CN112552396B - anti-African swine fever virus p54 protein monoclonal antibody, preparation method and application - Google Patents

Abstract

The invention relates to an anti-African swine fever virus p54 protein monoclonal antibody, a preparation method and an application. The invention designs and synthesizes a polypeptide capable of simulating the NTD of the conservative p54 protein, and couples it with BSA as an immunogen, and immunizes BALB/c mice by an immunological method to prepare an anti-ASFVp54 protein monoclonal antibody, and the antibody can specifically It recognizes and binds to the N-terminal region of p54 protein, and this recognition region (aa 1-29) is different from the existing commercial monoclonal antibodies. The present invention provides the heavy chain variable region and light chain variable region gene sequences of the above-mentioned antibodies. On this basis, conventional genetic engineering or protein engineering methods can be used to obtain the monoclonal antibody of the present invention. The monoclonal antibody has strong specificity and high sensitivity, and can specifically react with ASFV HLJ/18 strain viruses, but does not react with porcine-derived viruses such as CSFV, PCV2, PRRSV, PEDV and PRV. The research on the pathogenesis and clinical detection of ASFV pathogens have laid the foundation.

Description

Anti-African swine fever virus p54 protein monoclonal antibody, preparation method and application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to an anti-African swine fever virus p54 protein monoclonal antibody, a preparation method and an application.

Background technique

African swine fever virus (ASFV), a large and complex DNA virus, is the causative agent of African swine fever (ASF) and the only known member of the genus Asfarviridae. ASF is a highly contagious hemorrhagic disease in domestic pigs with up to 100% morbidity and mortality. ASFV was first discovered in Kenya in 1921, nearly a century ago. Since its spread to Georgia in 2007, ASFV has spread rapidly across multiple countries in Africa, Asia, and Europe, with recurring outbreaks in these regions. At the same time, ASFV has multiple natural hosts and reservoirs, which further increases the potential threat of ASF. Given the enormous risk and threat of this transboundary animal disease, it has been included in the Terrestrial Animal Health Code of the World Organization for Animal Health (OIE) and has been designated as an animal disease that must be reported to the OIE.

Due to the global economic importance of ASFV, attempts have been made since the late 1960s to develop an effective, safe vaccine to protect pigs from ASFV infection. However, due to the complex structure of ASFV and the limited understanding of viral protein functions, a safe and effective vaccine against ASF has not been developed in the world, and the control of African swine fever strictly relies on animal quarantine. Therefore, the detection and prevention of ASFV is very important.

ASFV possesses a large linear double-stranded DNA (dsDNA) genome of 170-194 kb, encoding more than 150 proteins. Although as many as 68 structural proteins have been identified from virions, the function of most proteins is unknown. ASFV virions have a unique multi-layered structure, and intracellular virions are composed of nucleosides (first layer), inner capsid (second layer), inner membrane (third layer) and capsid (fourth layer). Extracellular virions have an additional outer envelope (the fifth layer). Both intracellular and extracellular ASFV virions are infectious. Structural proteins that make up viral particles are distributed in these multilayered structures and play various roles in viral infection. Identification of the antigenic properties of viral structural proteins is essential for understanding virus-host interactions and is critical for improving ASFV serodiagnosis and designing subunit or viral vector vaccines. Currently, ASFV has 25 genotypes, and the genetic and antigenic diversity of certain genes is one of the main reasons for the lack of a cross-protective vaccine against this lethal swine disease. The diversity of antigenic structural proteins may also have a huge impact on the reliable diagnosis of ASFV infection.

In order to further study the role of antigenic structural proteins in virus infection and to achieve accurate diagnosis of ASFV virus infection, there is still a need in the art for more monoclonal antibodies that can recognize different structural proteins and different antigenic epitopes of ASFV. Provide new directions and new ideas. The coding sequence of African swine fever virus p54 protein is polymorphic among different strains, but its N-terminal region (NTD) is relatively conserved among strains and plays an important role in virus morphogenesis and invasion. However, there is no monoclonal antibody specific for NTD in the prior art and reports. Therefore, the present invention designs and synthesizes a polypeptide capable of simulating the p54 protein NTD, and couples it with BSA as an immunogen, in order to obtain a monoclonal antibody that specifically recognizes the p54 protein NTD, for further research on the characteristics of the antigenic structural protein p54 and The functions of different domains provide tools and provide new directions and ideas for the prevention and control of ASF.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an anti-ASFV p54 protein monoclonal antibody, the monoclonal antibody can specifically recognize the N-terminal region (aa 1-29) on the ASFV p54 protein.

The second object of the present invention is to provide a preparation method of African swine fever virus p54 protein monoclonal antibody.

The third object of the present invention is to provide the application of African swine fever virus p54 protein monoclonal antibody.

In order to achieve the above object, the present invention adopts the following technical solutions:

Anti-African swine fever virus p54 protein monoclonal antibody, the heavy chain variable region of the monoclonal antibody includes CDR1 whose amino acid sequence is shown in SEQ ID NO.1-3, and CDR2 whose amino acid sequence is shown in SEQ ID NO.2 , CDR3 whose amino acid sequence is shown in SEQ ID NO.3; the light chain variable region of the monoclonal antibody includes CDR1 whose amino acid sequence is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.5 CDR2, CDR3 whose amino acid sequence is shown in SEQ ID NO.6.

Specifically, the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.7; the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown in SEQ ID NO.8.

Specifically, the heavy chain constant region of the monoclonal antibody is of IgG1 type, and the light chain constant region is of Kappa type.

Specifically, the anti-ASFV p54 protein monoclonal antibody can specifically recognize the ASFV p54 protein; further, the above-mentioned anti-ASFV p54 protein monoclonal antibody can specifically recognize the ASFV p54 protein The N-terminal region of the ASFV is shown in SEQ ID NO.11; further, the above-mentioned anti-ASFV p54 protein monoclonal antibody can specifically recognize the peptide or table with the DSEFFQPV sequence on the ASFV p54 protein. bit.

It is obvious to those skilled in the art that, on the basis of the amino acid sequences of the heavy chain and light chain variable regions of the monoclonal antibodies specifically disclosed in the present invention, one or more amino acid additions, deletions, substitutions, etc. can be carried out by conventional protein engineering methods. Modified to obtain conservative variants or fragments thereof, while still maintaining specific binding to the peptide segment or epitope with DSEFFQPV sequence on the p54 protein of African swine fever virus.

A nucleic acid molecule encoding the above-mentioned monoclonal antibody against African swine fever virus p54 protein.

Specifically, the nucleotide sequence of the gene encoding the heavy chain variable region of the anti-African swine fever virus p54 protein monoclonal antibody is shown in SEQ ID NO: 9; the light chain variable region encoding the anti-African swine fever virus p54 protein monoclonal antibody The nucleotide sequence of the gene is shown in SEQ ID NO: 10.

The antibody nucleic acid molecules involved in the present invention can be obtained by genetic engineering recombinant technology or chemical synthesis method. It is obvious to those skilled in the art that the nucleotide sequence and/or light chain of the variable region of the heavy chain obtained after the above-mentioned nucleic acid molecule provided by the present invention is mutated by one or more nucleotide additions, deletions, substitutions, modifications, etc. The variant sequence of the nucleotide sequence of the variable region, the single-chain antibody or chimeric monoclonal antibody or modified monoclonal antibody or other forms of monoclonal antibody or antibody fragment composed of the encoded amino acid sequence, still retains the same relationship with African swine fever. The ability of a peptide or epitope with the DSEFFQPV sequence on the viral p54 protein to specifically bind.

A recombinant expression vector comprising the above-mentioned nucleic acid molecule.

Further, the recombinant expression vector is selected from prokaryotic or eukaryotic expression vectors; further, the recombinant expression vector is selected from bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, reverse transcription virus or other vector.

A host cell comprising the above-mentioned recombinant expression vector, or the above-mentioned nucleic acid molecule integrated into the genome.

Further, the expression system is bacteria, yeast, filamentous fungi, mammalian cells, insect cells, plant cells or cell-free expression systems.

A method for preparing African swine fever virus p54 protein monoclonal antibody, the method comprises the following steps: culturing the above host cell under appropriate conditions.

Application of the above African swine fever virus p54 protein monoclonal antibody in the preparation of African swine fever virus detection reagents or kits.

The anti-ASFV p54 protein monoclonal antibody of the present invention can specifically recognize the African swine fever virus p54 protein and the African swine fever virus, and the monoclonal antibody of the present invention can be used to detect the African swine fever virus p54 protein, and can also detect African swine fever virus p54 protein swine fever virus.

Specifically, the detection reagent or kit comprises an anti-ASFV p54 protein monoclonal antibody.

The beneficial effects obtained by the present invention:

The African swine fever virus p54 protein monoclonal antibody provided by the present invention is based on the design and synthesis of a polypeptide that can simulate the N-terminal region (NTD, aa 1-29) of the conserved p54 protein, and is coupled with BSA as an immunogen, Prepared by immunizing BALB/c mice with immunological methods, it can specifically recognize and bind to the NTD (aa 1-29) and peptide segments of p54 protein. This recognition site (DSEFFQPV) is different from the existing commercial monoclonal antibodies. . During this process, we analyzed the conservation of p54 using bioinformatics, and by reviewing previous studies and in-depth analysis, we found that NTD is a key antigenic region within the p54 protein. Tests have shown that antibodies induced against protein p54 mainly recognize linear epitopes, and anti-p54 protein-positive serum can specifically recognize NTD. Furthermore, dimerization of p54, required for mature virion formation, must occur through a unique cysteine residue located in the NTD. This indicates that NTD is a new target for the preparation of monoclonal antibodies (mAbs) against p54 protein and a key target for studying AFSV morphogenesis. The monoclonal antibody prepared by using ^NTD polypeptide coupled with BSA as the immunogen has strong specificity and high sensitivity. Other swine-derived viruses, such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV 2), porcine pseudorabies virus (PRV), etc. At the same time, it can be used in various immunological detection methods such as ELISA and IPMA, and has a good application prospect.

The present invention also provides the amino acid sequence and nucleotide sequence of the variable region of the heavy chain and light chain of the African swine fever virus p54 protein monoclonal antibody. On this basis, conventional genetic engineering or protein engineering methods can be used to obtain the present invention The monoclonal antibody can be further modified by adding, deleting, and replacing one or more amino acids to obtain its active fragment or conservative variant, which lays a foundation for further improving the specificity and affinity of the antibody.

Description of drawings

Fig. 1 is the serum titer of immunized mice;

In the figure, 21dpi is the mouse serum at 21 days after immunization; 42dpi is the mouse serum at 42 days after immunization.

Figure 2 shows the identification of monoclonal antibody by Western blotting;

In the figure, well 1 is the prokaryotic expression product of pET28a-P54; well 2 is the empty expression product of pET28a;

Fig. 3 is the IPMA identification of monoclonal antibody specificity;

In the figure, A is the IPMA identification result of 293T cells transiently transfected with pcDNA ^TM 3.1/myc-HisA/ASFV P54; B is the control of untransfected 293T cells

Figure 4 shows the cross-reactivity of the monoclonal antibody detected by IPMA with other swine-derived viruses;

In the figure, A is ASFV HLJ/18 strain; B is PCV2; C is CSFV; D is PRRSV; E is PEDV; F is PRV.

Figure 5 is a graph showing the results of indirect ELISA identification of the p54N ₂₉ B cell epitope of p54 protein;

In the figure, P1-P3 are overlapping polypeptides covering amino acids 1-29 of p54 protein; NC is SMCC-BSA as a negative control, and PC is a polypeptide p54N ₂₉ as a positive control.

Figure 6 is a truncated peptide library to determine the minimum epitope map;

In the figure, A is the reaction result of N-terminal truncated polypeptide and monoclonal antibody, and B is the reaction result of C-terminal truncated polypeptide and monoclonal antibody.

Figure 7 is a diagram showing the identification of the key amino acid sequence of the B cell epitope of p54 protein _p54N29 .

In the figure, E1 is the ² DSEFFQPV ⁹ epitope; F5A, F6A, Q7A, P8A, V9A are the polypeptides mutated into alanine at the corresponding site of the epitope.

Fig. 8 is the application of the monoclonal antibody provided by the present invention in IPMA detection;

In the figure, A is the monoclonal antibody described in the present invention; B is ASFV pig positive serum as a positive control; C is an anti-PCV2Cap protein monoclonal antibody as a negative control.

Fig. 9 is the application of the monoclonal antibody provided by the present invention in IFA detection;

In the figure, A is the monoclonal antibody described in the present invention; B is the anti-PCV2 Cap protein monoclonal antibody as a negative control.

Detailed ways

The present invention will be further described below in conjunction with the specific embodiments, but the protection scope of the present invention is not limited to this; the instruments and equipment involved in the following examples are conventional instruments and equipment unless otherwise specified; the reagents involved are not otherwise specified. , are commercially available conventional reagents; the test methods involved are conventional methods unless otherwise specified.

Example 1 Selection and preparation of immunogens

The viral structural protein p54 is encoded by the E183L gene and is located in the inner membrane of the viral envelope. The N-terminus of p54 is relatively conserved among virus strains and contains a unique cysteine. It is used in the process of virus morphogenesis, activity, and invasion. have an important role. In the preliminary test, a peptide was synthesized based on the NTD sequence of the p54 protein to simulate NTD. The peptide has a good reaction with the serum of clinically recovered pigs infected with ASFV, which proves that the NTD region of the p54 protein contains antigenic determinants, which can induce the body to produce antibodies against NTD. Therefore, the present invention uses p54 protein NTD coupled to carrier protein BSA as an immunogen. Specifically, the preparation of the immunogen includes the following steps:

(1) With reference to the p54 protein sequence of ASFV strain SY18 (GenBank: MH717102.1) reported in the first ASF outbreak in China, a peptide that can mimic the NTD of p54 protein was synthesized by solid-phase synthesis peptide technology and named NTD , and its amino acid sequence is shown in SEQ ID NO.2.

(2) Polypeptide coupled carrier protein BSA (bovine serum albumin)

Coupling was performed using the water-soluble amino-thiol crosslinker Sulfo-SMCC. Sulfo-SMCC has two reactive groups, sulfo-NHS ester and maleimide, which can react between primary amino groups and sulfhydryl groups. First, under the conditions of pH 7-9, Sulfo-SMCC reacts with the primary amine group of carrier protein BSA to form a stable amide bond to obtain activated carrier protein BSA. Second, the activated BSA was dialyzed against PBS (pH 7.2-7.4), and the dialysate was changed at least three times, with an interval of 6 hours. The dialyzed solution was collected, and the protein concentration was adjusted to 5 mg/ml with PBS. Finally, under the condition of pH 6.5-7.5, the activated BSA reacts with the sulfhydryl group of the polypeptide NTD to form a stable thioether bond to form an immunogenic carrier protein BSA and polypeptide NTD conjugate for antibody production.

Example 2 Preparation of monoclonal antibodies

1. Animal Immunization

(1) Freund's complete adjuvant is added to the immunogen NTD-BSA, and it is used for the first immunization after being emulsified;

(2) 2 female BALB/c mice aged 4-8 weeks were immunized by subcutaneous injection at multiple points on the back, and the immunization dose was 10 μg/mice;

(3) BALB/c mice were boosted with the same method and dose after emulsification with incomplete Freund's adjuvant and immunizing antigen every 3 weeks;

(4) Three weeks later, blood was collected from the tail vein to measure the specific antibody titer against NTD, and the mice with higher titer were selected (Figure 1). BALB/c mice were hyperimmunized with immunogen without adjuvant at a dose of 20 μg/mice.

2. Cell fusion and monoclonal antibody preparation

The spleen cells of the immunized mice were fused with the mouse myeloma cells SP2/0 in a ratio of 8:1 by the method of polyethylene glycol, and the fused cells were screened with HAT selection medium; After 12 days, the positive hybridoma cells were preliminarily screened by indirect ELISA method with the polypeptide NTD as the coating antigen;

Indirect ELISA method steps:

(1) Dilute the unconjugated NTD polypeptide with CBS solution into a coating solution with a concentration of 2 μg/mL to coat the ELISA plate, 100 μl/well, and block overnight at 4°C;

(2) The hybridoma supernatant (primary antibody) was diluted twice with 5% skim milk, and added to the ELISA plate in turn, 50 μl/well, the positive control was ASFV pig positive serum, and incubated at 37°C for 30 min;

(3) Discard the primary antibody, wash the plate with PBST, wash well, and pat dry;

(4) Add the diluted HRP-labeled goat anti-mouse IgG (secondary antibody) into the reaction well, 50 μl/well. Incubate at 37°C for 30min;

(5) Discard the secondary antibody, rinse with PBST, and pat dry;

(6) Add 100 μl of the ready-made TMB color developing solution to each well, and react in the dark room for 15 minutes;

(7) Add 50 μl of 2M H ₂ SO ₄ to each well to stop the reaction;

(8) The microplate reader reads the OD ₄₅₀ value of each well.

3. Subcloning of Hybridoma Cells by Limiting Dilution

Dilute the above-mentioned positive hybridoma cells to about 1.5 cells/ml with 1640/10 complete medium, add 100 μl per well to a 96-well plate pre-plated with 100 μl feeder cells, and culture in a 37°C, 5% CO ₂ incubator 6 to 8 days; further screen positive hybridoma cells by indirect ELISA; subcloning 2 to 3 times until a hybridoma cell line that stably secretes anti-P54 protein monoclonal antibody is obtained, the target hybridoma cells can be obtained. The positive monoclones obtained by screening were expanded and cultured, and the number of cells was 1-2×10 ⁶ /tube for cryopreservation.

4. Stability identification of monoclonal hybridoma cell lines

The established monoclonal hybridoma cell line was continuously cultured for 3 months and repeatedly cryopreserved and recovered in liquid nitrogen to identify the stability of the hybridoma cell; the results showed that the monoclonal hybridoma cell line had good stability.

5. Preparation of monoclonal antibodies by inducing ascites in vivo

Selected female Balb/c mice that had been born, injected 500 μl of sterilized paraffin intraperitoneally. One week later, the monoclonal hybridoma cells obtained were intraperitoneally injected again with an injection volume of 2 × 10 ⁵ cells. The ascites was extracted from the abdominal distension of the mice, and the supernatant was collected after centrifugation, and the ascites was purified by the ammonium caprylic sulfate method.

Example 3 Purification and identification of antibodies

1. Purify antibody by saturated ammonium sulfate precipitation method, the operation method is as follows:

(1) Take 5ml of monoclonal antibody ascites, add 5ml of PBS buffer, and then add 2.5ml of saturated ammonium sulfate solution dropwise to make a final concentration of 20% ammonium sulfate solution, stir while adding, mix well, and let stand 30min.

(2) 8000r/min, centrifuge for 20min, discard the precipitate to remove fibrin.

(3) Add 12.5 ml of saturated ammonium sulfate solution to the supernatant, mix well, and let stand for 30 minutes.

(4) Centrifuge at 8000 r/min for 20 min, and discard the supernatant.

(5) Add 10 ml of PBS buffer to the precipitate to dissolve it, then add 5 ml of saturated ammonium sulfate solution to make it a 33% ammonium sulfate solution, mix well, and let it stand for 30 minutes.

(6) Centrifuge at 8000 r/min for 20 min and discard the supernatant to remove albumin.

(7) Repeat step 5, 2 to 3 times.

(8) Dissolve the precipitate with 5 ml of PBS buffer, put it into a dialysis bag, dialyze it with PBS buffer at 4°C, and change the medium 4 times.

(9) 8000r/min, centrifuge for 20min, discard the precipitate, the supernatant is the purified antibody, measure the antibody concentration, and store it at -20°C after aliquoting.

2. Monoclonal Antibody Titer Measurement

The indirect ELISA assay method is carried out with reference to Example 2, and the primary antibody is slightly different: the purified monoclonal antibody is diluted 2 times from 1:1000 with 5% skim milk, and then added to the ELISA plate in turn, 50 μl/well , the positive control was ASFV pig positive serum, incubated at 37°C for 30 minutes; other steps were carried out with reference to Example 2, and the ELISA test results showed that the titer of the monoclonal antibody was 1:1.024×10 ⁶ .

3. Subtype identification

The subtype of the monoclonal antibody was identified by using the Mouse Monoclonal Antibody Isotyping Kit (Sigma, Mouse Monoclonal Antibody Isotyping Kit). The identification result showed that the monoclonal antibody belongs to IgG1, and the light chain type is Kappa type.

4. Monoclonal antibody specific identification

Western-blotting identification of monoclonal antibodies: The complete coding sequence (CDR) of the p54 gene (GenBank accession MH717102) was subcloned into the pET-28a plasmid to construct a prokaryotic expression vector pET28a-p54, and the recombinant clone pET28a-p54 was transformed into In Escherichia coli BL21 (DE3) cells, the monoclonal antibody was used for western blotting detection. The results are shown in Figure 2. The monoclonal antibody can specifically react with the ASFV p54 protein expressed in prokaryotic cells, but not with the empty vector;

The above monoclonal antibodies were diluted in a certain proportion and added to the 293T cells transiently transfected with the eukaryotic expression vector pcDNA ^TM 3.1/myc-HisA/ASFVP54. The results of the IPMA detection method were shown in Figure 3. ASFV P54 protein reacts specifically but not with untransfected cells.

5. Identification of cross-reactivity with porcine-derived viruses

At the same time, the monoclonal antibody ascites was diluted in a certain proportion and added to the cells infected with PCV2, CSFV, PRRSV, PRV, and PEDV, and the IPMA detection method was used to determine whether the monoclonal antibody had cross-reactivity with PCV2, CSFV, PRRSV, PRV, and PEDV. . The IPMA results are shown in Figure 4. The monoclonal antibody only reacts with the ASFV HLJ/18 strain, and the reaction results with several other viruses (PCV2, CSFV, PRRSV, PRV, PEDV) are all negative, which proves that the monoclonal antibody reacts with ASFV. Good specificity, no cross-reaction with several other common swine viruses.

Example 4 Amplification and Sequence Determination of Monoclonal Antibody Variable Region Gene

1. Primer Design

According to the sequence characteristics of the mouse monoclonal antibody, design the heavy chain variable region primer sequence:

P1: 5'-cctggtGAGGAGTCTGGACCTG-3';

P2: 5'-AAATCGAGAAGCACA-3'.

Design light chain variable region primer sequences:

P3: 5'-CCTGTCAGTCTTGGAGATCAA-3';

P4: 5'-TATCCGTTTGATTTCCAGCTT-3'.

2. PCR amplification

The variable region sequences of monoclonal antibodies were obtained by molecular cloning technology and sent to Shanghai Sangon Biological Co., Ltd. for sequencing.

The sequencing results are as follows: the heavy chain variable region and light chain variable region gene sequences of the monoclonal antibody of the present invention are respectively shown in SEQ ID NO.10 and SEQ ID NO.11, and the corresponding heavy chain variable regions deduced therefrom The amino acid sequences of the region and the light chain variable region are shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively. Further analysis obtained the amino acid sequences of the CDRs of the heavy chain variable region of the monoclonal antibody as shown in SEQ ID NO.1-3 respectively; the amino acid sequences of the CDRs of the light chain variable region of the monoclonal antibody were shown as SEQ ID NO.4-6 respectively. Show.

Example 5 Identification of antigenic epitopes recognized by monoclonal antibodies

1. Design of overlapping peptides

The peptide scanning method was used to design and synthesize 3 peptides, covering the entire NTD, and the adjacent peptides overlapped by 7 amino acids. The amino acid sequence of the polypeptide segment is shown in Table 1.

Table 1

name position amino acid sequence P1 1-15aa MDSEFFQPVYPRHYG P2 9-23aa VYPRHYGECLSPVTT P3 18-29aa LSPVTTPSFFST

2. Indirect ELISA to screen short peptides that can bind to antibodies

Dilute the polypeptide with CBS buffer, coat the ELISA plate, 37°C, 2h; wash the plate 2-3 times with PBST; block with 5% skim milk, 37°C, 2h; use the monoclonal antibody obtained in Example 3 of the present invention as the The primary antibody was used for indirect ELISA, and incubated at 37°C for 1 h; after plate washing, HRP-labeled goat anti-mouse IgG (H+L) was added, and incubated at 37°C for 30 minutes; after plate washing, TMB substrate chromogenic solution was added, and color development was terminated after 5 minutes. Read with a microplate reader. The results showed that the screened monoclonal antibody could react specifically with the polypeptide P1 ( ¹ MDSEFFQPVYPRHYG ¹⁵ ) ( FIG. 5 ).

3. Monoclonal antibody recognition epitope identification.

A truncation library was constructed by systematically truncating the positive polypeptide P1 at both ends, and the results are shown in Figure 6, where picture A shows the reaction between the N-terminal truncated polypeptide and the monoclonal antibody, and picture B shows the C-terminal truncated polypeptide and the monoclonal antibody. Anti-reaction results, it can be seen from Figure 6 that the minimum epitope peptide required for epitope activity is " ² DSEFFQPV ⁹ ", after determining the minimum epitope peptide, an alanine scanning library is designed to identify the key residues in the epitope base. The purity of all synthetic peptides was equal to or greater than 95%. The reactivity of each peptide in the truncated peptide library and the alanine scanning peptide library with the monoclonal antibody was determined by ELISA, and the key amino acid of the epitope " ² DSEFFQPV ⁹ " was determined to be ⁶ FQPV ⁹ (Fig. 7).

Example 6 Application of monoclonal antibodies in the preparation of African swine fever virus detection reagents or kits

This embodiment provides an African swine fever virus detection reagent, the detection reagent comprises the anti-African swine fever virus p54 protein monoclonal antibody provided by the present invention. The specificity of the detection reagent was verified by IPMA and IFA detection methods respectively.

Pig primary alveolar macrophages were infected with African swine fever virus HLJ/18 strain, fixed with methanol, and prepared into monolayer cell reaction plates. The monolayer cell reaction plate infected with ASFV HLJ/18 strain was kindly provided by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

1. IPMA method to detect African swine fever infected cells, the specific steps are as follows:

(1) The monolayer cell reaction plate prepared above was taken out of the refrigerator, equilibrated to room temperature, and washed once with PBST.

(2) Using the above-mentioned detection reagent (including the anti-ASFV p54 protein monoclonal antibody provided by the present invention) as the primary antibody, dilute it with 5% skim milk 1:1000, and set the positive control as ASFV pig positive serum, The negative control was anti-PCV2 Cap protein monoclonal antibody, and the sample volume was 50 μl per well. Incubate in a 37°C incubator for 30min.

(3) PBST was washed three times, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody. HRP-labeled goat anti-pig IgG was added to the positive control wells as the secondary antibody. Incubate in a 37°C incubator for 30min.

(4) Wash three times with PBST, add AEC substrate chromogenic solution, develop color at room temperature for 10 min, and observe the staining results under an optical microscope.

Determination of results: Typical reddish-brown staining was observed in cells infected with African swine fever virus, but no reddish-brown staining was observed in uninfected cells and negative controls. The results are shown in Fig. 8, indicating that the anti-ASFV p54 protein monoclonal antibody of the present invention can specifically detect ASFV-infected cells.

2. The IFA detection method can be performed with reference to the IPMA detection method. The secondary antibodies are slightly different. The FITC-labeled goat anti-mouse secondary antibody is diluted 1:500 and added to the corresponding wells. Other steps are performed with reference to IPMA. No chromogenic substrate is required, and results are observed under a fluorescence microscope. Determination of results: typical green fluorescence can be observed in cells infected with African swine fever virus, but no fluorescence in uninfected cells and negative controls. The results are shown in Fig. 9, indicating that the anti-ASFV p54 protein monoclonal antibody of the present invention can specifically detect cells infected with ASFV.

Meanwhile, the monoclonal antibodies of the present invention can also be used in ELISA detection reagents or ELISA detection kits, as in Example 2, or the monoclonal antibodies of the present invention can be used in ELISA detection reagents or kits after being labeled with a marker to detect ASFV or ASFVp54 protein; the monoclonal antibody of the present invention can also be used as a primary antibody in Western blotting to detect ASFV or ASFV p54 protein, as in Example 3; or the monoclonal antibody of the present invention can be used as a gold-labeled antibody or a capture antibody for the immune layer Analyze the test paper to detect ASFV or ASFV p54 protein.

The present invention has been described in detail above in conjunction with the accompanying drawings and the embodiments, but those skilled in the art can understand that, without departing from the purpose of the present invention, each specific parameter in the above-mentioned embodiments can also be changed, Forming a plurality of specific embodiments is the common variation range of the present invention, and will not be described in detail here.

<110> Henan Zhongze Biological Engineering Co., Ltd.

<120> Anti-ASFV p54 protein monoclonal antibody, preparation method and application

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<170> PatentIn version 3.5

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<211> 315

<212> DNA

<213> Artificial sequences

<221> Heavy chain variable region

<400> 9

cctggtgagg agtctggacc tggcctggtg aaaccttctc agtctctgtc cctcacctgc 60

actgtcactg gcttctcaat cacccgtgat tatggctgga actggatccg gcagtttcca 120

ggaaataaac tggagtggat ggcctatata agttatagtg gtagtaatag ctataaccca 180

tctctcaaaa gtcgaatctc tatcactcga gacacatcca agaaccagtt cttcctggag 240

ttgaattctg tgactactga ggacacagcc acatattact gtgcccttat gatgacgacg 300

tgtgcttctc gattt 315

<210> 10

<211> 312

<212> DNA

<213> Artificial sequences

<221> Light chain variable region

<400> 10

cctgtcagtc ttggagatca agcctccatc tcttgcagat ctagtcagag ccttgtacac 60

agtaatggaa acacctattt agattggtac ctgcagaagc caggccagtc tccaaagctc 120

ctgatcaacg actcaagttt ccaccgattt tctggggtcc cagacaggtt cagtggcagt 180

ggatcaggga cagatttcac actcaagatc agcagagtgg aggctgagga tctgggaatt 240

tatttctgct ctcaaagtac acttcttcct ccgacgttcg gtggaggcac caagctggaa 300

atcaaacgga ta 312

<210> 11

<211> 29

<212> PRT

<213> Artificial sequences

<221> NTD

<400> 11

Met Asp Ser Glu Phe Phe Gln Pro Val Tyr Pro Arg His Tyr Gly Glu

1 5 10 15

Cys Leu Ser Pro Val Thr Thr Pro Ser Phe Phe Ser Thr

20 25

<210> 12

<211> 22

<212> DNA

<213> Artificial sequences

<221> Heavy chain variable region primer P1

<400> 12

cctggtgagg agtctggacc tg 22

<210> 13

<211> 15

<212> DNA

<213> Artificial sequences

<221> Heavy chain variable region primer P2

<400> 13

aaatcgagaa gcaca 15

<210> 14

<211> 21

<212> DNA

<213> Artificial sequences

<221> Light chain variable region primer P3

<400> 14

cctgtcagtc ttggagatca a 21

<210> 15

<211> 21

<212> DNA

<213> Artificial sequences

<221> Light chain variable region primer P4

<400> 15

tatccgtttg atttccagct t 21

<210> 16

<211> 15

<212> PRT

<213> Artificial sequences

<221> P1 peptide

<400> 16

Met Asp Ser Glu Phe Phe Gln Pro Val Tyr Pro Arg His Tyr Gly

1 5 10 15

<210> 17

<211> 15

<212> PRT

<213> Artificial sequences

<221> P2 peptide

<400> 17

Val Tyr Pro Arg His Tyr Gly Glu Cys Leu Ser Pro Val Thr Thr

1 5 10 15

<210> 18

<211> 12

<212> PRT

<213> Artificial sequences

<221> P3 peptide

<400> 18

Leu Ser Pro Val Thr Thr Pro Ser Phe Phe Ser Thr

1 5 10

<210> 19

<211> 8

<212> PRT

<213> Artificial sequences

<221> Epitope

<400> 18

Asp Ser Glu Phe Phe Gln Pro Val

1 5

Claims (10)

1. anti-African swine fever virus p54 protein monoclonal antibody, is characterized in that, the heavy chain variable region of described monoclonal antibody comprises aminoacid sequence such as CDR1 shown in SEQ ID NO.1, aminoacid sequence such as SEQ ID NO.2 Shown CDR2, amino acid sequence are CDR3 shown in SEQ ID NO.3; The light chain variable region of the monoclonal antibody includes CDR1 amino acid sequence shown in SEQ ID NO.4, and amino acid sequence is shown in SEQ ID NO.5 The shown CDR2, the amino acid sequence is the CDR3 shown in SEQ ID NO.6. 2. The anti-ASFV p54 protein monoclonal antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.7; the monoclonal The amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.8. 3. The anti-ASFV p54 protein monoclonal antibody according to claim 1 or 2, wherein the heavy chain constant region of the monoclonal antibody is IgG1 type, and the light chain constant region is Kappa type. 4. The anti-ASFV p54 protein monoclonal antibody according to claim 1 or 2, wherein the monoclonal antibody specifically recognizes the N-terminal region of ASFV p54 protein as SEQ ID NO.11 sequence shown. 5. A nucleic acid molecule, wherein the nucleic acid molecule encodes the anti-ASFV p54 protein monoclonal antibody according to claim 1 or 2. 6. The nucleic acid molecule according to claim 5, wherein the nucleotide sequence of the gene encoding the heavy chain variable region of the anti-African swine fever virus p54 protein monoclonal antibody is shown in SEQ ID NO: 9; The gene nucleotide sequence of the variable region of the light chain of the monoclonal antibody against the swine fever virus p54 protein is shown in SEQ ID NO: 10. 7. A recombinant expression vector, wherein the recombinant expression vector comprises the nucleic acid molecule according to claim 5 or 6. 8. A host cell, characterized in that the host cell comprises the recombinant expression vector of claim 7, or the nucleic acid molecule of claim 5 or 6 is integrated into the genome. 9 . The method for preparing the African swine fever virus p54 protein monoclonal antibody according to claim 1 or 2 , wherein the method comprises the steps of: culturing the host cell according to claim 8 under appropriate conditions. 10 . 10. The application of the African swine fever virus p54 protein monoclonal antibody according to claim 1 or 2 in the preparation of an African swine fever virus detection reagent or kit.

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