Pep4, a polypeptide sequence that binds to pathogenic serum type 4 FAdV Fiber-2 antibody, and its application.

Abstract

This invention provides a polypeptide sequence Pep4 that binds to antibodies against pathogenic serum type 4 FAdV Fiber-2 and its application. The polypeptide sequence Pep4 is linear, and its amino acid sequence is PSTATLTDFEPMANRSVTSPWTYSANGYYEPSI. The polypeptide sequence Pep4 is stable, readily soluble in water, targets the B-cell epitopes of pathogenic serum type 4 FAdV Fiber-2, and has a strong binding affinity to antibodies against pathogenic serum type 4 FAdV Fiber-2. Pep4 exhibits high specificity.

Description

Technical Field

[0001] This invention relates to B-cell epitopes of avian adenovirus Fiber-2 protein, and more particularly to a polypeptide sequence Pep4 that binds to pathogenic serum type 4 FAdV Fiber-2 antibody and its applications. Background Technology

[0002] Avian adenovirus (FAdV) is classified into subgroups I, II, and III based on its sequence characteristics. Subgroup I can be further divided into five species (AE) based on serocross-neutralization assays, with serotype 4 FADV belonging to species C. It primarily causes pericardial effusion and inclusion body hepatitis in chickens. Since 2015, it has experienced large-scale outbreaks in most parts of my country, with a mortality rate reaching 30%.

[0003] Avian adenovirus particles have an icosahedral spatial structure and lack an envelope. The viral capsid contains three main structural proteins: hexon, penton, and fiber proteins. Among them, the fiber protein is the main associated virulence factor, participating in the body's major protective antigen components. Its carboxyl-terminal and knob-terminal domains are responsible for initiating infection, promoting viral attachment and binding to cell receptors, and enabling mutual adhesion between the two. Furthermore, the knob-terminal domain contains type-specific antigenic epitopes, generating corresponding immune responses to achieve the purpose of resisting viral invasion.

[0004] Since the 1990s, the unprecedented development of bioinformatics and computer molecular simulation technology has provided a broader platform for the analysis of protein function and higher-order structure. More and more researchers are able to screen functional peptides at the molecular level and further verify their findings. Compared with traditional peptide screening methods, computer screening utilizes homology modeling technology to predict the spatial structure of target proteins based on amino acid homology according to the resolved protein structures. This saves a significant amount of time and manpower, representing a major breakthrough in immunology and becoming one of the important and commonly used techniques. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a polypeptide sequence Pep4 that binds to antibodies against pathogenic serum type 4 FAdV Fiber-2 and its applications. The polypeptide sequence Pep4 is stable, readily soluble in water, targets the B-cell epitopes of pathogenic serum type 4 FAdV Fiber-2, and exhibits strong binding affinity to antibodies against pathogenic serum type 4 FAdV Fiber-2. Pep4 also demonstrates high specificity.

[0006] The object of the present invention is achieved in the following manner: a polypeptide sequence Pep4 that binds to a pathogenic serum type 4 FAdV Fiber-2 antibody, wherein the polypeptide sequence Pep4 is linear and the amino acid sequence of the polypeptide sequence Pep4 is PSTATLTDFEPMANRSVTSPWTYSANGYYEPSI, as described in SEQ ID NO: 1.

[0007] The peptide sequence Pep4 that binds to the pathogenic serum type 4 FAdV Fiber-2 antibody includes any corresponding adjustments or modifications to the Pep4 sequence, with the Pep4 sequence as the core. Modifying materials include, but are not limited to, biotin, avidin, magnetic beads, nanomaterials, fluorescent materials, enzymes, and specific proteins.

[0008] The application of the peptide sequence Pep4, which binds to the pathogenic serum type 4 FAdV Fiber-2 antibody.

[0009] The peptide sequence Pep4, which binds to the pathogenic serum type 4 FADV Fiber-2 antibody, is used for the rapid qualitative or quantitative detection of serum type 4 FADV. This includes, but is not limited to, enzyme-linked immunosorbent assay (ELISA), plasmon resonance assay (PRA), and polyacrylamide gel electrophoresis (PAG).

[0010] The application of the peptide sequence Pep4, which binds to the pathogenic serum type 4 FAdV Fiber-2 antibody, in the preparation of rapid detection reagents or kits for avian adenovirus type 4.

[0011] The aforementioned peptide sequence Pep4, which binds to the pathogenic serum FADV Fiber-2 antibody, is used to detect serum FADV 4. Applications include, but are not limited to, agar diffusion assays, indirect ELISA, double-antibody sandwich ELISA, and rapid test strip immunochromatographic techniques.

[0012] The aforementioned peptide sequence Pep4, which binds to pathogenic serum type 4 FADV Fiber-2 antibody, is used to detect serum type 4 FADV antibodies. Applications include, but are not limited to, agar diffusion assays, indirect ELISA, double-antibody sandwich ELISA, and rapid test strip immunochromatographic techniques.

[0013] A vaccine or immunogen comprising the polypeptide sequence Pep4, which binds to an antibody against pathogenic serum type 4 FAdV Fiber-2. The vaccine or immunogen can be prepared using conventional techniques such as solid-phase carrier conjugation.

[0014] Compared to existing technologies, the peptide sequence Pep4 provided by this invention is stable, readily soluble in water, and possesses the following properties: (1) it targets the B-cell epitope of pathogenic serum type 4 FADV Fiber-2; (2) it exhibits strong binding affinity to antibodies against pathogenic serum type 4 FADV Fiber-2; and (3) the peptide has high specificity. In qualitative detection applications, due to the well-established technology for synthesizing peptides, the peptide sequence Pep4 is easier and faster to obtain than purified virus, reducing the associated detection costs. The peptide Pep4 of this invention can be modified, such as by labeling, to achieve rapid qualitative and quantitative detection of pathogenic serum type 4 FADV. Attached Figure Description

[0015] Figure 1 The secondary structure diagram of the WZ strain Fiber-2-knob protein shows: A. α-helix region B. β-sheet region C. β-turn region D. random coil.

[0016] Figure 2 Analytical diagrams of the hydrophilicity index, flexible region, antigen index, and surface accessibility index of the Fiber-2-knob region.

[0017] Figure 3 Linear structural diagrams showing the tertiary and quaternary structures of the Fiber-2-knob protein and the location of the Pep4 antigenic epitope.

[0018] Figure 4 Electron cloud structure diagram of the tertiary and quaternary structures of the Fiber-2-knob protein and the location of the Pep4 antigenic epitope. Detailed Implementation

[0019] The present invention will now be described in detail with reference to specific embodiments. It should be noted that these embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the above description of the present invention.

[0020] This invention utilizes protein expression, purification and crystallization, X-ray diffraction and structural analysis, as well as bioinformatics and immunology techniques to analyze the amino acid differences in the Knob domain of the Fiber-2 protein from pathogenic serum type 4 FADV WZ strain and non-pathogenic serum type 4 FADV ON1 strain (GenBank: GU188428.1). The analysis systematically examines the primary, secondary, tertiary, and quaternary structures of the protein, studying its hydrophilicity, surface accessibility, flexibility, and antigenicity. Based on this, a B-cell epitope with the best binding affinity was artificially synthesized and named Pep4. This polypeptide has a linear sequence, and its amino acid sequence is: PSTATLTDFEPMANRSVTSPWTYSANGYYEPSI, as described in SEQ ID NO: 1.

[0021] The artificially synthesized peptide Pep4 was evaluated by enzyme-linked immunosorbent assay (ELISA), which confirmed that the peptide has high affinity and strong specificity for the target protein and is a B-cell antigen epitope. This indicates that the peptide Pep4 designed in this invention can be used for research such as antibody detection and protein purification related to pathogenic serum type 4 FADV.

[0022] This invention provides a polypeptide sequence Pep4 that can bind to pathogenic serum type 4 FAdV Fiber-2 antibody, with the amino acid sequence: PSTATLTDFEPMANRSVTSPWTYSANGYYEPSI, as described in SEQ ID NO: 1.

[0023] The polypeptide Pep4 of the present invention has the following characteristics: (1) the polypeptide is a B cell epitope targeting pathogenic serum type 4 FAdV Fiber-2; (2) the polypeptide has a strong binding affinity to pathogenic serum type 4 FAdV Fiber-2 antibody; and (3) the polypeptide has strong specificity.

[0024] Compared to existing technologies, the present invention provides a specific polypeptide against pathogenic serum type 4 FADV, which has significant characteristics. It can specifically detect the presence of pathogenic serum type 4 FADV, and can also use existing immunological techniques to identify the presence of pathogenic serum type 4 FADV antibodies, determine the positive or negative level of pathogenic serum type 4 FADV antibodies, and can also be used for labeling and other purposes for other antigen-antibody reactions or immunotherapy.

[0025] The peptide sequence Pep4 that binds to the pathogenic serum type 4 FAdV Fiber-2 antibody includes any corresponding adjustments or modifications to the Pep4 sequence, with the Pep4 sequence as the core. Modifying materials include, but are not limited to, biotin, avidin, magnetic beads, nanomaterials, fluorescent materials, enzymes, and specific proteins.

[0026] The application of the peptide sequence Pep4, which binds to the pathogenic serum type 4 FAdV Fiber-2 antibody.

[0027] The peptide sequence Pep4, which binds to the pathogenic serum type 4 FADV Fiber-2 antibody, is used for the rapid qualitative or quantitative detection of serum type 4 FADV. This includes, but is not limited to, enzyme-linked immunosorbent assay (ELISA), plasmon resonance assay (PRA), and polyacrylamide gel electrophoresis (PAG).

[0028] The application of the peptide sequence Pep4, which binds to the pathogenic serum type 4 FAdV Fiber-2 antibody, in the preparation of rapid detection reagents or kits for avian adenovirus type 4.

[0029] The aforementioned peptide sequence Pep4, which binds to the pathogenic serum FADV Fiber-2 antibody, is used to detect serum FADV 4. Applications include, but are not limited to, agar diffusion assays, indirect ELISA, double-antibody sandwich ELISA, and rapid test strip immunochromatographic techniques.

[0030] The aforementioned peptide sequence Pep4, which binds to pathogenic serum type 4 FADV Fiber-2 antibody, is used to detect serum type 4 FADV antibodies. Applications include, but are not limited to, agar diffusion assays, indirect ELISA, double-antibody sandwich ELISA, and rapid test strip immunochromatographic techniques.

[0031] A vaccine or immunogen comprising the polypeptide sequence Pep4, which binds to an antibody against pathogenic serum type 4 FAdV Fiber-2. The vaccine or immunogen can be prepared using conventional techniques such as solid-phase carrier conjugation.

[0032] Example 1: Design and screening of peptide sequences

[0033] The inventors used the DNAStar software Protein system to analyze the secondary structure and biological and physicochemical properties of the Fiber-2-knob protein of strain WZ (GenBank: MZ508442), including hydrophilicity, surface accessibility, flexibility and antigenicity.

[0034] like Figure 1 As shown, the Garnier-Robson method found that residues 105-111 and 119-122 are β-turn regions; residues 94-97 are irregularly coiled regions.

[0035] Analysis using the Chou-Fasman method revealed that residues 100-107 form an α-helix structure, residues 98-102 and 129-138 form β-sheet regions, and residues 94-97, 108-121, and 124-127 form β-turn regions.

[0036] The two analytical methods yielded similar results, with differences in secondary structure predictions. However, both methods predicted that the knob protein contains β-sheets or β-turns, while α-helices and random coils are less common.

[0037] The β-turn structure and random coil structure of the secondary structure of proteins are relatively soft, which is conducive to antibody chimerism. They are often highly hydrophilic and are often displayed on the surface of the protein. This region often contains the B cell epitope of the protein.

[0038] The Kyte-Doolittle method was used to predict the hydrophilicity of the Fiber 2-knob protein, such as... Figure 2 It was found that residues 104-129 are hydrophilic regions, while other regions are less hydrophilic and may be embedded in the protein's internal structure.

[0039] Analysis of the flexible region using the Karplus-Schulz method revealed that amino acid residues at positions 83-104, 109-114, and 124-128 are favorable for antibody binding.

[0040] Using the Jameson-Wolf method to analyze antigenicity, we found amino acid regions with high antigenicity at amino acid residues 83-114 and 118-130.

[0041] Analysis of antigen surface accessibility using the Emini method revealed that residues 106-116 and 118-124 have high plasticity and are easy to form antigenic epitopes.

[0042] Analysis revealed a correlation between the flexible regions, hydrophilicity index, antigenic index, and surface accessibility index. Short peptides containing amino acids 11-28, 53-69, 84-115, and 155-165 were particularly prominent and represent potential antigenic epitopes.

[0043] The inventors relied on X-ray diffraction and homology modeling techniques to analyze the Fiber-2-knob domain of FAdV-4. Using the Fiber-2-knob amino acid sequence as a template, their tertiary and quaternary structures were reconstructed using SWISS-MODEL, and the tertiary and quaternary structures of the protein were observed and analyzed using PyMol software.

[0044] The B-cell epitopes of the Fiber 2-knob protein were analyzed using the online website ABCpred Prediction Server (https: / / webs.iiitd.edu.in / raghava / abcpred / index.htmL). Combining secondary, tertiary, and quaternary structural analysis, physicochemical property analysis, modeling, and amino acid substitution analysis of the B-cell epitopes, a comprehensive analysis of the existing B-cell epitopes was conducted. Residues 95-127 were identified as potential B-cell epitopes of the Fiber-2 protein. Subsequently, Pep4 was synthesized by Jier Biochemical (Shanghai) Co., Ltd.: its amino acid sequence is Pep4: PSTATLTDFEPMANRSVTSPWTYSANGYYEPSI, as described in SEQ ID NO: 1.

[0045] The spatial structure and localization of the peptide Pep4 were visualized using Pymol software. For example... Figure 3 As shown, loop 1 (blue) represents the β-turn structure of peptide Pep4, and α-helix 2 (red) represents the α-helix structure of peptide Pep4. These two structures demonstrate... Figure 3 The location of the peptide Pep4. Figure 4 The mid-surface cloud layer 3 is peptide Pep4, indicating that peptide Pep4 is located on the surface of the Fiber-2-knob spatial structure.

[0046] Example 2: Identification of Screening Peptides

[0047] The synthesized peptide Pep4 was used as the coating antigen. Chicken positive sera against WZ strain Fiber-2 protein and WZ strain Fiber-2-knob protein were used as primary antibodies, with SPF chicken serum as a negative control. The Fiber 2 protein epitope peptides were identified using an indirect ELISA method. In this example: the coating buffer was carbonate buffer at pH 9.6, the blocking buffer was 5% skim milk, PBST was phosphate buffer at pH 7.4 containing 0.05% Tween 20, and the stop solution was 2M H2SO4.

[0048] The method is as follows:

[0049] (1) The polypeptide dissolved in dimethyl sulfoxide (DMSO) was diluted with coating buffer to 20 μg / mL and added to 96 wells of an ELISA plate, 50 μL per well, and coated overnight at 4°C;

[0050] (2) Wash the plate three times with PBST, 300 μL / well, and let it stand for 3 min each time. After drying, add 300 μL of blocking solution and incubate at 37℃ for 2 h;

[0051] (3) Wash the plate 3 times with PBST, 300 μL / well, and let it stand for 3 min each time. After drying, add 50 μL of positive serum and negative control serum diluted 1:1000 with blocking buffer. Repeat each sample 3 times and incubate at 37℃ for 1 h.

[0052] (4) Wash the plate 3 times with PBST, 300 μL / well, and let it stand for 3 min each time. After drying, add rabbit anti-chicken IgG diluted 1:7000 (diluent is blocking solution) as secondary antibody, 50 μL / well, and incubate at 37℃ for 1 h;

[0053] (5) Wash the plate three times with PBST, 300 μL / well, and let it stand for 3 min each time. After drying, add 50 μL / well of the substrate single-component TMB colorimetric solution and incubate at 37°C in the dark for 15 min;

[0054] (6) Add 50 μL of stop solution (2M H2SO4) to each well, immediately place it on the microplate reader to measure OD450nm, record the data and calculate the average value of each test sample.

[0055] Table 1. ELISA results of detecting serum FADV type 4 chicken serum antibodies using peptide Pep4 as the coating antigen.

[0056]

[0057] As shown in Table 1, the OD450nm values ​​of peptide Pep4 with anti-WZ strain Fiber-2 serum were 1.888 and 1.427, respectively. These ratios compared to negative serum were 26.6 and 16.6, indicating a strong and specific binding reaction between peptide Pep4 and positive serum. Based on these results, it can be determined that peptide Pep4 is a linear B-cell epitope targeting the Fiber 2 protein.

[0058] Example 3: Peptide Pep4 can specifically detect highly pathogenic serum type 4 FADV antibodies.

[0059] Using peptide Pep4 as the coating antigen, positive sera against pathogenic FADV WZ strain, non-pathogenic FADV ON1 strain (GenBank: GU188428.1) Fiber-2 protein, non-pathogenic FADV ON1 strain (GenBank: GU188428.1) Fiber-2-knob protein, E-type 8a FADV TR95 strain (GenBank: KT862810.1) Fiber protein, A-type 1 FADV CELO strain Fiber-2 protein (GenBank: U46933.1), and group III FADV egg drop syndrome virus (EDSV) Jing911 strain (patent number: CN200610018066.9) were used as primary antibodies. The OD450nm results were detected by indirect ELISA, and the method and steps were the same as in Example 2.

[0060] As shown in Table 2, when using the peptide Pep4 as the coating antigen to detect different subgroups and serotypes of FADV chicken serum, only the positive serum against the pathogenic FADV WZ strain showed a positive reaction. The positive serum / negative serum ratio of the non-pathogenic FADV ON1 strain, which also belongs to serotype 4, was <2.1 regardless of whether it was against Fiber-2 protein or Fiber-2-konb protein (in ELISA, a positive serum / negative serum ratio >2.1 is considered an opposing relationship between positive and negative serotypes). This indicates that the ELISA method does not produce a positive reaction with the non-pathogenic strain of FADV against serotype 4. Simultaneously, this invention also detected positive chicken sera against type E serum 8a / 8b FADV-8aTR59 strain Fiber protein, positive chicken sera against type A serum 1 FADV-1 CELO strain Fiber-2 protein, and positive chicken sera against group III FADV egg drop syndrome virus (EDSV) Jing911 strain. The positive serum / negative serum ratio was <2.1, indicating a negative reaction, further demonstrating that this method can only detect antibodies against highly pathogenic type 4 FADV serum and has good specificity.

[0061] Table 2. Results of Pep4-specific ELISA validation

[0062]

[0063] Example 3: Comparison of Pep4

[0064] Fiber-2 sequences of pathogenic and non-pathogenic serotype 4 FADV were compared, with a focus on the similarity and differences between the protein sequences at the Pep4 position in this patent. The results are shown in Table 3. All pathogenic serotype 4 FADV protein sequences are identical at the Pep4 position, while non-pathogenic serotype 4 FADV shows differences at six sites (underlined in Table 3). This indicates that Pep4 is a conserved sequence present in pathogenic serotype 4 FADV, meaning that Pep4 is present in all sequences of pathogenic serotype 4 FADV. This provides a strong primary structural basis for demonstrating that Pep4 specifically recognizes both pathogenic and non-pathogenic FADV. Furthermore, experimental data from Example 2 show that Pep4 specifically reacts only with pathogenic serotype 4 FADV and not with other serotypes of FADV. Therefore, Pep4 is a common B-cell antigen epitope for pathogenic serotype 4 FADV. Based on the above theoretical foundation and experimental data, the Pep4 of this patent can specifically identify and distinguish between pathogenic and non-pathogenic FADV.

[0065] Table 3. Amino acid sequence comparison of Pep4 with other pathogenic and non-pathogenic serum types 4 FADV.

[0066]

[0067] Note: Underlined amino acid sites that differ from Pep4.

[0068] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. It should be noted that for those skilled in the art and any person skilled in the art, any equivalent substitutions or changes made to the technical solution and inventive concept of the present invention without departing from the overall concept of the present invention, as well as any changes and improvements made, should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide Pep4 that binds to pathogenic serum type 4 FAdV Fiber-2 antibody, characterized in that: The polypeptide Pep4 is linear, and its amino acid sequence is as described in SEQ ID NO:

1.

2. A modified polypeptide Pep4 that binds to a pathogenic serum type 4 FAdV Fiber-2 antibody, characterized in that: The polypeptide Pep4 is linear, and its amino acid sequence is as described in SEQ ID NO:

1. Based on the Pep4, the polypeptide Pep4 is modified accordingly. The modification materials include biotin, avidin, magnetic beads, nanomaterials, fluorescent materials, or enzymes.

3. The use of the peptide Pep4, which binds to the pathogenic serum type 4 FAdV Fiber-2 antibody according to claim 1 or 2, in the preparation of a rapid qualitative or quantitative detection reagent or kit for serum type 4 FAdV.

4. The use of the peptide Pep4, which binds to the pathogenic serum type 4 FADV Fiber-2 antibody according to claim 1 or 2, in the preparation of a detection reagent or kit for detecting serum type 4 FADV.

5. The use of the peptide Pep4, which binds to pathogenic serum type 4 FADV Fiber-2 antibody according to claim 1 or 2, in the preparation of a detection reagent or kit for detecting serum type 4 FADV antibody.

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