CN116987155B

CN116987155B - African swine fever virus epitope peptide and application thereof

Info

Publication number: CN116987155B
Application number: CN202211121266.2A
Authority: CN
Inventors: 庄国庆; 孙爱军; 张帅; 张改平
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2022-09-15
Filing date: 2022-09-15
Publication date: 2024-08-27
Anticipated expiration: 2042-09-15
Also published as: CN116987155A

Abstract

The invention relates to the fields of immune technology, molecular biology and molecular virology, in particular to an African swine fever virus epitope peptide and application thereof. The amino acid sequence of the African swine fever virus epitope peptide is shown as SEQ ID NO:1 to 9. The African swine fever virus antigen epitope peptide and the antigen epitope peptide with the same function derived from the antigen epitope peptide can be used for preparing specific antibodies of the African swine fever virus and detecting reagents, kits or biochips of the African swine fever virus, and provides support for developing vaccines for preventing and/or treating the African swine fever virus infection.

Description

African swine fever virus epitope peptide and application thereof

Technical Field

The invention relates to the fields of immune technology, molecular biology and molecular virology, in particular to an African swine fever virus epitope peptide and application thereof.

Background

African Swine Fever Virus (ASFV) is the only member of African swine fever virus family and genus African swine fever virus, and can cause acute and hemorrhagic clinical symptoms after infection of domestic pigs and wild pigs, and the infection can cause death of infected pigs for 3-7 days and the death rate of the infection is as high as 100%.

ASFV is a double-stranded DNA virus with a genome length of 170-193Kb, a viral particle diameter of 260-300nm, encoding more than 150 proteins. The ASFV has five layers of capsule structures of an outer membrane, an outer shell, an inner membrane, an inner garment shell and a core shell, and the structure is complex. Antigens of different structure during infection of ASFV exert different roles during infection.

The African swine fever is used as a major monitored class I animal epidemic disease, no safe and effective vaccine exists at present, the existing vaccine research comprises an inactivated vaccine, a subunit vaccine, a carrier vaccine, a natural attenuated vaccine, a genetic engineering deletion vaccine and the like, the vaccine has certain defects in the aspects of protection rate, broad spectrum and safety, and main reasons for restricting the research and development of ASFV vaccine comprise undefined ASFV key protective antigens and unknown functions of natural immunity and adaptive immunity in the ASFV protection process. Existing studies indicate that p72, p30, p54, CD2v of ASFV may be protective antigens of ASFV. Strains with partial gene deletions of I177L, DP, R, A L, 9GL, DP96R and MGF families have protection effect on ASFV.

The linear epitope of the antigen has broad-spectrum protective effect, is an important antigen element of virus structural protein, is generally 3-20 amino acid sequences, and plays an important role in inducing humoral immunity and cellular immune response of an organism. The research of the epitope has great significance for perfecting the research of the virus protein structure and the relation between the virus and the host, and simultaneously provides a material foundation for the detection of the virus and the preparation of the epitope vaccine. Therefore, continuous research on new effective, safe and broad-spectrum ASFV epitopes has important significance for developing ASFV infection vaccines.

Disclosure of Invention

In view of the above technical problems, the invention utilizes ASFV infection positive inactivated serum combined with amplicon high throughput sequencing technology to screen and obtain ASFV antigen, and verifies the screened antigen fragment.

In a first aspect of the invention, there is provided an african swine fever virus epitope peptide, wherein the amino acid sequence of the epitope peptide is as shown in SEQ ID NO:1 to 9.

In a second aspect, the invention provides an application of the African swine fever virus epitope peptide in preparation of a reagent, a kit and/or a biochip for detecting African swine fever virus.

In a third aspect of the invention, an african swine fever virus antigen is provided, which is prepared by coupling the epitope peptide with a carrier protein.

In a fourth aspect, the invention provides an african swine fever virus specific antibody, which is a monoclonal antibody or a polyclonal antibody prepared from the african swine fever virus antigen.

In a fifth aspect, the invention provides a medicament for preventing or treating african swine fever virus, which comprises the african swine fever virus epitope peptide and/or a product of the african swine fever virus epitope peptide after modification or reformation.

Preferably, the modified form is selected from the group consisting of phosphorylation, acetylation, glycosylation, amidation and/or derivatization with known protecting groups, blocking groups; the modification is selected from the group consisting of proteolytic hydrolysis and/or production of recombinant proteins.

In a sixth aspect of the present invention, there is provided a nucleic acid molecule comprising a sequence encoding said african swine fever virus epitope peptide.

In a seventh aspect of the invention, there is provided an expression vector comprising said nucleic acid molecule.

Compared with the prior art, the invention has the beneficial effects that:

According to the invention, the African swine fever virus infection positive inactivated serum is purified by ProtinA +G magnetic beads, a commercial phage random dodecapeptide library is utilized to obtain the African swine fever virus epitope peptide through subtractive panning, amplicon high-throughput sequencing and localized Short-blastp analysis screening, and the African swine fever virus epitope peptide obtained through screening is verified, so that the African swine fever virus epitope peptide and the antigen epitope peptide with the same function derived from the African swine fever virus epitope peptide can be used for preparing specific antibodies of the African swine fever virus and detecting reagents, kits or biochips of the African swine fever virus, and have important significance for serological detection of the African swine fever virus, and support is provided for research and development of vaccines for preventing and/or treating African swine fever virus infection.

Drawings

FIG. 1 is a schematic diagram showing SDS-PAGE results of ProtinA +G magnetic beads purified serum;

FIG. 2 is a schematic representation of the results of identifying ProtinA +G magnetic beads purified serum by Westernbloting using Goat anti pig-HRP;

FIG. 3 is a graphical representation of the results of a four-round phage subtractive panning titer assay, wherein ①～④ represents four rounds of panning;

FIG. 4 is a schematic representation of amplicon results from PCR amplification of phage random peptide regions;

FIG. 5 is a schematic diagram of amplicon high throughput sequencing data processing;

FIG. 6 is a schematic diagram showing the result of amplicon fragment length distribution;

FIG. 7 is a schematic representation of amplicon high throughput sequencing reads data QC results;

FIG. 8 is a schematic representation of structural display of a localized Short-blastp result overlay analysis screening portion of epitopes;

FIG. 9 is a schematic diagram showing the results of ASFV positive serum Dot-blot detection of ASFV antigen segments; wherein 1 to 40 are epitope peptides corresponding to numbers 1 to 40 in Table 5, and 41 to 45 are epitope peptides of P72 protein in Table 6.

Detailed Description

In order to clearly illustrate the technical features of the present solution, the present invention will be described in detail below with reference to the accompanying drawings. The scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.

The instruments, reagents, materials, etc. used in the examples described below are conventional instruments, reagents, materials, etc. known in the art, and are commercially available. The experimental methods, detection methods, and the like in the examples described below are conventional experimental methods, detection methods, and the like that are known in the prior art unless otherwise specified.

Example 1

Screening of African Swine Fever Virus (ASFV) humoral immunity-related antigen

1. Test materials

Commercial phage random dodecapeptide library is purchased from NEB organism, ASFV positive serum is purchased from Chinese veterinary medicine monitoring institute, ASFV antigen region polypeptide is synthesized by Shanghai peptide valley biotechnology company, PRIMER STAR Max is purchased from Bao Ri medical organism, and amplicon sequencing, on-machine sequencing, splitting and analysis are all completed by Suzhou Jin Weizhi biotechnology company.

2. Solution preparation

(1) TBS buffer: weighing 8.77g NaCl and 7.88g Tris, dissolving in 800mL deionized water, regulating the pH value of the solution to 7.4 by using concentrated HCl, adding water to a constant volume of 1L, sterilizing at 121 ℃ for 20min under high pressure, and preserving at room temperature for later use;

(2) 0.1% tbst buffer: on the basis of TBS buffer solution, adding 1mL Tween-20, sterilizing at 121 ℃ for 20min under high pressure, and preserving at room temperature for later use;

(3) 0.3% tbst buffer: 3mL of Tween-20 is added on the basis of TBS buffer solution, and the mixture is autoclaved for 20min at 121 ℃ and stored at room temperature for standby;

(4) 0.5% tbst buffer: 5mL of Tween-20 is added on the basis of TBS buffer solution, and the mixture is autoclaved for 20min at 121 ℃ and stored at room temperature for standby;

(5) Tetracycline hydrochloride stock solution: weighing 0.5g of tetracycline hydrochloride powder, adding deionized water to a volume of 100mL, filtering and sterilizing by using a 0.22 mu m filter membrane, and sub-packaging and preserving at-20 ℃;

(6) glycine-HCL eluent: 1.5g glycine and 0.1g BSA were weighed, dissolved in 80mL deionized water, and stored at 2.2,4 ℃with the pH adjusted to concentrated hydrochloric acid.

(7) Tris-HCl buffer (pH 9.2): weighing 15.8g of Tris, dissolving in 80mL of deionized water, regulating the pH to 9.2 by concentrated hydrochloric acid, fixing the volume of the deionized water to 100mL, sterilizing at 121 ℃ for 20min under high pressure, and preserving at room temperature for later use;

(8) NaI solution: dissolving a proper amount of NaI in a buffer solution of Tris-HCL (10 mM) with pH of 8.0 and adding EDTA (1 mM) to make the final concentration of NaI be 4M/L;

(9) PEG/NaCl solution: 10g of PEG 8000 and 7.3g of NaCl are weighed, dissolved and fixed to 50mL, and after autoclaving, the mixture is preserved at room temperature, the PEG mass ratio is 20%, and the NaCl concentration is 2.5M.

(10) PBS buffer: weighing 8.0g of NaCl, 0.2g of KCl and 1.44g of Na ₂HPO₄、0.24g KH₂PO₄, dissolving in 800mL of deionized water, regulating the pH value of the solution to 7.4 by using HCl, adding water to a constant volume of 1L, sterilizing at 121 ℃ under high pressure for 20min, and preserving at room temperature for later use;

(11) PBST buffer: adding 500 mu L of Tween-20 into 1L of PBS after autoclaving, mixing well, and preserving at 4 ℃;

(12) Sealing liquid: weighing 5.0g of skimmed milk, adding into 100mL of PBST buffer solution, dissolving, and storing at 4deg.C;

(13) Coating buffer solution: 8.58g of Na ₂CO₃·10H₂ O and 5.8g of NaHCO ₃ are weighed and added into deionized water, the volume is fixed to 1L, the PH is regulated to 9.5,4 ℃ for preservation;

(14) IPTG: 2g of IPTG was dissolved in 8mL of ultrapure water, the volume was set to 10mL, and the solution was sterilized by filtration through a 0.22 μm filter. Subpackaging into 1.5mL EP tube, and preserving at-20deg.C;

(15) 20mg/ml X-gal: 200mg of X-gal powder was weighed and dissolved in Dimethylformamide (DMF) solution, and the solution was packed in sterile 1.5mL EP tubes and stored at-20 ℃;

(16) Kanamycin: dissolving 100mg kanamycin in enough ultrapure water, fixing the volume to 10mL, filtering and sterilizing by using a 0.22 mu m filter membrane, split charging into 1.5mL EP pipes, and preserving at-20 ℃;

(17) LB medium: weighing 10g of tryptone, 5g of yeast extract and 5g of NaCl, dissolving in 800mL of deionized water, regulating the pH to 7.4 with NaOH, fixing the volume to 1L, sterilizing at 121 ℃ for 20min under high pressure, and preserving at room temperature for later use;

(18) LB/IPTG/X-gal plates: 15g of agar powder is added into each 1L of LB culture medium, after autoclaving, the temperature is reduced to about 55 ℃, 1mL of IPTG, 2mL of 20mg/mL of X-gal and antibiotics with corresponding concentrations are added, and LB plates are prepared.

3. Test method

3.1 ProteinA+G magnetic beads purification of antibodies in serum

Gently beating resuspended ProteinA+G magnetic beads with a pipette, taking appropriate amount of ProteinA+G magnetic beads into a clean centrifuge tube according to 10 μl or 20 μl of magnetic bead suspension per 500 μl of sample, and adding 1 XTBS to a final volume of about 0.5ml; the resuspended ProteinA+G beads were gently blown with a pipette. The mixture was placed on a magnetic rack and separated for 10 seconds, and the supernatant was removed. Repeating the above steps twice; protein A+G magnetic beads were resuspended in 1 XTBS according to the amount of initial volume, the supernatant was blotted off, 500. Mu.L of antibody working solution or normal IgG working solution was added, and after resuspension, incubation was reversed on a room temperature tumble mixer for 15-60 min. Normal IgG of the same antibody species was formulated for removal of non-specific binding or as a negative control. mu.L of 1 XTBS was added and the resuspended ProteinA+G beads were gently blown with a pipette. The mixture was placed on a magnetic rack and separated for 10 seconds, and the supernatant was removed. The wash was repeated three times. ProteinA+G beads were resuspended in 1 XTBS in the amount of initial volume.

3.2 Identification of ProteinA+G magnetic bead purified serum by Polyacrylamide gel electrophoresis (SDS-PAGE)

The purified serum was loaded with 5 x protein loading buffer, denatured by heating, subjected to 12.5% SDS-PAGE (voltage 80V,30min line, 150V,60 min), stained with 0.05% Coomassie blue staining solution at room temperature for 1h, and shake-stained overnight.

3.3 Westernblotting evaluation of ProteinA+G magnetic bead purified serum

ProteinA+G magnetic bead purified serum was separated by electrophoresis on a 12.5% SDS-PAGE gel, proteins were transferred to PVDF membrane, blocked with 5% skim milk for 1h, and goat anti-porcine IgG was labeled with HRP at a dilution of 1:5000 and incubated as secondary antibody. After incubation for 1h at room temperature, PBST was washed 3 times for 10min each, followed by the addition of Super SignalWest Pico chemiluminescent film substrate (Thermo FISHER SCIENTIFIC, USA) and luminescent development using AMERSHAM IMAGER 680.

3.4 Phage random dodecapeptide pool subtractive panning

(1) E.coli ER2738 glycerol bacteria preserved at-80 ℃ are streaked on LB-Tet plates and cultured overnight at 37 ℃. The streak plate was stored at 4℃for a period of 1 month.

(2) E.coli ER2738 is selected and inoculated in 30mL LB liquid medium for 5h at 37 ℃ and 170rpm, and OD ₆₀₀ value is 0.3-0.8 in logarithmic phase. And (5) placing the cultured bacterial liquid in a refrigerator at the temperature of 4 ℃ for storage.

(3) E, culturing the coll ER2738 until the OD ₆₀₀ in the early logarithmic growth phase is stored at 0.01-0.05,4 ℃ for standby.

(4) Adding the blocking solution into the antibody-coated magnetic beads or the magnetic beads, and blocking for 1h at room temperature.

(5) Taking out the sealed magnetic beads, separating the magnetic frame, and removing the sealing liquid.

(6) For each round of screening, 10. Mu.L of the original library containing 10 ¹¹ phages or amplified phage library after the previous round of panning was diluted with 500. Mu.L of TBS buffer and added to uncoated ProteinA+G magnetic beads and incubated at room temperature with gentle shaking. Magnetic rack separation unbound dilution supernatant was added to ASFV negative serum coated proteona+g magnetic beads and incubated at room temperature with gentle shaking. Magnetic rack separation unbound dilution supernatant was added to ASFV positive serum coated proteona+g beads and incubated with gentle shaking at room temperature (see table 1 for details).

(7) Separating unbound phage in unbound diluent by using a magnetic rack, and washing 5-10 times by using TBST.

(8) Adding 100 mu L glycine-hydrochloric acid (0.2 moL/L, pH 2.2) into a magnetic bead coated with proteoA+G of ASFV positive serum to elute phage bound to ASFV positive serum target molecule, adding, placing on an oscillator, slightly shaking for 10min, separating supernatant by a magnetic rack, and sucking eluent into a clean sterile 1.5ml centrifuge tube; then adding 15 mu LTris-HCL (pH 9.2) to neutralize the eluent, mixing, storing at 4deg.C overnight, adding equal amount of sterile glycerol, and storing at-20deg.C for a long time.

3.5 Phage titer assay

Phage titers were measured on LB agar plates containing IPTG and X-gal by taking 10. Mu.L of eluate and 10-fold gradient dilution of eluted phage using E.coil ER 2738 (OD ₆₀₀ value 0.5) in logarithmic growth phase. Phage titers were calculated by plating LB-IPTG-X-gal plates at appropriate dilutions, incubating overnight at 37℃and counting the number of blue plaques and the corresponding dilutions. The phage with well quantified titer can be stored in a refrigerator at 4 ℃ and stored for a long time in a refrigerator at-20 ℃ after the equal amount of sterile glycerol is added.

3.6 Amplification and enrichment of phage eluate

(1) Half of the remaining eluate after titer was measured was added to 30mL E.coli ER2738 cultures which had been cultivated to be in the log phase, at 37℃and 300rpm for 5 hours.

(2) The cultures were collected in sterile 50mL centrifuge tubes, centrifuged at 10,000rmp at 4℃for 10min; transferring the supernatant to a new sterile centrifuge tube; centrifuge at 4℃and 10,000rmp for 6min.

(3) 80% Of the volume of the supernatant was transferred to a fresh centrifuge tube, added to PEG/NaCL in 1/6 of the volume of the transfer liquid, and left to stand at 4℃for 4-8h or allowed to settle overnight.

(4) Centrifugation was performed at 10,000rmp for 10min at 4℃and the supernatant was discarded, phage pellet was dissolved in 1mLTBS, 1/6 volume of PEG/NaCL was added and allowed to stand at 4℃for 2h.

(5) Centrifugation at 15,000rmp for 15min at 4℃and removal of supernatant, phage pellet was dissolved in 150. Mu.L TBS, centrifuged at 15,000rmp for 1min at 4℃and the supernatant was transferred to a fresh clean centrifuge tube. Post-amplification phage titers were determined with reference to 3.5.

TABLE 1 summary of phage multiple round panning conditions

3.7 Phage DNA extraction

(1) The amplified phage was added with an equal volume of PEG/NaCL and allowed to stand at room temperature for 10-20min.

(2) Centrifuging at 4deg.C for 15,000rmp for 15min, discarding supernatant, re-suspending the precipitate in 100 μL of 4M NaI solution, adding 250 μL of absolute ethanol, and standing at room temperature for 10-20min.

(3) Centrifugation at 15,000rmp at 4℃for 15min, discarding supernatant, re-suspending with 500. Mu.L of 70% ethanol pre-chilled at-20℃and centrifugation. The DNA precipitate was dissolved by adding 30. Mu.L of deionized water after repeating 2 times of air drying.

3.8 Phage random peptide region amplicon PCR amplification

Using extracted phage single-chain DNA as a template, amplifying for 30 cycles at 98 ℃,10 s,58 ℃, 5s,72 ℃ and 30s under the conditions of Primer STARMax, and carrying out agarose gel electrophoresis on the amplified product to recover target fragments. High throughput sequencing pooling and sequencing was performed by Jin Weizhi organisms.

3.9 Sequencing data Mass analysis

And (3) performing image base recognition (Base calling) on the raw image data of the sequencing result by using software CASAVA (v1.8.2), performing preliminary mass analysis to obtain raw data (PASS FILTER DATA, PF) of a sequencing sample, and storing the sequencing data in a FASTQ (fq) file format.

In high-throughput sequencing, some sequencing errors such as point mutation usually occur, and the quality of the sequence end is relatively low, so that in order to obtain a higher-quality and more accurate biological information analysis result, the sequencing original data needs to be optimized. Optimizing the treatment by Cutadapt 1.9.1 software, 1) removing the primer and the adaptor sequence; 2) Removing bases with a two-terminal mass value lower than 20; 3) Sequences with a ratio of N bases greater than 10% were removed.

3.10, Target sequence abundance statistics

And grabbing the target sequence according to 10nt on the upstream and downstream of the target sequence, converting the target sequence into amino acid sequences in batches according to a codon table, and carrying out abundance statistics.

3.11 Localized Blast analysis

After high-throughput sequencing of the amplicon, intercepting a random peptide region DNA sequence, removing a low-quality sequence and a non-effective reading length sequence, converting the sequence into a Fasta file format, establishing a comparison library by using an amino acid sequence of an ASFV Pig/HLJ/2018 strain, performing Blastp analysis on the Fasta format of the amino acid sequence obtained by sequencing and the comparison library, wherein the analysis mode is short-Blastp, E-value is less than or equal to 0.05, and the maximum matching degree of the sequence is 5. The correspondence analysis code is as follows: fasta-db hlj-out alignment -outfmt"7qacc sacc sstart send qseq sseq length pident score evalue"-task blastp-short-word_size 2-evalue 0.05-max_target_seqs 5-num_threads 4. was subjected to subsequent merging using Notepad++, excel software.

TABLE 2 primer sequence listing

Primer name	Primer sequence (5 '-3')	Primer length (nt)
			PhageM13-seq-R	CCCTCATAGTTAGCGTAACGA	21
PhageM13-seq-F	CCGATACAATTAAAGGCT	18

3.12 ASFV antigen segment overlap analysis

According to the sequence comparison result, the positions of the beginning (ascending arrangement) and the ending (descending arrangement) of the amino acid matching regions are compared, the antigen region with a wide containing region is removed, and the corresponding statistical abundance is superimposed to be used as the abundance value of the ASFV antigen epitope region.

3.13, Dotblotting experiments

ASFV antigen polypeptides were synthesized based on the ASFV antigen segment information obtained, and the polypeptides were diluted to 2mg/mL using appropriate solutes. mu.L of each well was spotted on a cellulose acetate film (NC), air-dried at room temperature, blocked with 5% skimmed milk for 1 hour, and ASFV pig positive serum, pig negative serum (negative control) (1:500) were used as primary antibodies, incubated at room temperature for 1 hour, then washed with PBST 3 times for 10 minutes each, and incubated with HRP-labeled goat anti-pig IgG antibody at a dilution of 1:5000 as secondary antibodies. Incubation for 1h at room temperature, pbst wash 3 times, followed by addition of Super SignalWestPico chemiluminescent film substrate (Thermo FISHER SCIENTIFIC, USA) and detection using AMERSHAM IMAGER 680 luminescence.

3.14, Data analysis

All statistical analyses were performed using GraphPadPrism 9.3.0 software (GraphPad software Inc, san Diego, CA, USA) and P <0.05 was statistically significant for differences. Short-blastp aligned ASFV strain to pin/HLJ/2018, genbank accession No. using localized Blast ⁺ software, version No. 2.11: MK333180.1. Protein structural analysis and epitope segment labelling were performed using the Pymol 2.4 educational edition.

4. Results

4.1 Purification and identification of ASFV Positive and negative serum by ProteinA+G magnetic beads

After purification of serum by protein A+G magnetic beads, protein samples were prepared, and SDS-PAGE results showed that protein A+G bands appeared at 25kDa and were the heavy chain region of the antibodies at 45-60kDa (FIG. 1). Western blotting analysis showed that the heavy chain region of the antibodies in the purified serum was recognized by HRP-labeled goat anti-pig IgG antibodies (FIG. 2). At 25kDa is the binding band of ProteinA+G to the antibody.

4.2 Results of panning of phage random dodecapeptide pool and results of random peptide region amplification

Phage titers were measured by four rounds of phage panning and amplification (Table 3, FIG. 3), and the target phage was effectively enriched by a factor of 3.27X10 ³.

The target region was amplified using random peptide region amplification universal primers, and the results showed (FIG. 4) that PCR gave a DNA fragment of the target region, which was 256bp in size to the expected. The target fragment region is recovered for the next step of amplicon high throughput sequencing and library building.

Table 3 phage four-round panning elution phage titer assay

Panning times	Phage titer (pfu/mL)	Panning ratio
			1	1.1×10⁵	9.09×10⁵
2	1.3×10⁶	7.69×10⁴
			3	3.2×10⁷	3.13×10³
4	3.6×10⁸	2.78×10²

4.3 High throughput sequencing of amplicon results QC analysis

The high throughput sequencing and flow chart of the amplicon are shown in fig. 5, and the sequencing data CLEAN DATA result is shown in table 4, so that the sequence information of the target region can be better obtained. The average quality of CLEAN DATA base is shown in figures 6-7, the error rate is lower than 0.05%, the quality of the sequence obtained by high-flux sequencing of the amplicon is better, the fragment length of the amplicon library is 5-298bp, and the length is mainly concentrated at about 250 bp.

TABLE 4 Clean data statistics

Sequencing sample name	Average length of reads	Reads number of	Total number of bases	Q20(％)	Q30(％)	GC(％)	N(ppm)
								Phage4-1	149.13	99437588	14828874914	95.74	88.95	40.57	5.99
Phage4-2	149.10	122768982	18304663438	95.63	88.70	40.16	6.00
								Phage4-3	149.11	91731414	13677770588	95.60	88.77	40.34	5.97

Note that: each sample CLEAN DATA statistics: 01_qc/. Trim. Stat

Q20 (%): a percent number of bases with a sequencing error rate of less than 1%;

Q30 (%): sequencing error rate less than 0.1% base number percent;

GC (%): the base of C+G accounts for the number percent of all bases;

N (ppm): the base N, which cannot be determined by sequencing, is contained in every megabase.

4.4 ASFV high abundance antigen segment analysis

The first 40 antigen segment information with the top abundance is screened out through localized Short-Blast analysis according to the abundance of antigens, and the first 40 antigen segment information with the top abundance is possibly an antigen segment with higher antibody abundance in the ASFV infection process, thereby being beneficial to the establishment and application of an ASFV serological diagnosis method.

TABLE 5 high abundance antigen segment Top 40 summary table

The amino acid sequence of the antigen epitope peptide with the number of 2 in the table is shown as SEQ ID NO:1, the amino acid sequence of the antigen epitope peptide with the number of 7 is shown as SEQ ID NO:2, the amino acid sequence of the antigen epitope peptide with the number of 21 is shown as SEQ ID NO:3, the amino acid sequence of the antigen epitope peptide with the number of 25 is shown as SEQ ID NO:4, the amino acid sequence of the antigen epitope peptide with the number of 26 is shown as SEQ ID NO:5, the amino acid sequence of the antigen epitope peptide with the number of 31 is shown as SEQ ID NO:6, the amino acid sequence of the antigen epitope peptide with the number of 36 is shown as SEQ ID NO:7, the amino acid sequence of the antigen epitope peptide with the number of 37 is shown as SEQ ID NO:8, the amino acid sequence of the antigen epitope peptide with the number 38 is shown as SEQ ID NO: shown at 9.

Example 2

Partial antigen segment structural display

The epitope segments obtained by screening are marked and displayed on the surface of a part of the antigen of the analyzed structure, and p72 (PDB accession number: 6KU 9), S273R (PDB accession number: 6 LJB), p15 (PDB accession number: 6 LNL), A238L (PDB accession number: 4F 0Z), NP419L (PDB accession number: 6 IMK), p35 (PDB accession number: 7 BQA), O174L (PDB accession number: 1 JQR), NP868R (PDB accession number: 7D 8U) and EP296R (PDB accession number: 6KI3 are marked. Since the crystal structure of the G1340L protein has not been resolved, the above epitope peptide exhibits no G1340L epitope information.

The results show that the antigen segments obtained from the screening are all located at surface positions of the protein structure (fig. 8), which is mostly related to the linear structure of the proteins displayed by the phage random peptide region.

TABLE 6 protein structural surface display antigen segment information table

Example 3

ASFV epitope segment identification

The synthetic epitope peptides numbered 1-40 in Table 5 and ASFV antigen segment peptides of p72 protein in Table 6 (i.e. epitope peptides numbered 41-45 in Table 6) were used to perform DotBuot detection with ASFV positive serum and ASFV negative serum obtained from the intermediate test, p72 protein was used as positive control, and random polypeptide was used as negative control.

The results of the test showed (FIG. 9) that the average effective reactivity of the selected epitope peptides numbered 1 to 40 in Table 5 and the ASFV epitope region of the p72 protein in Table 6 with positive serum was 86.67%. Wherein, the antigen epitope of ASFVCHACD01760 protein corresponds to the 02 number polypeptide in table 5, the antigen epitope of C257L protein corresponds to the 07 number polypeptide in table 5, the antigen epitope of F1055L protein corresponds to the 21 number polypeptide in table 5, the antigen epitope of G1340L protein corresponds to the 25 number and 26 number polypeptides in table 5, the antigen epitope of K421R protein corresponds to the 31 number polypeptide in table 5, the antigen epitope of MGF360-4L protein corresponds to the 36 number polypeptide in table 5, the antigen epitope of MGF505-10R protein corresponds to the 37 number and 38 number polypeptides in table 5, which can be specifically combined with ASFV positive serum, wherein, the reactivity of the 02 number, 21 number, 25 number, 26 number, 31 number, 36 number and 38 number polypeptides with ASFV positive serum is strong, which indicates that the antigen epitope peptide has better reactivity with ASFV positive serum; no. 07 and No. 37 polypeptides can specifically bind ASFV positive serum, but the reactivity is weaker than that of other polypeptide epitopes, which is possibly related to insufficient exposure of No. 07 and No. 37 polypeptides in the developing exposure process, and the effective reaction rate of the polypeptides and ASFV positive serum from different sources is 100%. Polypeptide epitopes No. 11, no. 12, no. 16, no. 27, no. 30 in table 5 were non-reactive with ASFV positive serum in Dotblot assays; the No. 17 polypeptide epitope can also react with negative serum, indicating that the No. 17 polypeptide epitope may belong to an epitope polypeptide that non-specifically binds to an antibody.

The above embodiments are not to be taken as limiting the scope of the invention, and any alternatives or modifications to the embodiments of the invention will be apparent to those skilled in the art and fall within the scope of the invention.

The present invention is not described in detail in the present application, and is well known to those skilled in the art.

Claims

1. An african swine fever virus epitope peptide, which is characterized in that the amino acid sequence of the epitope peptide is shown in SEQ ID NO:1.

2. Use of an african swine fever virus epitope peptide of claim 1 in the preparation of a reagent, kit and/or biochip for detecting african swine fever virus.

3. An african swine fever virus antigen, which is characterized in that the antigen is prepared by coupling the african swine fever virus epitope peptide of claim 1 with a carrier protein.

4. A medicament for preventing or treating african swine fever virus, comprising the african swine fever virus epitope peptide of claim 1.

5. A nucleic acid molecule encoding the african swine fever virus epitope peptide of claim 1.

6. An expression vector comprising the nucleic acid molecule of claim 5.