Senecavirus VP2 protein B cell neutralizing epitope polypeptide and monoclonal antibody and application thereof

Abstract

The invention relates to a linear B-cell neutralizing epitope polypeptide of Senecavirus VP2 protein and application of the linear B-cell neutralizing epitope polypeptide. The amino acid sequence of the linear B cell neutralizing epitope of the Seneca virus VP2 protein is SLQELN. A BALB / c mouse is immunized by the VP2 recombinant protein, two monoclonal antibodies 1A1F6 and 7H10C3 for identifying the neutralizing epitope are obtained through virus neutralization test screening by utilizing a hybridoma cell technology, the monoclonal antibodies have neutralizing activity on Seneca virus infection, and the median inhibitory concentrations of the monoclonal antibodies are 0.64 mu g / mL and 1.21 mu g / mL respectively. The highly-conservative linear B cell neutralizing epitope of the VP2 protein is accurately positioned through segmented expression of the VP2 protein and scanning of synthetic polypeptide, and bioinformatics analysis shows that the neutralizing epitope is highly conservative in an SVA epidemic strain and is a potential Seneca virus novel vaccine antigen target; the B cell neutralizing epitope and the monoclonal antibody enrich immunological functions of Seneca virus VP2 protein, and lay a good foundation for research and development of subsequent antiviral drugs, vaccines and detection products.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Seneca virus VP2 protein B cell neutralizing epitope polypeptide and a monoclonal antibody thereof and application thereof. Background Art

[0002] Senecavirus A (SVA), also known as Seneca Valley virus, is a member of the Picornaviridae family and the only member of the Senecavirus genus. This virus is associated with vesicular disease in pigs. Its clinical symptoms are often difficult to distinguish from those of diseases such as foot-and-mouth disease (FMD) and swine vesicular disease (SVD), which may lead to misdiagnosis and pose a significant risk to the pig industry. The clinical manifestations of SVA infection in pigs usually begin with mild symptoms such as lethargy and lameness, and then may progress to vesicular lesions on the snout, hooves or coronary bands. In addition to vesicular disease, SVA can also cause epidemic neonatal mortality in piglets. Diagnostic methods for SVA include reverse transcription-PCR (RT-PCR) and serological testing, which are essential for identifying infected animals and managing outbreaks. Therefore, it is necessary to explore the development of monoclonal antibodies against SVA proteins and identify specific antigenic epitopes to design and develop new vaccines.

[0003] The SVA genome is approximately 7,300 nucleotides long and consists of a single open reading frame encoding a polyprotein that is subsequently cleaved into structural and nonstructural proteins. Structural proteins include VP1, VP2, VP3, and VP4, which form the viral capsid, while nonstructural proteins are involved in viral replication and immune evasion. The VP2 protein of SVA plays an important role in the structure and function of the virus. Studies have shown that VP2 is the main target of the immune response, contains multiple dominant B cell epitopes, and can trigger the production of neutralizing antibodies, providing effective protective immunity for the pig herd. This shows that VP2 is not only essential for the structural integrity of the virus, but also plays a key role in the host's immune response, making it a prime candidate for vaccine development. In addition, the VP2 protein interacts with the anthrax toxin receptor 1 (ANTXR1) to mediate viral invasion, which is essential for the virus's ability to infect host cells.

[0004] Therefore, the VP2 protein of Senecavirus A is not only a key structural component, but also an important target for vaccine development and therapeutic intervention. Identifying its B cell neutralizing epitopes and screening for corresponding neutralizing monoclonal antibodies are of great significance for SVA antiviral research and vaccine development. Summary of the invention

[0005] To solve the above problems, the present invention provides a linear B cell neutralizing epitope associated with the SVAVP2 protein, and two neutralizing monoclonal antibodies that specifically recognize the B cell neutralizing epitope, providing a theoretical basis and core antibodies for the detection of Seneca virus and the development of related vaccines.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A B cell neutralizing epitope polypeptide of Seneca virus VP2 protein, wherein the amino acid sequence of the B cell neutralizing epitope polypeptide is SLQELN.

[0008] A monoclonal antibody to Seneca virus VP2 protein, wherein the monoclonal antibody is 1A1F6, which can specifically recognize the B cell neutralizing epitope polypeptide according to claim 1, and the half inhibition concentration of the antibody to Seneca virus is 0.64 μg / mL.

[0009] A monoclonal antibody to the VP2 protein of Seneca virus, wherein the monoclonal antibody is 7H10C3, which can specifically recognize the B cell neutralizing epitope polypeptide according to claim 1, and the half inhibitory concentration IC 50 It is 1.21μg / mL.

[0010] The application of the B cell neutralizing epitope polypeptide of the Seneca virus VP2 protein in neutralizing antibody detection reagents and immune evaluation.

[0011] The application of the B cell neutralizing epitope polypeptide of the Seneca virus VP2 protein in the design of Seneca virus antigens and novel vaccines.

[0012] The monoclonal antibody is used in neutralizing antibody detection reagents and immune evaluation.

[0013] Beneficial effects of the present invention

[0014] (1) The present invention uses molecular biology and cell biology techniques to identify the linear B cell neutralizing epitope 151-SLQELN-156 associated with the Seneca virus VP2 protein. The VP2 protein was successfully expressed and purified in an insect baculovirus expression system, BALB / c mice were immunized by immunological methods, monoclonal antibodies against the VP2 protein were prepared by cell fusion technology, and monoclonal antibodies with virus neutralizing activity were screened by virus neutralization tests; the antigenic epitope recognized by the neutralizing monoclonal antibody was identified by full-length segmented expression of the VP2 protein and overlapping peptide scanning, and sequence comparison analysis showed that the epitope was highly conserved in the SVA epidemic strain.

[0015] (2) The neutralizing monoclonal antibodies of the present invention are 1A1F6 and 7H10C3, wherein the IC 50 The IC value of 7H10C3 is 0.64 μg / mL. 50 It was 1.21 μg / mL. Both monoclonal antibodies specifically recognized the B cell epitope SLQELN.

[0016] (3) The B cell neutralizing epitopes identified in the present invention enrich the immunological function of the Seneca virus VP2 protein, laying a good foundation for the subsequent research and development of antiviral drugs, vaccines and detection products. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 IFA was used to detect the expression of VP2 protein in Sf21 cells infected with baculovirus Bac-VP2 cosmid.

[0018] Figure 2 Immunogenicity analysis of SVA recombinant VP2 protein and identification of purified VP2 recombinant protein.

[0019] Figure 3 The neutralization effect of hybridoma cell supernatant on SVA was analyzed by IFA method.

[0020] Figure 4 IFA was used to determine the neutralizing antibody titers of monoclonal antibodies 1A1F6 and 7H10C3 against SVA.

[0021] Figure 5 Schematic diagram of truncated VP2 protein and synthetic peptides.

[0022] Figure 6 Antibodies identify neutralizing linear epitopes on the VP2 protein.

[0023] Figure 7 Dot-blot and Peptide-ELISA were used to detect the binding of monoclonal antibodies to P1-P10 peptides.

[0024] Figure 8 Dot-blot and Peptide-ELISA were used to detect the binding of monoclonal antibodies to P6.1~P6.4 peptides.

[0025] Fig. 9 Dot-blot and Peptide-ELISA were used to detect the binding of monoclonal antibodies to P6.5~P6.9 peptides.

[0026] Fig.10 Identification of key amino acid sites in the B cell neutralizing epitope of VP2 protein.

[0027] Fig.11 Conservation analysis of the B cell neutralizing epitope of VP2 protein in different SVA virus strains.

[0028] Fig.12 Structural analysis of the B cell neutralizing epitope of VP2 protein. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments.

[0030] Example 1. Expression and purification of SVAVP2 protein

[0031] 1.1 Construction and expression of insect baculovirus expression vector of SVAVP2 protein

[0032] With reference to the VP2 gene sequence of the SVA-HeNNY-1-2018 virus strain (GenBank accession number: MK357116), the gene coding sequence was optimized according to the codon usage preference of the baculovirus expression system, and a signal peptide was added to the N-terminus. The codon-optimized VP2 gene was synthesized by General Biotechnology Co., Ltd. and subcloned into the pFastBac1 baculovirus expression vector. The recombinant plasmid was named: pFastBac1-VP2. The pFastBac1-VP2 plasmid was used to transform Escherichia coli DH10Bac competent cells, and Bac-VP2 cosmid was obtained by blue-white screening. Bac-VP2cosmid was transfected into Sf21 insect cells using Cellfection II Reagent transfection reagent and cultured for 72 hours. The supernatant was collected and cultured for three generations. The expression of VP2 in Sf21 cells infected with recombinant baculovirus was detected by indirect immunofluorescence. Sf21 cells infected with recombinant baculovirus showed specific red fluorescence, indicating that the SVA VP2 recombinant baculovirus successfully expressed the SVAVP2 protein. The results are shown in Figure 1 .

[0033] Figure 1 In the figure, A is IFA detection of baculovirus Bac-VP2 cosmid infected Sf21 insect cells, where the red fluorescence is VP2 protein expressed in Sf21 insect cells; B is IFA detection of normal Sf21 insect cells, where the blue fluorescence is cell nucleus staining.

[0034] 1.2 Protein purification

[0035] 72 hours after Sf21 cells were infected, the cell supernatant was collected by centrifugation. The supernatant was first filtered through a 0.22μm filter membrane and then purified by nickel affinity chromatography. The nickel column was installed on the AKTA purifier, and the sample was loaded after the column was equilibrated with the equilibrium solution (20mmol / LTris-HCl, 150mol / LNaCl). After washing with a buffer containing 50mmol / L imidazole, the SVAVP2 protein was eluted with a 20mol / LTris-HCl, 150mmol / LNaCl buffer containing 300mmol / L imidazole, and the purified protein was identified by SDS-PAGE and Western blot. The VP2 recombinant protein in the culture supernatant of the transfected cells was eluted with 300mmol / L imidazole to obtain a high-purity VP2 purified protein, which was specifically recognized by a rabbit anti-VP2 polyclonal antibody (made in this laboratory) with a molecular weight of approximately 38kDa. It was used for mouse immunization and monoclonal antibody screening. The results are shown in Figure 2 .

[0036] Figure 2 In the figure, A is Western blot analysis of rabbit anti-VP2 polyclonal antibody, and B is SDS-PAGE analysis. M: protein marker; 1: transfected cell culture supernatant; 2: flow-through solution; 3: 50mmol / L imidazole washing; 4: 300mmol / L imidazole elution.

[0037] Example 2. Screening and identification of monoclonal antibodies

[0038] 2.1 Animal immunization

[0039] 50 μg of the purified VP2 protein obtained in Example 1 was fully emulsified with an equal amount of Freund's complete adjuvant, and BALB / c mice were first immunized by subcutaneous injection at multiple points on the back. After the first immunization, the same dose of purified VP2 protein and an equal amount of Freund's incomplete adjuvant were used to immunize once every 14 days, and a total of 4 6-week-old female BALB / c mice were immunized. After the fourth immunization, the tail vein blood of the immunized mice was collected, and the serum antibody titer was detected using an ELISA plate coated with VP2 protein. The primary antibody was the mouse venous blood continuously diluted after the fourth immunization, and the enzyme-labeled goat anti-mouse IgG antibody was the secondary antibody. The ELISA results showed that the serum antibody titer range of the four mice was 1:6.4×10 4 to 1:1.28×10 5 The mice with the highest serum antibody titer were selected and given a booster immunization by intraperitoneal injection of 50 μg VP2 protein.

[0040] 2.2 Screening of hybridomas

[0041] One week before fusion, the revived SP2 / 0 cells were subcultured and cell fusion was performed with the spleen cells of mice 3 days after booster immunization. The spleen cells of mice were separated and then gently mixed and fused with SP2 / 0 myeloma cells through the fusion agent PEG 1500. The fused cells were plated in a 96-well plate, HAT selection medium was added to the fused cells, and cultured in a 37°C CO2 incubator. After 7 days, the culture medium was discarded and replaced with HT medium. When the cell clusters in the 96 wells filled 1 / 3 of the wells, the supernatant was aspirated as the primary antibody, and preliminary screening was performed by enzyme-linked immunosorbent assay (ELISA). The positive hybridoma cells were transferred to a 24-well cell plate, and then the hybridoma cells with neutralizing activity were screened by neutralization test. The positive hybridoma cells were subcloned by limiting dilution method to obtain hybridoma cell lines that stably secreted neutralizing monoclonal antibodies. Positive hybridoma cells were injected intraperitoneally into BALB / c mice sensitized with liquid paraffin (16-week-old female BALB / c mice were intraperitoneally inoculated with 500 μL sterile liquid paraffin) to produce ascites. The ascites was collected from the mice and purified using the caprylic acid-ammonium sulfate method.

[0042] (1)ELISA

[0043] The VP2 recombinant protein purified in Example 1 (1 μg / mL, 100 μL / well) was used to coat a 96-well plate. The protein was placed in 0.05 mol / L carbonate buffer (CBS, pH 9.6) and coated at 37°C for 2 h or at 4°C overnight. After washing 5 times with PBST (1×PBS, 0.05% Tween20, pH 7.4), the cells were placed in blocking solution (PBST containing 5% skim milk), blocked at 37°C for 2h, and washed 5 times with PBST; 100 μL of hybridoma culture supernatant was added to each well, the cells were incubated at 37°C for 30min, the serum of mice immunized with VP2 protein was used as the positive control, the serum of non-immunized mice was used as the negative control, and the cells were washed 5 times with PBST; 100 μL of horseradish peroxidase-labeled goat anti-mouse IgG antibody (1:5000 dilution) was added, the cells were incubated at 37°C for 30min, and the cells were washed 5 times with PBST; 100 μL of TMB was added to react at room temperature for 5min, and the reaction was terminated with 2mol / LH2SO4; the absorbance at 450nm was read using an enzyme reader to screen hybridoma cell lines that stably secrete anti-SVAVP2 protein monoclonal antibodies.

[0044] (2) Neutralization test

[0045] The hybridoma cell supernatant was mixed with 200 TCID 50SVA-HeNNY-1-2018 were mixed in equal amounts and incubated in a CO2 incubator at 37°C for 2 hours; the virus-antibody mixture was inoculated into a monolayer of BHK-21 cells in a 96-well plate at 100 μL / well and incubated in a 37°C incubator for 24 hours. The virus fluorescent spots were analyzed by IFA method to screen monoclonal antibodies with neutralizing activity. SVAVP2 protein rabbit polyclonal antibody serum (1:1000 dilution) was added as the primary antibody, and Alexa fluor 647-labeled donkey anti-rabbit IgG (1:500 dilution) was added as the secondary antibody (3E2C3 was used as the non-neutralizing antibody negative control, and PC was used as the positive serum neutralization control); after staining with DAPI, the neutralizing activity of the monoclonal antibody was detected under a fluorescence microscope. The results are shown in Figure 3 , where 3E2C3 is a non-neutralizing antibody negative control, and PC is a positive serum neutralization control. It can be seen that the fluorescence signals in the reaction wells of monoclonal antibodies 1A1F6 and 7H10C3 are significantly lower than those in the negative control, indicating that 1A1F6 and 7H10C3 both exhibit significant neutralizing activity against SVA virus infection.

[0046] 2.3 Characterization of monoclonal antibodies

[0047] The titers of the two monoclonal antibodies were determined by indirect ELISA. The titers of the two monoclonal antibodies were 1:5.12×10 5 to 1:1.02×10 6 The subtypes of the two monoclonal antibodies were identified using a commercial kit, and the heavy chains of the two monoclonal antibodies were both IgG2b, and the light chains were both kappa.

[0048] Neutralization activity of monoclonal antibodies 1A1F6 and 7H10C3 against SVA was further determined by IFA-based neutralization assay. The purified antibodies were diluted to 1 mg / mL and then diluted 1:2, 1:4, 1:8, ... 1:2048 in serum-free DMEM. Each antibody dilution was repeated 3 times. A volume of 100 μL of 200 TCID 50 The virus was mixed with an equal volume of antibody and incubated in a 37°C incubator for 1 hour. The mixture was then added to the monolayer cells and incubated for 24 hours. The supernatant was discarded and the cells were fixed with anhydrous ethanol for 10 minutes at room temperature. PBST was washed three times. Anti-VP2 rabbit polyclonal antibody was added and incubated at 37°C for 1 hour; Alexa Fluor 647 donkey anti-rabbit fluorescent secondary antibody was added and incubated at 37°C for 1 hour; after PBST washing, DAPI was used for staining at room temperature for 10 minutes. Observed under a fluorescence microscope and photographed and recorded.

[0049] The following formula was used to calculate: virus inhibition rate (%) = (average number of spots in virus control - average number of spots in each antibody concentration) / average number of spots in virus control × 100%. The data were analyzed using the nonlinear regression dose-response model in GraphPad Prism software. The half inhibitory concentration (IC 50 ), the results are shown in Figure 4 .

[0050] Figure 4 It can be seen that the IC of 1A1F6 50 The IC value of 7H10C3 is 0.64 μg / mL. 50 It was 1.21μg / mL, proving that both monoclonal antibodies have good ability to neutralize SVA virus, providing a theoretical basis for the design of new SAV vaccine antigens.

[0051] Example 3. Identification of linear B cell neutralizing epitopes of SVAVP2 protein

[0052] 3.1 Precise localization of the linear B cell neutralizing epitope of SVAVP2 protein

[0053] First, the antigenic epitope regions of the VP2 protein recognized by two neutralizing monoclonal antibodies 1A1F6 and 7H10C3 were located. Figure 5 The segmented expression strategy shown designs three partially overlapping VP2 protein fragments V1 (1-100aa), V2 (100-200aa) and V3 (190-284aa). Figure 5 , the numbers 1-284 represent the length of the amino acid sequence of VP2 protein, lines of different lengths represent different regions within the sequence, and P1-P10 represent different synthetic peptide sequences.

[0054] The gene encoding VP2 protein fragment was subcloned into pCGGS vector, and HEK293T cells were transfected with recombinant plasmids to express three VP2 protein fragments. The reactivity of VP2 protein fragments with monoclonal antibodies was analyzed by Western blot. The VP2 fragment expression protein was separated by 12% SDS-PAGE, and then transferred to PVDF membrane; placed in blocking solution, blocked at 37℃ for 1h, and washed thoroughly with PBST; added SVAVP2 protein monoclonal antibody and incubated on a shaker at room temperature for 1h, and washed thoroughly with PBST; added goat anti-mouse IgG / HRP and incubated at 37℃ for 1h, and washed thoroughly with PBST; used ECL substrate for chemiluminescent signal development, and analyzed the reactivity of VP2 protein fragments with monoclonal antibodies.

[0055] Results Figure 6, wherein the plasmid encoding V1, V2 or V3 was transfected into 293T cells, and then the cell lysate was subjected to Western blot analysis using antibodies. The results of Western blot detection of VP2 fragment transfected cells using HA-tag antibody showed that VP2 fragments V1, V2 and V3 were successfully expressed in HEK293T cells, showing specific color bands ( Figure 6 A); monoclonal antibody 1A1F6 ( Figure 6 B) and 7H10C3( Figure 6 C) Western blot results of cells transfected with VP2 fragments showed that the V2 fragment specifically reacted with monoclonal antibodies 1A1F6 and 7H10C3, showing specific color bands, while no obvious color bands were observed for V1 and V3 fragments, indicating that monoclonal antibodies 1A1F6 and 7H10C3 specifically recognized the V2 region (100-200aa) of VP2 protein.

[0056] To further locate the antigen epitope recognized by the monoclonal antibody, continuous overlapping peptides were designed for the V2 epitope region of the VP2 protein. The peptides were synthesized by Gill Co., Ltd. The peptide schematic diagram and sequence are shown in Figure 5 As shown in Table 1, each peptide overlapped with the previous and next peptides by 5 amino acids, and a cysteine ​​was added at the C-terminus for carrier protein coupling. The peptides were coupled to IgG-free BSA as detection antigens, and the reactivity of the peptides with monoclonal antibodies 1A1F6 and 7H10C3 was detected by Dot-blot and Peptide-ELISA.

[0057] (1) Dot-blot: Dilute the peptide with ultrapure water to the recommended concentration of 0.1 mg / mL, and spot 1 μg / spot on nitrocellulose membrane. Set VP2 recombinant protein and BSA as positive and negative controls, respectively. Block with 5% skim milk at 37°C for 1 h. Use two monoclonal antibodies as primary antibodies and HRP-labeled goat anti-mouse IgG as secondary antibodies. Incubate at 37°C for 30 min, and perform chemiluminescence detection with ECL chemiluminescence solution.

[0058] (2) Peptide-ELISA: The peptide was diluted to 5 μg / mL with 0.05 mol / L CBS (pH 9.6), coated on a 96-well ELISA plate at 37°C for 2 h, and detected using the aforementioned indirect ELISA method.

[0059] Dot-blot and Peptide-ELISA test results are shown in Figure 7 , recombinant VP2 protein was used as a positive control, and BSA was used as a negative control.

[0060] Dot-blot results showed that P6 peptides were all bound to monoclonal antibody 1A1F6 ( Figure 7 A) and 7H10C3( Figure 7B) Specific reaction, with obvious color spots, while other polypeptides have no obvious color spots; Peptide-ELISA ( Figure 7 C) Consistent with the results of Dot-bot, monoclonal antibodies 1A1F6 and 7H10C3 specifically bound to the P6 polypeptide and showed obvious color development, while other polypeptides did not show obvious color development, indicating that monoclonal antibodies 1A1F6 and 7H10C3 can recognize the antigen epitope 147-GKAKSLQELNEEQWV-161 (SE Q ID NO: 2).

[0061] To further locate the antigen epitopes recognized by monoclonal antibodies 1A1F6 and 7H10C3, two consecutive amino acids were truncated from both ends of the P6 polypeptide, and the polypeptides were synthesized as above, and Dot-blot and Peptide-ELISA tests were performed. The results are shown in Figure 8 , P6 peptide was used as a positive control, and BSA was used as a negative control.

[0062] Dot-blot results showed that the truncated peptides P6.1, P6.2, P6.3, and P6.4 all interacted with the monoclonal antibody 1A1F6 ( Figure 8 A) 7H10C3( Figure 8 B) Specific reaction, showing obvious color spots; Peptide-ELISA ( Figure 8 C) Consistent with the results of Dot-blot, the deletion of 4 amino acids at the N-terminus and C-terminus of P6 polypeptide did not significantly affect the specific binding of polypeptide with monoclonal antibodies 1A1F6 and 7H10C3, indicating that the antigen epitope recognized by monoclonal antibodies 1A1F6 and 7H10C3 is located at 151-SLQELNE-157 (SEQ ID NO: 3). The N-terminus and C-terminus of P6.8 polypeptide were truncated one by one, and Dot-blot and Peptide-ELISA tests were performed. The results are shown in Fig. 9 , P6 peptide was used as a positive control, and BSA was used as a negative control.

[0063] Dot-blot results showed that the C-terminal was missing Glu 157 It did not significantly affect the specific binding of polypeptide 151-SLQELNE-157 (SEQ ID NO: 3) with monoclonal antibodies 1A1F6 and 7H10C3, showing obvious color spots, while the N-terminal deletion of Ser 151 Or C-terminal deletion of Asn 156 It completely lost the ability to bind to monoclonal antibodies 1A1F6 and 7H10C3, and no obvious color spots were observed; Peptide-ELISA ( Fig. 9 C) Consistent with the Dot-blot results, it showed that the linear B cell neutralizing epitope recognized by monoclonal antibodies 1A1F6 and 7H10C3 was 151-SLQELN-156 (SEQ ID NO: 1).

[0064] Table 1 SVAVP2 series truncated polypeptide sequences

[0065]

[0066] 3.2 Identification of key amino acids in the neutralizing epitope of VP2 protein in B cells

[0067] Based on the linear B cell neutralizing epitope peptide 151-SLQELN-156 (SEQ ID NO: 1) recognized by monoclonal antibodies 1A1F6 and 7H10C3, the amino acids of the peptide were replaced sequentially with alanine (Ala). The neutralizing epitope mutant peptide sequence is shown in Table 2 and was synthesized by Gill Biochemical (Shanghai) Co., Ltd. The reactivity of the mutant epitope with monoclonal antibodies 1A1F6 and 7H10C3 was detected by Dot-blot and Peptide-ELISA. The results are shown in Fig.10 , Dot-blot results show that Ser 151 ,Leu 152 ,Leu 155 or Asn 156 When mutated to Ala, the neutralizing antigen epitope peptide was completely lost and the monoclonal antibody 1A1F6 ( Fig.10 A) and 7H10C3( Fig.10 B) reactivity, no obvious color spots were observed, while Gln 153 or Glu 154 The mutation had no obvious effect, and the color spots appeared; Peptide-ELISA ( Fig.10 C) is consistent with the Dot-blot results, indicating that Ser 151 ,Leu 152 ,Leu 155 and Asn 156 It is the key amino acid for the VP2 linear neutralizing antigen epitope binding monoclonal antibodies 1A1F6 and 7H10C3.

[0068] Table 2 Alanine (A) mutant polypeptide sequences of neutralizing antigen epitope polypeptides

[0069]

[0070] 3.3 Conservative analysis and spatial structural characteristics of linear neutralizing antigen epitopes

[0071] In order to systematically analyze the conservation of the neutralizing antigen epitopes recognized by monoclonal antibodies 1A1F6 and 7H10C3 in different genotypes of SVA VP2 proteins, the amino acid sequences of the structural protein VP2 genes of 30 domestic and foreign SVA isolates were downloaded from GenBank, and the amino acid sequence variation of 151-SLQELN-156 (SEQ ID NO: 1) was analyzed using the biological software DNAMAN. The results are shown in Figure 2. Fig.11 The neutralizing antigen epitope 151-SLQELN-156 (SEQ ID NO: 1) was highly conserved among the SVA strains analyzed, and only the 152nd amino acid of the G103_SV_1 / 2016 / Thailand strain underwent a Leu→Ile mutation, indicating that the B cell neutralizing antigen epitope sequence 151-SLQELN-156 (SEQ ID NO: 1) recognized by monoclonal antibodies 1A1F6 and 7H10C3 was highly conserved among the SVA epidemic strains and was an ideal target for the development of new SVA vaccines, detection reagents and broad-spectrum neutralizing antibody preparations.

[0072] The spatial structure of the VP2 epitope was visualized and analyzed using PyMol software, a three-dimensional protein structure model of the VP2 protein was constructed, and the structural characteristics of the antigen epitope 151-SLQELN-156 (SEQ ID NO: 1) were analyzed. The results are shown in Fig.12 .

[0073] Fig.12 A is the visualization of the epitope on the VP2 protein backbone, epitope 151-SLQELN-156 (SEQ ID NO: 1) (blue), which mainly exists in a helical form. Fig.12 B is the visualization of the epitope on the solid surface of the VP2 protein. The epitope 151-SLQELN-156 (SEQ ID NO: 1) is located on the surface of the VP2 protein (blue), indicating that monoclonal antibodies 1A1F6 and 7H10C3 recognize the B cell neutralizing antigen epitope 151-SLQELN-156 (SEQ ID NO: 1) exposed on the surface of the VP2 protein, making it easier to be recognized by antibodies or the immune system, which is the key to vaccine design, antibody development and immune response initiation.

Claims

1. A B cell neutralizing epitope polypeptide of Seneca virus VP2 protein, characterized in that: The amino acid sequence of the B cell neutralizing epitope polypeptide is SLQELN.

2. A monoclonal antibody against Seneca virus VP2 protein, characterized in that: The monoclonal antibody is 1A1F6, which can specifically recognize the B cell neutralizing epitope polypeptide according to claim 1, and the half inhibition concentration of the antibody to Seneca virus is 0.64 μg / mL.

3. A monoclonal antibody against Seneca virus VP2 protein, characterized in that: The monoclonal antibody is 7H10C3, which can specifically recognize the B cell neutralizing epitope polypeptide according to claim 1, and the half inhibitory concentration IC 50 It is 1.21μg / mL.

4. Use of the B cell neutralizing epitope polypeptide of the Seneca virus VP2 protein according to claim 1 in neutralizing antibody detection reagents and immune evaluation.

5. Use of the B cell neutralizing epitope polypeptide of the Seneca virus VP2 protein according to claim 1 in the design of Seneca virus antigens and new vaccines.

6. Use of the monoclonal antibody according to claim 2 or 3 in neutralizing antibody detection reagents and immune evaluation.

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