OD ₄₅₀ value by adopting an enzyme-labeled instrument, wherein the P/N is more than or equal to 2.0 and positive.

9. A two-sandwich ELISA detection method of african swine fever virus antigen based on the monoclonal antibody of claim 2.

10. The method of double sandwich ELISA detection according to claim 9 comprising the steps of:

(1) Coating an ELISA plate by using the African swine fever virus p72 protein monoclonal antibodies 2F3 and 1D7 as capture antibodies, wherein the coating concentration is 1 mug/mL, incubating overnight at 4 ℃, sealing and washing;

(2) Diluting biotin-labeled monoclonal antibodies 2F3 and 1D7 according to the concentration of 1 mg/mL, and incubating as detection antibodies;

(3) Adding a color development liquid for developing color, and adding a stopping liquid for stopping the reaction;

(4) And (3) measuring the OD ₄₅₀ value by adopting an enzyme-labeled instrument, and selecting the pairing of the capture antibody with the maximum P/N value and the detection antibody to establish the optimal sandwich ELISA method.