Linear B-cell antigenic epitopes of African swine fever virus NP419L protein and their applications

Abstract

This invention belongs to the field of biological immunology and discloses the linear B-cell antigenic epitope of the African swine fever virus (ASFV) NP419L protein and its applications. The epitope sequence was verified by ASFV-positive swine serum, exhibiting good immunoreactivity. ELISA experiments using the B-cell epitope identified in this invention can identify ASFV-positive serum, with sensitivity superior to the full-length NP419L protein and other truncated fragments. The epitope identified in this invention is highly conserved in reference ASFV strains in multiple endemic areas of China, possessing broad applicability and adaptability. This invention can be widely applied to the etiological diagnosis, serological diagnosis, immunological detection, and disease prevention and control of ASFV, as well as to the preparation of ASFV vaccines and research on pathogenic mechanisms.

Description

Technical Field

[0001] This invention belongs to the field of biological immunology, specifically relating to the linear B-cell antigenic epitope of the African swine fever virus NP419L protein and its application. Background Technology

[0002] African swine fever virus (ASFV) belongs to the family Asfarviridae and the genus Asfivirus. Its genome is a linear double-stranded DNA, approximately 170-190 kb in size, encoding over 150 structural and non-structural proteins. This large and complex proteome endows ASFV with sophisticated immune evasion strategies, enabling it to effectively antagonize the host's innate and adaptive immune responses. This is the core technological bottleneck that has repeatedly hindered ASF vaccine development. Although studies have identified p72, p54, and p30, major capsid and membrane proteins, as having strong immunogenicity and widely used in serological diagnostics, subunit vaccines based on these single or a few proteins have so far failed to induce robust and effective protective immunity. The functions of many proteins in the ASFV proteome, particularly their roles in inducing humoral immune responses and their key B-cell antigenic epitopes, have not yet been systematically elucidated. Therefore, discovering and identifying more viral proteins with high immunogenicity that can induce broad-spectrum neutralizing antibodies or become key immune markers is a necessary prerequisite for promoting breakthroughs in basic research and application development of ASFV.

[0003] Antigenic epitopes, as chemical determinants on antigen molecules that can be specifically recognized and bound by immune cell receptors, are the smallest functional units of the immune response. Precise epitope mapping of viral proteins is crucial for a deep understanding of virus-host interactions and elucidating the mechanisms of protective immunity. Furthermore, developing diagnostic reagents and peptide vaccines based on dominant B-cell linear epitopes offers significant advantages over whole-protein antigens: firstly, they possess high specificity, effectively avoiding cross-reactivity; secondly, from a production perspective, chemically synthesized peptides exhibit high purity, batch stability, and significantly improved production convenience and standardization. Most antigenic epitopes are conformational epitopes, which typically depend on the three-dimensional structure of proteins. Obtaining them requires maintaining the protein's complete conformation, leading to complex preparation, poor stability, high cost, and limited applications. In contrast, linear epitopes offer significant advantages in synthesis and application, as they can be directly obtained through chemical synthesis, making them more suitable for immunoassay and vaccine design. However, the distribution of linear epitopes in proteins is irregular, and only a few fragments are immune-associated, making screening challenging. Under current technological conditions, traditional peptide scanning screening methods often have low signal-to-noise ratios and high false-positive rates, and the epitopes obtained may not necessarily have application value. Linear epitopes recognized by protein-based monoclonal antibodies are highly random and accidental, inefficient and costly, making it difficult to accurately obtain functional linear epitopes with application value, and thus failing to meet current diagnostic and vaccine development needs.

[0004] NP419L is a highly conserved non-structural protein encoded by ASFV. This protein is expressed early in the viral course, possesses a typical nucleic acid-binding domain, and plays a role in viral replication and transcriptional regulation. Studies have shown that specific antibodies against the NP419L protein exist in the serum of convalescent pigs infected with ASFV, suggesting that this protein may be exposed to the host immune system and has the potential to become a vaccine candidate or diagnostic antigen. Currently, the dominant B-cell linear epitope region of the NP419L protein has not been precisely identified. Summary of the Invention

[0005] The purpose of this invention is to provide a linear B-cell antigenic epitope of the African swine fever virus NP419L protein, wherein the antigenic epitope is a linear polypeptide with the amino acid sequence shown in SEQ ID NO.1.

[0006] Another object of the present invention is to provide applications of the above-mentioned B-cell antigenic epitopes, including but not limited to their use in the preparation of African swine fever virus antibody detection reagents and African swine fever virus subunit vaccines.

[0007] To achieve the above objectives, the present invention employs the following technical measures:

[0008] This invention screened the NP419L protein, which strongly reacts with positive serum, from multiple African swine fever proteins. Through multiple attempts at different truncated expression of the ASFV NP419L protein, the region that strongly reacts with African swine fever positive serum was screened, and its dominant linear B cell epitope region was gradually located. The amino acid sequence of the antigenic epitope is GYNNYHSAHLAKLKPLLDAEFILVDYT, as shown in SEQ ID NO.1.

[0009] The scope of protection of this invention also includes:

[0010] The fusion protein obtained by fusing the antigenic epitope polypeptide described in SEQ ID NO.1 with a protein tag.

[0011] The gene encoding the antigenic epitope polypeptide or fusion protein described in SEQ ID NO.1.

[0012] Expression cassettes, recombinant vectors, recombinant microorganisms, or isolated recombinant eukaryotic cells containing the above-mentioned coding genes.

[0013] The application of the above-mentioned antigenic epitope polypeptide, fusion protein, the encoding gene of the antigenic epitope polypeptide or fusion protein described in SEQ ID NO.1, expression cassettes having the above-mentioned encoding gene, recombinant vectors, recombinant microorganisms or ex vivo recombinant eukaryotic cells in the preparation of African swine fever virus antibody detection reagents.

[0014] The application of the above-mentioned antigenic epitope polypeptide, fusion protein, the encoding gene of the antigenic epitope polypeptide or fusion protein described in SEQ ID NO.1, expression cassettes having the above-mentioned encoding gene, recombinant vectors, recombinant microorganisms or ex vivo recombinant eukaryotic cells in the preparation of African swine fever virus detection kits.

[0015] The application of the above-mentioned antigenic epitope polypeptide, fusion protein, the encoding gene of the antigenic epitope polypeptide or fusion protein described in SEQ ID NO.1, expression cassettes having the above-mentioned encoding gene, recombinant vectors, recombinant microorganisms or ex vivo recombinant eukaryotic cells in the preparation of African swine fever virus subunit vaccines.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] This invention is the first to identify a specific dominant linear B-cell epitope in the ASFV NP419L protein. The identified epitope sequence is highly conserved and versatile, exhibiting high conservation across multiple ASFV strains and demonstrating good versatility and cross-strain applicability.

[0018] The identified epitope peptides can be used to develop ASFV serological diagnostic reagents, pathogen detection methods and immunological detection reagents, as well as for antibody preparation or vaccine adjuvant research and other fields.

[0019] The short and precise B-cell antigen epitope sequence provides candidate sites for the future development of ASFV candidate vaccines such as peptide vaccines, multi-protein fusion vaccines, and multi-epitope chimeric vaccines. It is expected that by optimizing the combination of epitopes, the efficiency of immune protection can be improved and immune interference or immune escape can be avoided.

[0020] This invention uses ASFV-infected serum to assess the immunogenicity of NP419L truncated protein. Compared with traditional mouse immunization models, this method is closer to the actual viral infection state. The obtained antigenic epitopes have higher immunorelevance and practical value in the context of natural infection, thus improving the clinical relevance and practicality of the screening results.

[0021] The dominant B-cell antigenic epitopes screened in this invention exhibit strong immune responses to African swine fever-positive serum, demonstrating excellent specificity and sensitivity. This provides strong support for the development of broad-spectrum, highly sensitive, and highly specific ASFV diagnostic reagents and candidate vaccines, and has high practical value and promising prospects for promotion. Attached Figure Description

[0022] Figure 1 This is a graph showing the results of a Western blot experiment identifying the reaction between the dominant African swine fever antigen protein and serum from African swine fever-positive pigs.

[0023] Figure 2 This is a schematic diagram of the NP419L truncated protein.

[0024] Figure 3 This is a diagram identifying the protein expression of NP419L protein with truncated segments A1, A2, and A3.

[0025] Figure 4 This is a graph showing the reaction results of NP419L protein truncated segments A1, A2, and A3 with African swine fever-positive pig serum.

[0026] Figure 5 This is a protein expression identification diagram of the truncated B1 and B2 segments of the NP419L protein.

[0027] Figure 6 This is a graph showing the reaction results of NP419L protein truncated segments B1 and B2 with African swine fever-positive pig serum.

[0028] Figure 7 This is a graph showing the results of reactions between different NP419L protein antigenic epitope peptides and African swine fever positive and negative swine serum.

[0029] Figure 8 This is an ELISA result of the reaction between the African swine fever virus NP419L protein antigenic epitope polypeptide EP1 and African swine fever positive and negative sera.

[0030] Figure 9This is a comparison chart of the sensitivity of antigenic epitope peptides.

[0031] Figure 10 This is a schematic diagram showing the conservation of the antigenic epitope polypeptide of the present invention. Detailed Implementation

[0032] The present invention will be further described below with reference to the following embodiments, but the present invention is not limited to the following embodiments. Those skilled in the art should understand that modifications, substitutions, alterations, combinations, and simplifications can be made to the details and form of the technical solutions of the present invention without departing from the spirit and principle of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0033] Example 1:

[0034] Validation of the antigenicity of the African swine fever virus NP419L protein:

[0035] Using the prevalent ASFV genotype II strain SY18 (GenBank: MH766894) as a template, specific primers were designed and synthesized. Using ASFV inactivated nucleic acid as a template, DNA sequence fragments of multiple African swine fever protein genes were amplified by high-fidelity PCR. The amplified fragments were then inserted into pCAGGS-HA to obtain the pCAGGS-HA recombinant plasmid that can express different African swine fever proteins.

[0036] HEK-293T cells were seeded in 6-well cell culture plates, and pCAGGS-HA recombinant plasmid was added to each well for transfection. The supernatant was collected as a cell protein sample for subsequent Western blotting experiments. The results are as follows: Figure 1 As shown, among several screened recombinant ASFV proteins, the NP419L protein exhibited a specific and strong immune response to ASFV-positive porcine serum, displaying a clear protein band at the expected molecular weight position. In contrast, other proteins showed weaker or no response. The NP419L protein demonstrated good immunogenicity and is a key immunogenic antigen of ASFV.

[0037] Example 2:

[0038] Determination of the dominant B-cell epitope of NP419L protein

[0039] To precisely locate its dominant B-cell epitopes, this invention conducted multiple attempts at truncating and expressing the full-length sequence of the NP419L protein (Gene ID: 22220329) using different methods, followed by immunoblotting analysis. First, attempts were made to truncate the full-length protein into segments of 1aa-296aa, 125aa-419aa, and 60aa-353aa. Each segment was successfully expressed, but the truncated protein failed to react with positive serum. Second, attempts were made to truncate the full-length protein into two segments: 1aa-235aa and 185aa-419aa. Protein expression was successful, but the protein was not recognized by positive serum. Based on these results, this invention ultimately adopted a three-segment truncation strategy, dividing the NP419L protein into A1, A2, and A3 segments (…). Figure 2 The amino acid sequences of segments A1 to A3 are shown in SEQ ID NO.3 to SEQ ID NO.5. These three protein segments were expressed separately. Analysis with ASFV-positive serum initially located the dominant B-cell epitope in the A2 region. Subsequently, segment A2 was further truncated into segments B1 and B2. Reaction with ASFV-positive serum confirmed that the dominant B-cell epitope region was in segment B2. Finally, peptide synthesis verification was performed on segment B2, confirming the amino acid sequence of its dominant antigenic epitope as: GYNNYHSAHLAKLKPLLDAEFILVDYT. The gene encoding this peptide is shown in SEQ ID NO.2.

[0040] Using pCAGGS-HA plasmid as a template, primers were designed to amplify the full-length NP419L gene and truncated fragments A1-A3, which were then inserted between the restriction enzyme sites Xho I and EcoRI in the pCAGGS-HA vector to construct truncated NP419L plasmids. The recombinant plasmids were transformed into competent DH5α cells. After screening and identification, plasmids were extracted using an endotoxin-free plasmid extraction kit for transient transfection. Plasmids were transfected at 2 μg / well into six-well cell culture plates containing HEK-293T cells. After 24 h of transfection, the cells were washed twice with PBS, and 250 μL of Western blot and IP lysis buffer was added to each well. The cells were sonicated, centrifuged, and the supernatant was collected for Western blotting. Successful expression of the recombinant protein was confirmed using an HA-tagged antibody. The results are shown below. Figure 3 As shown, all proteins were successfully expressed. Subsequently, the epitope regions of NP419L dominant B cells were identified using three ASFV-positive porcine serum samples from different sources. The results are as follows. Figure 4 As shown, all three different positive sera were able to recognize the A2 segment protein, suggesting that dominant B cell antigenic epitopes are concentrated in the A2 region.

[0041] Furthermore, to narrow down the range of the dominant epitope, segment A2 was divided into segments B1 and B2, covering the entire length of A2 and overlapping with each other. The amino acid sequence of segment B1 is: LQWIAGQANAKTDSSELHFYVFDCFWSDQLQMPS NKRQQLLTNIFKQKEDLTFIHQVENFSVKNVDEALRLKAQFIKEGYEGAIVRNANGPYEPG YNNYH; the amino acid sequence of segment B2 is: GYNNYHSAHLAKLKPLLDAEFILVDYTQGKKGKDLG AILWVCELPNKKRFVVTPKHLTYADRYALFQKLTPALFKKHLYGKELTVEYAELSPKTGIPL QARAVGFREPISVLEII.

[0042] To facilitate subsequent detection, primers were designed using the pmCherry-C1 plasmid as the expression backbone to amplify the full-length NP419L protein and truncated fragments B1 and B2. These fragments were then cloned into the restriction enzyme sites Xho I and BamHI of the pmCherry-C1 vector to construct the full-length NP419L and truncated fragments B1 and B2 plasmids, respectively. The recombinant plasmids were transformed into competent DH5α cells, and the plasmids were extracted using an endotoxin-free plasmid extraction kit and used for transfection into HEK-293T cells. After transfection, cell lysis, SDS-PAGE electrophoresis, and Western blot detection were performed, and expression was verified using an anti-Cherry tag antibody. The results are as follows: Figure 5 As shown, all proteins were successfully expressed. Subsequently, the dominant B-cell epitope regions of the NP419L protein B1 and B2 segments were identified using three different ASFV-positive sera. The results are as follows. Figure 6 As shown, the B2 segment was stably and strongly recognized by the three positive sera, while the B1 segment showed no significant response. This result further pinpoints the dominant B-cell epitope region of NP419L within the B2 segment.

[0043] Furthermore, to determine specific, least dominant B-cell epitopes, this invention synthesized a series of seven 25-amino acid peptides with overlapping adjacent peptides based on the B2 segment sequence. The synthesis of the antigenic epitope peptides was completed by Nanjing Genscript Biotech Co., Ltd. The peptide sequences are as follows:

[0044] EP1: GYNNYHSAHLAKLKPLLDAEFILVDYT;

[0045] EP2: LLDAEFILVDYTQGKKGKDLGAILWVC;

[0046] EP3:KGKDLGAILWVCELPNKKRFVVTPKHL;

[0047] EP4:NKKRFVVTPKHLTYADRYALFQKLTPA;

[0048] EP5: DRYALFQKLTPALFKKHLYGKELTVEY;

[0049] EP6:KHLYGKELTVEYAELSPKTGIPLQARA;

[0050] EP7: ELSPKTGIPLQARAVGFREPISVLEII.

[0051] The synthesized EP1-EP7 peptides were spotted onto nitrocellulose (NC) membranes, 2 μg per membrane. After the membranes were completely dry, they were blocked with TBST containing 5% BSA at 37°C for 2 h, followed by Western blotting. Dot-blot experiments were performed using ASFV positive and negative sera as primary antibodies, and the results are shown below. Figure 7 As shown, peptide EP1 exhibited a strong positive reaction signal with all ASFV-positive sera, but no cross-reactivity with negative sera. Peptides EP2 to EP7 showed no significant reaction with either positive or negative sera. The core immunodominant linear B-cell epitope EP1 of the NP419L protein was ultimately identified as follows: 305 GYNNYHSAHLAKLKPLLDAEFILVDYT 331 As shown in SEQ ID NO.1.

[0052] Example 3:

[0053] Application of the antigenic epitope polypeptide EP1 prepared in Example 2 in the preparation of African swine fever virus antibody detection reagent:

[0054] The synthesized NP419L protein dominant B-cell antigenic epitope peptide (SEQ ID NO.1) was dissolved in ELISA coating buffer (0.1 mol / L carbonate buffer, pH 9.6, 1.59 g Na2CO3, 2.93 g NaHCO3 dissolved in an appropriate amount of ddH2O and then brought to a final volume of 1 L) to a final concentration of 100 ng / well. 100 μL was added to each well of the plate and coated at 4°C for 16 h. The coating buffer was discarded, and the plate was washed three times with PBST (PBS containing 0.1% Tween-20), 300 μL / well each time. Subsequently, 5% skim milk prepared with PBS was used as the blocking buffer, and the plate was blocked at 37°C for 2 h. The blocking buffer was discarded, and the plate was washed three more times with PBST. African swine fever virus positive and negative sera were used as primary antibodies, diluted 1:100, and 100 μL was added to each well of the ELISA plate and incubated at 37°C for 1.5 h. After incubation, discard the serum and wash three times with PBST. Then add 100 μL of rabbit anti-porcine HRP-IgG enzyme-labeled secondary antibody (1:8000) to each well and incubate at 37°C for 1 hour. Discard the enzyme-labeled secondary antibody, wash three times with PBST, and shake off as much liquid as possible from the wells on the last wash. Add 50 μL of TMB chromogenic buffer to each well and incubate at 37°C in the dark for 10 minutes. Then add 50 μL / well stop solution to terminate the reaction. Read the OD values ​​at 450 nm using a microplate reader. Results are as follows. Figure 8 As shown, compared with the negative serum group, the NP419L protein antigenic epitope polypeptide exhibited significant and specific binding to the positive serum, with OD... 450 The significantly elevated value indicates that the epitope peptide can be recognized by specific antibodies produced by ASFV-infected animals, exhibiting good immunogenicity and antigenicity, supporting its potential application in the serological diagnosis and detection of African swine fever virus infection.

[0055] The dominant B-cell antigenic epitope EP1 (SEQ ID NO.1) of the NP419L protein identified in this invention, as well as the full-length NP419L protein, A2 segment protein, and B2 segment protein as comparative controls, were dissolved in ELISA coating buffer (0.1 mol / L carbonate buffer). Then, coating was performed overnight at 4°C with a final concentration of 100 ng / well. Blocking was performed at 37°C for 2 h using 5% skim milk as the blocking buffer. Three different sera obtained from different times and regions and exhibiting African swine fever virus positive sera were serially diluted from 1:25 to a maximum of 1:51200, and incubated at 37°C for 1.5 h. Then, rabbit anti-porcine HRP-IgG enzyme-labeled secondary antibody diluted 1:8000 was added, and the reaction was carried out at 37°C for 1 h. TMB chromogenic solution was added, and the mixture was incubated at 37°C in the dark for 10 min. Finally, 50 μL / well of stop solution was added, and the OD value was read at 450 nm.

[0056] Experimental results are as follows Figure 9 ( Figure 9 As shown in the average of the detection values ​​of three positive serum strains of African swine fever virus (ASFV), the detection sensitivity of epitope EP1 is similar to that of the full-length NP419L protein, and even slightly higher than that of the full-length protein at some serum dilutions. Meanwhile, the detection sensitivity of epitope EP1 is significantly higher than that of truncated fragments A2 and B2 containing its sequence. These results indicate that the dominant B-cell epitope EP1 of the NP419L protein identified in this invention not only effectively maintains its immunorecognition performance with the full-length protein, but also avoids interference from other non-dominant regions of the protein, concentrating and amplifying the immunodominant signal, thus exhibiting higher sensitivity. Epitope EP1 can still be detected at a serum dilution of 1:6400.

[0057] Therefore, the dominant B-cell antigenic epitope EP1 of the NP419L protein identified in this invention has high immunoreactivity, high sensitivity and specificity, and has high application potential in the development of ASFV serological diagnostic reagents, and can be used as a preferred antigen for the construction of related products.

[0058] Example 4:

[0059] Conservation of the antigenic epitope peptides prepared in Example 2 among different ASFV strains:

[0060] Furthermore, to verify the conservation of the screened antigenic epitope sequence among different ASFV strains, the publicly available amino acid sequence of the NP419L protein was downloaded from the NCBI database and multiple sequence alignment analysis was performed using MEGA-X software. The results showed that this antigenic epitope... 305 GYNNYHSAHLAKLKPLLDAEFILVDYT 331 (SEQ ID NO.1) exhibits high conservation across multiple ASFV genotypes, showing 100% amino acid sequence identity in the NP419L protein sequences of all major ASFV genotypes (including genotypes I, II, IV, VIII, IX, XX, XXII, and some undetermined genotype strains). Partial sequence alignment results are shown below. Figure 10 As shown, the NP419L protein sequence is 100% conserved in currently published sequences. This indicates that the epitope has good stability and universality across different ASFV strains, providing strong support for its application in cross-strain diagnostic detection and vaccine design.

Claims

1. A synthetically produced linear B-cell antigenic epitope polypeptide of African swine fever virus NP419L protein, the polypeptide being shown in SEQ ID NO.

1.

2. The fusion protein obtained by fusing the antigen epitope polypeptide and the protein tag according to claim 1.

3. The gene encoding the antigenic epitope polypeptide of claim 1 or the fusion protein of claim 2.

4. An expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant eukaryotic cell having the gene encoding as described in claim 3.

5. The use of the antigenic epitope polypeptide of claim 1, the fusion protein of claim 2, the encoding gene of claim 3, and the expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant eukaryotic cell of claim 4 in the preparation of African swine fever virus antibody detection reagents.

6. The use of the antigenic epitope polypeptide of claim 1, the fusion protein of claim 2, the encoding gene of claim 3, and the expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant eukaryotic cell of claim 4 in the preparation of an African swine fever virus detection kit.

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