African swine fever virus p30 protein antigen epitope peptide, monoclonal antibody and application
By preparing African swine fever virus p30 protein antigenic epitope peptides and monoclonal antibodies, the shortcomings of existing technologies in using p30 protein as a detection and vaccine target have been overcome, achieving efficient detection and vaccine development.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
- Filing Date
- 2026-02-09
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies struggle to effectively utilize the African swine fever virus p30 protein as a target for detection and vaccine development, lacking detailed information on its antigenic epitopes, resulting in deficiencies in detection methods and vaccine development.
We provide the African swine fever virus p30 protein antigenic epitope peptide (140-155aa) and its monoclonal antibody. By preparing the nucleotide sequence and expression vector, we can express the monoclonal antibody in host cells to achieve specific recognition and application of the p30 protein.
This enriches the epitope information of the p30 protein, provides an efficient detection method and a basis for novel vaccines, and lays the foundation for the research and development of antiviral drugs and detection products.
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Figure CN122145586A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to an African swine fever virus p30 protein antigenic epitope peptide, its monoclonal antibody, and its applications. Background Technology
[0002] African swine fever (ASF) is an acute, highly contagious disease of pigs caused by the African swine fever virus (ASFV), which has devastated the global pig industry. ASF is listed as a notifiable animal disease by the World Organisation for Animal Health (OIE) and is classified as a Class A animal disease in my country. ASFV belongs to the family African swine feverviridae and the genus African swine fevervirus, and is currently the only known arbovirus-borne DNA virus. ASFV is a multi-layered enveloped double-stranded DNA virus with a large genome encoding more than 150 proteins, exhibiting a complex viral structure and pathogenic mechanism.
[0003] The p30 protein, encoded by the CP204L gene, is a key structural protein expressed in the early stages of ASFV infection. It is expressed in large quantities at the initial stage of ASFV infection and exhibits good immunogenicity. Studies have shown that the p30 protein can induce the host to produce high titers of early antibodies. Furthermore, the p30 protein participates in viral internalization and plays an important role in the process of viral entry into host cells. Antibodies against the p30 protein can inhibit more than 95% of the viral internalization process in porcine macrophages and Vero cells. In addition, a recombinant fusion protein of p30 and p54 proteins can induce the production of neutralizing antibodies. These characteristics of the p30 protein make it one of the more ideal detection targets for African swine fever virus. Simultaneously, this protein has also been screened as an effective component of vaccines. By preparing antibodies against the p30 protein and identifying the protein epitopes it recognizes, data support and research materials can be provided for the development of subunit vaccines or epitope-deficient vaccines. Summary of the Invention
[0004] In view of this, the present invention provides an African swine fever virus p30 protein antigenic epitope peptide, a monoclonal antibody, and its application.
[0005] To achieve the above objectives, the technical solution claimed by this invention is as follows:
[0006] This invention provides an antigenic epitope peptide of African swine fever virus p30 protein, wherein the antigenic epitope peptide is located at 140-155aa of p30 protein, and its amino acid sequence is shown in SEQ ID NO:1.
[0007] The present invention also provides a nucleotide encoding the aforementioned antigenic epitope peptide, the sequence of which is shown in SEQ ID NO:2.
[0008] The present invention also provides the use of the said antigenic epitope peptide in the preparation of monoclonal antibodies against African swine fever virus p30 protein.
[0009] The present invention also provides an expression vector containing the aforementioned nucleotides.
[0010] The invention also provides a host cell containing the expression vector described above.
[0011] This invention also provides a monoclonal antibody comprising a heavy chain and a light chain; the heavy chain subtype is IgG2b; the light chain is a κ chain; the nucleotide sequence of the variable region of the heavy chain of the monoclonal antibody is shown in SEQ ID NO:3; the nucleotide sequence of the variable region of the light chain is shown in SEQ ID NO:4. The monoclonal antibody is capable of specifically recognizing the African swine fever virus p30 protein antigenic epitope peptide.
[0012] This invention also provides the application of the African swine fever virus p30 protein antigenic epitope peptide in the preparation of antibody products for detecting African swine fever virus, and the application of monoclonal antibodies in the design and preparation of novel vaccines using African swine fever virus antigens.
[0013] Beneficial effects:
[0014] This invention provides an African swine fever virus (ASFV) p30 protein antigenic epitope peptide, a monoclonal antibody, and their applications. This invention identifies a novel antigenic epitope peptide of the ASFV p30 protein, enriching the epitope information of this protein. The specific recognition of this antigenic epitope peptide provides a basis for establishing an efficient method for detecting ASFV, and also lays the foundation for studying the structure and function of the p30 protein. This invention also provides the application of the ASFV p30 protein antigenic epitope peptide in the preparation of antibody products for detecting ASFV and its application in the design of ASFV antigens and novel vaccines, laying a good foundation for the subsequent development of antiviral drugs, vaccines, and detection products. Attached Figure Description
[0015] Figure 1 This is a schematic diagram illustrating the indirect immunofluorescence identification of the reaction between p30 monoclonal antibody and African swine fever virus in an embodiment of the present invention; wherein: Mock represents uninoculated primary alveolar macrophages, and ASFV represents virus-inoculated primary alveolar macrophages.
[0016] Figure 2 This is a schematic diagram of Western blot identification of the reaction between p30 monoclonal antibody and virus in an embodiment of the present invention; wherein: Marker is a protein molecular weight standard, Mock is lysate of primary alveolar macrophages that have not been inoculated with the virus, and ASFV is lysate of primary alveolar macrophages inoculated with the virus.
[0017] Figure 3 This is a schematic diagram of Western blot identification of the antigenic epitope recognized by the p30 monoclonal antibody in an embodiment of the present invention; wherein: Marker is the protein molecular weight standard, pGEX-6p-1 is an empty vector, and 1-70aa, 60-130aa, 120-194aa, 120-150aa, 140-170aa, 160-194aa, 140-155aa, 150-160aa, and 155-170aa are amino acids 1-70, 60-130, 120-194, 120-150, 140-170, 160-194, 140-155, 150-160, and 155-170 of the p30 protein expressed by the GST tag fusion, respectively.
[0018] Figure 4 This is a schematic diagram of the amplification results of the light and heavy chains of the p30 monoclonal antibody in an embodiment of the present invention; where M is the marker, a nucleotide molecular weight standard.
[0019] Figure 5 This is a schematic diagram illustrating the reactivity of p30 monoclonal antibody expressed with eukaryotic p30 protein as identified by indirect immunofluorescence in an embodiment of the present invention; wherein: pCMV-myc is HKE293T cells transfected with empty vector, and myc-p30 is HEK293T cells expressing myc-tagged p30 protein.
[0020] Figure 6 This is a schematic diagram illustrating the reactivity of p30 monoclonal antibody expressed in Western blot with p30 protein expressed in eukaryotes in an embodiment of the present invention; wherein: pCMV-myc is the lysate of HKE293T cells transfected with empty vector, and myc-p30 is the lysate of HEK293T cells expressing myc-tagged p30 protein. Detailed Implementation
[0021] To further illustrate the present invention, the technical solutions provided by the present invention are described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention. The embodiments provided below can serve as a guide for further improvements by those skilled in the art, and do not constitute a limitation on the present invention in any way.
[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0023] Example 1: African swine fever virus p30 protein antigenic epitope peptide
[0024] This embodiment provides an African swine fever virus p30 protein antigenic epitope peptide, which is located at 140-155 amino acids of the p30 protein, and its amino acid sequence is shown in SEQ ID NO:1: LAQKTVQHIEQYGKAP.
[0025] Example 2: A nucleotide
[0026] This embodiment provides a nucleotide that encodes the African swine fever virus p30 protein antigenic epitope peptide described in Example 1. The sequence of the nucleotide is shown in SEQ ID NO:2: CTTGCACAAAAGACTGTGCAACATATTGAACAATATGGAAAGGCACCT.
[0027] Example 3: An expression vector
[0028] This embodiment provides an expression vector containing the nucleotides described in the second set of embodiments.
[0029] Example 4: A host cell
[0030] This embodiment provides a host cell containing the expression vector described in Example 3.
[0031] Example 5: A monoclonal antibody
[0032] This embodiment provides a monoclonal antibody comprising a heavy chain and a light chain; the heavy chain subtype is IgG2b; the light chain is a κ chain; the nucleotide sequence of the variable region of the heavy chain of the monoclonal antibody is shown in SEQ ID NO:3; the nucleotide sequence of the variable region of the light chain is shown in SEQ ID NO:4. The monoclonal antibody can specifically recognize the African swine fever virus p30 protein antigenic epitope peptide described in Example 1; the nucleotide sequence of the variable region of the heavy chain of the monoclonal antibody is shown in SEQ ID NO:3, and the nucleotide sequence of the variable region of the light chain is shown in SEQ ID NO:4.
[0033] Example 6: An Application
[0034] This embodiment provides the application of the antigenic epitope peptide described in Example 1 in the preparation of antibody products for detecting African swine fever virus and in the preparation of monoclonal antibodies against African swine fever virus p30 protein.
[0035] This set of examples also provides the application of the monoclonal antibody described in Example 5 in the design and preparation of novel vaccines against African swine fever virus antigens.
[0036] Experimental Example
[0037] 1. Method
[0038] (1) Preparation of p30 protein monoclonal antibody
[0039] Purified recombinant p30 protein preserved in the applicant's laboratory was mixed with an equal volume of adjuvant (Frederick complete adjuvant for the first immunization, and Frederick incomplete adjuvant for the second and third immunizations). The mixture was administered intraperitoneally to 6-week-old SPF-grade BALB / c female mice at a dose of 100 μg / mouse, with immunizations every two weeks. Seven days after the third immunization, blood was collected from the tail and serum antibody levels were detected using an indirect ELISA method. Three days before cell fusion, mice with the highest serum titers were selected for booster immunization.
[0040] Using PEG, healthy myeloma cells were fused with spleen cells from boosted mice, and the fused cells were seeded into 96-well cell culture plates. When the fused cells reached 1 / 3-1 / 2 of the well volume, the cell supernatant was collected as a primary antibody, and positive clones were screened using an indirect ELISA method. For positive clones, subcloning was performed using a limiting dilution method, specifically as follows: cells from the positive clone wells were pipetted to prepare a single-cell suspension, and the cell concentration was determined using a hemocytometer. Cells were diluted at a concentration of 1 cell / 100 μL, and the diluted solution was seeded at 100 μL / well into 96-well plates containing a feeder cell layer and incubated at 37°C and 5% CO2. Three consecutive subcloning cycles were performed to obtain hybridoma cell lines stably secreting specific p30 monoclonal antibodies.
[0041] (2) Identification of the characteristics of p30 protein monoclonal antibodies
[0042] Hybridoma cell lines stably secreting p30 monoclonal antibodies were intraperitoneally injected into 6-week-old SPF-grade BALB / c female mice sensitized with Freund's incomplete adjuvant to prepare ascites. Cell plates and cell lysates infected with the inactivated African swine fever virus strain Pig / HLJ / 2018, provided by the Harbin Veterinary Research Institute, were used to identify the reactivity of positive cell supernatant and ascites with the virus by indirect immunofluorescence and Western blot assays. Furthermore, the monoclonal antibody subtypes were identified using a mouse monoclonal antibody subclass identification kit.
[0043] The indirect immunofluorescence (IFA) assay was performed as follows: 0.2% Triton X-100 was used for permeation at room temperature for 10 min. After washing with PBS, 2% BSA was added and the cells were blocked at room temperature for 30 min. Hybridoma cell supernatant was added and incubated overnight at 4°C. The cells were washed three times with PBST, 5 min each time. FITC-labeled anti-mouse fluorescent secondary antibody was added in the dark and incubated at room temperature for 1 h. After washing with PBST, a small amount was left in the air for observation and image acquisition under a fluorescence microscope.
[0044] The Western blot assay was performed as follows: Cell samples were mixed with protein loading buffer and incubated at 100°C for 5 min, followed by SDS-PAGE gel electrophoresis for separation and wet transfer to an NC membrane. The cells were blocked with 5% skim milk at room temperature for 1 h, then incubated overnight at 4°C with hybridoma cell supernatant. The cells were washed three times with PBST for 5 min each time, and then incubated with HRP-labeled anti-mouse secondary antibody at room temperature for 1 h. After three washes with PBST, protein bands were visualized using ECL chemiluminescence.
[0045] (3) Identification and conservation analysis of p30 protein antigenic epitope peptides
[0046] The p30 protein was divided into three segments: amino acids 1-70, 60-130, and 120-194. Primers were designed (primers and their sequences are shown in Table 1) to amplify the three truncated fragments of the p30 protein, which were then ligated into the pGEX-6p-1 expression vector for segmented protein expression. Western blot was used to identify the reactivity of the segmented expressed protein with the prepared monoclonal antibody. Based on the identification results, the fragments that reacted with the monoclonal antibody were again divided into three segments for further identification.
[0047] Table 1. Primers for p30 protein truncation
[0048]
[0049] The amino acid sequences of all uploaded p30 proteins were downloaded from the NCBI database, and the sequences were aligned using the ClustalW method in MEGA-X software to analyze the conservation of amino acids at the corresponding positions of the antigenic epitopes.
[0050] (4) Sequencing and in vitro expression of p30 protein monoclonal antibody
[0051] RNA was extracted from hybridoma cell lines that stably secrete p30 monoclonal antibodies and reverse transcribed. Based on the heavy chain subtype and light chain type of the monoclonal antibody, nested PCR was used to amplify the heavy and light chain sequences, respectively. After gel recovery, the sequences were sequenced, and the obtained sequences were analyzed using an Ig database to determine the rearrangement of the variable regions in the heavy and light chains.
[0052] The obtained monoclonal antibody heavy and light chain sequences were cloned into mouse antibody heavy and light chain expression vectors via enzyme digestion and ligation. Recombinant plasmids containing the monoclonal antibody heavy and light chain sequences were co-transfected into HEK293T cells for in vitro antibody expression. Antibody expression was assessed using indirect immunofluorescence and Western blot assays.
[0053] 2. Results
[0054] (1) Preparation of p30 protein monoclonal antibody
[0055] Following three immunizations with p30 protein, mice with the highest serum antibody titers were selected for booster immunization. Spleens were aseptically harvested from the booster-immunized mice, and spleen cells were extracted and fused with SP2 / 0 cells. The cell supernatant was collected, and positive hybridoma cells were screened using indirect ELISA and then subcloned. After three rounds of screening and subcloning, a stable hybridoma cell line, 4D9, secreting p30 monoclonal antibody was obtained.
[0056] (2) Identification of the characteristics of p30 protein monoclonal antibodies
[0057] Hybridoma cell lines secreting p30 monoclonal antibodies were injected into the peritoneal cavity of sensitized mice to prepare ascites. The ascites was subtyped using a mouse monoclonal antibody subtype identification kit, revealing that the heavy chain subtype was IgG2b and the light chain was κ. ASFV was inoculated into primary alveolar macrophages, with a control group not receiving the virus. Cells were fixed after 48 h. Indirect immunofluorescence assays were performed using diluted ascites as the primary antibody. Results showed that... Figure 1 As shown, compared with uninfected cells, the p30 protein monoclonal antibody produced a specific green fluorescent signal when incubated with infected cells, indicating that it can specifically recognize ASFV infection. Cells infected with ASFV for 48 h and blank control cells were collected and lysed, and the reactivity of the monoclonal antibody with the virus was identified using Western blot. The results are as follows. Figure 2 As shown, compared with blank cells, the infected cell lane has a specific band that is the same size as the p30 protein, indicating that the monoclonal antibody can specifically recognize the p30 protein expressed by the virus.
[0058] (3) Identification and conservation analysis of p30 protein antigenic epitope peptides
[0059] To analyze the epitope peptides targeted by p30 monoclonal antibodies, the p30 protein was truncated into fragments of different lengths and fused with GST tags for expression. Western blot analysis was used to identify the reactivity of the segmented protein with the prepared monoclonal antibody. Results showed that the p30 monoclonal antibody recognized amino acid segments 120-194 (aa) of the p30 protein. Based on this result, the fragment was truncated to 120-150 aa, 140-170 aa, and 160-194 aa. Western blot analysis showed that the p30 monoclonal antibody specifically bound to segments 140-170 aa. Further truncation experiments showed that the p30 monoclonal antibody recognized segments 140-155 aa (aa). Figure 3Using MEGA-X software to compare the published amino acid sequences of the p30 protein, it was found that the antigenic epitopes 140-155aa are highly conserved in the p30 protein.
[0060] (4) Sequencing and in vitro expression of p30 protein monoclonal antibody
[0061] RNA was extracted from hybridoma cells and reverse transcribed. Nested PCR was then used to amplify the heavy and light chain sequences of monoclonal antibodies, respectively. The amplified products were identified by agarose gel electrophoresis, and the results are as follows: Figure 4 As shown in the figure, a distinct band appears in the PCR product between 250bp and 500bp, consistent with expectations. The recovered product was then sequenced. Analysis of the sequenced data revealed the heavy chain variable region nucleotide sequence as shown in SEQ ID NO:3, and the light chain variable region nucleotide sequence as shown in SEQ ID NO:4.
[0062] The heavy and light chain variable regions were cloned into the pCDNA3.1-mG2a and pCDNA3.1-Mk vectors, respectively. These two recombinant plasmids were extracted and co-transfected into HEK293T cells, where the expressed antibody was found in the cell supernatant. The reaction between the expressed antibody and p30 protein was detected using indirect immunofluorescence and Western blot. The results are as follows: Figure 5 and 6 As shown, the antibody expressed by HEK293T cells can recognize the p30 protein expressed in eukaryotes, indicating that the antibody was successfully expressed in vitro.
Claims
1. An epitope peptide of African swine fever virus p30 protein, the amino acid sequence of which is: LAQKTVQHIEQYGKAP.
2. The antigenic epitope peptide according to claim 1, characterized in that, The antigenic epitope peptide is located at 140-155aa of the p30 protein.
3. A nucleotide, characterized in that, The nucleotide encodes the antigenic epitope peptide of claim 1; the sequence of the nucleotide is shown in SEQ ID NO:
2.
4. The use of the antigenic epitope peptide according to claim 1 in the preparation of monoclonal antibodies against African swine fever virus p30 protein.
5. An expression carrier, characterized in that, The expression vector contains the nucleotides described in claim 2.
6. A host cell, characterized in that, The host cell contains the expression vector as described in claim 4.
7. A monoclonal antibody, characterized in that, The monoclonal antibody comprises a heavy chain and a light chain; the heavy chain subtype is IgG2b; the light chain is a κ chain; the nucleotide sequence of the variable region of the heavy chain of the monoclonal antibody is shown in SEQ ID NO:3; the nucleotide sequence of the variable region of the light chain is shown in SEQ ID NO:
4.
8. The monoclonal antibody according to claim 6 or 7, characterized in that, The monoclonal antibody can specifically recognize the antigenic epitope peptide of claim 1.
9. The use of the antigenic epitope peptide according to claim 1 in the preparation of products for detecting antibodies against African swine fever virus.
10. The application of the monoclonal antibody according to claim 8 in the design and preparation of novel vaccines against African swine fever virus antigens.