NL2029106B1

NL2029106B1 - Ctl epitope peptide of african swine fever and use thereof

Info

Publication number: NL2029106B1
Application number: NL2029106A
Authority: NL
Inventors: Wei Xiaohui; Li Zhonghua; Zhang Nianzhi; He Junlei
Original assignee: Univ China Agricultural
Priority date: 2021-09-02
Filing date: 2021-09-02
Publication date: 2023-03-20

Abstract

The disclosure provides CTL epitopes of African swine fever (ASF) and applications thereof, belonging to the technical field of peptide vaccine preparation. According to the 5 disclosure, African swine fever virus (ASFV) peptides that can be stably bound to MHC class I are screened by bioinformatics methods and in vitro methods for identifying MHC Ibinding motifs. The screened peptides are capable of initiating CD8+ T cells to generate immune response and stimulating the body to speciﬁcally generate CTL. The amino acid sequence of the peptides is set forth in any one of SEQ ID No: 1-18. The peptide provided 10 by the disclosure can be used for preparing specific epitope vaccine or peptide vaccine of ASF. It has high safety and good specificity, and has potential application value in preventing and controlling African swine fever.

Description

CTL EPITOPE PEPTIDE OF AFRICAN SWINE FEVER AND USE THEREOF

TECHNICAL FIELD

[01] The disclosure belongs to the technical field of peptide vaccine preparation, and particularly relates to a CTL epitope peptide of African swine fever and use thereof.

BACKGROUND ART

[02] African swine fever (ASF) is an acute, hemorrhagic and virulent infectious disease caused by African swine fever virus (ASFV) infecting domestic pigs and various wild boars (such as African wild boar, European wild boar, etc.). The ASF has a short pathogenic process, generally 5-14 days, and the mortality rate of the most acute and acute infections is as high as 100%. African swine fever is caused by double-stranded

DNA virus and is the only member of African swine fever virus family. The serotype of

ASFV is classified based on haemadsorption inhibition test (HAI), and 32 ASFV strains can be divided into 8 serum groups. The genotype of ASFV is determined by the single gene sequence encoding VP-72 protein, and the ASFV strains are divided into 24 genotypes. The different strains have different genome sizes, ranging from 170 to 190 kb, and encode about 150-167 proteins, including proteins needed for virus replication, structural proteins and proteins related to inhibition and escape of host defense system.

[03] At present, there is still a lack of effective preventing and treating measures for

African swine fever. The main preventing measure is to strengthen the isolation of pig farms, and the infected pigs are conducted with harmless treatment, then the pig farms are performed with thorough disinfection. Many countries have tried to develop ASFV inactivated vaccine, recombinant protein vaccine, DNA vaccine, DNA+ protein vaccine, live vector vaccine and attenuated live vaccine, but due to the dissatisfactory safety and protection effectiveness, these vaccines still can not be widely used.

[04] Cytotoxic T lymphocyte (CTL) response mediated by major histocompatibility complex class I (MHC I) molecule is an important means for the body to clear away diseased cells ir vivo and resist virus infection. MHC I molecules are expressed on the surface of all nucleated cells and platelets, bind and present intracellular antigen peptides (such as viral peptides degraded by proteasome) for the specific recognition of T cell receptor (TCR) on the surface of CD8* T cells. Under the synergistic action of coreceptor molecules (CD8 molecules, etc.), CD8* T cells are activated to specifically kill target cells. This process is called CTL response, and the peptides presented by MHC 1 molecule which can induce CTL response are called CTL epitopes. Over years, it has been proved that ASF cannot achieve effective control through humoral immunity, and it has been reported that CTL is a necessary mean to eliminate ASFV. The combination of MHC I molecule and peptide epitope is a prerequisite for initiating CTL response, which has become the basis of cellular antiviral immune response research, thus providing conditions for the development of novel peptide epitope vaccines.

SUMMARY

[05] Currently, there is no effective vaccine against ASF, and the objective of the present disclosure is to provide a CTL epitope of African swine fever with the potential to be developed as an African swine fever epitope vaccine and use thereof.

[06] In order to achieve the above objectives, the present invention uses bioinformatics methods and methods to identify MHC 1 binding motifs to screen peptides that can be stably bound to MHC I in ASFV, and develop and identify a new epitope vaccine based on this potential peptide epitope. The greatest advantage of the method is that the epitope prediction accuracy is greatly improved, and the developed novel epitope vaccine has high safety. In order to achieve the above purpose, the invention adopts the following technical solutions to realize the screening and identification of CTL epitopes of African swine fever. The method for screening CTL epitopes of African swine fever mainly includes the following steps:

[07] (1) In view of the existing research, the protein differences among different genotype strains are analyzed and compared, and the proteins with great variation and structural proteins are selected;

[08] (2) Through bioinformatics methods, the epitope prediction and statistics of the protein in step (1) are carried out through a plurality of swine leukocyte antigen class (SLA T) molecules to screen peptides with strong binding ability;

[09] (3) Through bioinformatics methods, the frequency of the peptides screened in the step (2) is calculated to screen peptides with the frequency higher than 50%;

[10] (4) A random peptide library is synthesized in vitro, and the amino acid distribution of the random peptide library was identified and analyzed by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) and

Protein De Novo Sequencing.

[11] (5) Identify the SLA I molecules that are mainly expressed in multiple pig herds and express them in vitro. Refold and purify SLA I complexes with random peptide library, and separate peptides by thermal denaturation;

[12] (6) The in vitro binding motif of SLA I molecule is identified by LC-MS/MS and De Novo Sequencing, and the peptide in step (3) is further screened by the motifs;

[13] (7) The peptides screened in step (6) are synthesized 7 vitro and purified; thus obtaining peptide with biological activity.

[14] Then, the peptide vaccine is studied, and a corresponding adjuvant is selected to emulsify the quantitative peptides. A reasonable number of experimental animals are selected for programmed immunization. Blood samples are collected at appropriate time before and after immunization, and the peripheral blood lymphocytes are isolated. Then the changes of CD8* T cells induced by peptide vaccine are detected by flow cytometry, and the peptide specific CTLs are detected by the ELISPOT method.

[15] In step (1) of the above screening process, strains of different ASFV genotypes and Chinese type II ASFV virus, preferably type I/II strong and weak strains, are selected. Genomic sequences of type I/ II strong and weak strains have been published in GenBank.

[16] In step (1), the non-isotype differential proteins are preferably the proteins with large difference, more preferably the P-Value <0.01.

[17] In step (2), ASF epitope prediction is carried out through archived SLA-I in multiple bioinformatics websites, such as NetMHCpan 4.0 (http://www.cbs.dtu.dk/services/NetMHC pan/).

[18] In step (2), the binding peptides, including strong binding and weak binding are screened, preferably the Affinity Threshold > 500 or Rank Threshold > 0.5.

[19] In step (3), the walking peptide is merged and counted, and the peptide with the largest span is screened.

[20] In step (4), the length of the random peptide is preferably 8-10 amino acids, more preferably 9 amino acids.

[21] In step (4), the synthesis method of the random peptide can choose the conventional method, preferably the Fmoc method.

[22] In steps (4) and (6), the FDR value is set to 1%, and the ALC (%) of De Novo analysis is greater than or equal to 50 points.

[23] In step (5), through clone expression analysis, a chains with high homology of

SLA-I are screened and identified for expression.

[24] In step (5), the method of protein renaturation and purification: the molar ratio of heavy chain, light chain and random peptide is 1: 1: 5; the conventional protein renaturation solution includes 100mM Tris, 400mM L-arginine, 2mM EDTA, 1.5306g/L reduced glutathione and 0.3064g/L glutathione, with a pH of 8.0; the conventional separating and purifying methods include such as molecular sieve chromatography, ion exchange chromatography.

[25] In step (5), the peptide can be separated and purified by the method commonly used in the field, such as membrane dialysis and solid phase extraction desalination, preferably the molecular weight cut-off of the filter membrane is 3KDa.

[26] In step (6), the judging method of the main anchor site and amino acid preference thereof is: through the De Novo analysis, the main anchors are directly judged by using Weblogo or icelogo plotting, the number of which is 3-4.

[27] In step (6), FDR value is set to 1%, De Novo ALC(%)>50, motif analysis is performed by using Weblogo and/or icelogo plotting, and the peptides are screened by scoring the anchor location sites.

[28] In step (7), the peptide is synthesized by Fmoc method and purified by HPLC with a purity of 99%.

After screening by the above method, 18 peptides with higher scores are obtained, and the amino acid sequences of these CTL epitopes of ASFV are set forth in any one of

SEQ ID No: 1-18, or the amino acid sequence set forth in any one of SEQ ID No:1-18 that is substituted and/or deleted by one or several amino acid residues; and/or the amino acid sequence 1s added with an amino acid sequence that is derived from SEQ ID No:1- 18 and keeps the function of the protein set forth in SEQ ID No:1-18.

[29] The coding gene of the CTL epitope of ASF belongs to the protection scope of the present disclosure.

[30] The disclosure provides a biomaterial containing the CTL epitopes of ASF, and the biomaterial is a recombinant expression vector, an expression cassette, a recombinant bacterium or a host cell. [BI] According to the disclosure, 6 pigs aged 2 months are randomly selected for the experiment, wherein 3 pigs are experimental groups and the other 3 pigs are control groups. Conventional immunization program is selected, all immunization programs are cancelled before immunization, and intensive immunization is carried out 2 weeks after the first immunization. Flow cytometry is used to detect CD3, CD4 and CD8 markers by specific monoclonal antibodies, and the changes of CD4*/CD8* positive T lymphocytes and their corresponding ratios are observed. The ELISPOT kit for detecting

IFN-y is selected to detect the cell number of IFN-y produced by peptide stimulation. It is found that the CTL epitope peptide of ASF or its coding gene or biomaterial containing it can increase the number of CD8* T cells and promote the secretion of IFN-y.

[32] Based on the above findings, it can be seen that these CTL epitopes of ASF have potential ability to prepare peptide vaccines. The disclosure further provides a drug containing the CTL epitopes of ASF.

[33] Preferably, the drug is a peptide vaccine.

[34] Furthermore, the peptide vaccine of the present disclosure further contains adjuvants, which are chitosan and carrier protein, preferably MONTANIDE ISA 50 V2,

MONTANIDE ISA 61 VG and/or MONTANIDE ISA 201 VG.

[35] The disclosure provides use of the CTL epitopes of ASF or its coding gene or biomaterial containing it in preparing a vaccine for preventing ASFV infection.

[36] The disclosure provides use of the CTL epitopes of ASF or its coding gene or biomaterial containing it in preparing a drug for treating ASFV infection.

[37] The disclosure provides use of the CTL epitopes of ASF or its coding gene or biomaterial containing it in preparing a reagent or a kit for detecting ASF.

[38] The present invention is based on vaccine safety, combining existing SLA I molecular bioinformatics and random peptide library methods to identify specific SLA

I motifs to predict corresponding ASFV epitopes, and develop a epitope vaccine against

ASFV. In the disclosure, a random peptide library is used to identify pigs that mainly express SLA I alleles and their corresponding binding motifs at one time, and complete the identification of the pigs that mainly express multiple SLA I alleles peptide-binding motifs. This greatly reduces research costs and time costs and improves the accuracy of virus potential epitope prediction, and the results obtained can be used for long-term screening of species-specific T cell epitopes, especially T cells for ASF epitope screening. The screened peptides have the ability to specifically stimulate the body to produce CTL, and can initiate CD8" T cells to produce immune response, and promote to secrete IFN-y. Based on the corresponding animal experiments, the safety of peptide vaccine and the reliability of activating CTL immune response are verified. The peptide provided by the disclosure can be used for preparing specific antigen epitope vaccine or peptide vaccine of ASF, which has high safety and good specificity, and has potential application value in preventing and controlling ASF.

BRIEF DESCRIPTION OF THE DRAWINGS

[39] Fig 1 shows the scanning and screening of proteins of ASFV Chinese strain by comparative proteins.

[40] Fig. 2A shows the total ion current (TIC) chromatogram (left image) of bound nonapeptide.

[41] Fig 2B is a Basepeak diagram of bound nonapeptide.

[42] Fig 3A is a weblogo diagram of the motif identification result of SLA-1*0401.

[43] Fig. 3B is a weblogo diagram of the motif identification result of SLA-1*1301.

[44] Fig. 4, Fig. SA, Fig. 5B, Fig. 6A and Fig. 6B are identification results of

CD4*CD8 and CD4 CDS" T cells detected by flow cytometry at different times before and after immunization.

[45] Fig 7 is a comparison diagram of CD4CD8" T cells changes between the control group and the immunization group before and after immunization.

[46] Fig. 8 is an identification result of peptide specificity ELISPOT on the third day after immunization, the ELISPOT detects IFNy secretion in PBMC before and after immunization.

[47] Fig 9 is an identification result of peptide specificity ELISPOT on the 14™ day after immunization, the ELISPOT detects IFNy secretion in PBMC before and after immunization.

[48] Fig. 10 is an identification result of peptide specificity ELISPOT on the 28! day after immunization, the ELISPOT detects IFNy secretion in PBMC before and after immunization.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[49] The following examples further illustrate the present disclosure, but should not be construed as limiting the present disclosure. Without departing from the spirit and essence of the disclosure, modifications or substitutions made to the methods, steps or conditions of the disclosure belong to the scope of the disclosure. Unless otherwise specified, the technical solutions used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in the examples are commercially available.

EXAMPLEL1 Screening and use of ASFV-derived potential T cell epitopes

[50] 1 Preliminary screening of virus epitopes

IS1] 1.1 Selection of viral proteins [S2] Based on previous studies, ASFV attenuated infection can infection of the same type of virulent strain , but cannot resist infection of different types of virulent strains.

Therefore, in order to find the specific CTL epitopes against Chinese strain, i.e. ASFV

China/2018/AnhuiXCGQ (GenBank: MK 128995.1), the protein differences among different genotypes of I/II strains were analyzed, and the proteins with large variation and structural proteins were screened. The specific steps were as follows: [S3] (1) According to the research of Linda Dixon and other scientists, the following four strains with different genotypes were selected for analysis and comparison, see Fig. 1 for details:

[54] ASFV China/2018/AnhuiXCGQ (GenBank: MK 128995 1) type II virulent,

ASFV OURT 88/3 (avirulent field isolate) (GenBank: AM 712240.1) type I attenuated,

ASFV NHV (GenBank: KM 262845.1) type II attenuated, and ASFV strain 160 (GenBank: KM 262844.1) type I virulent.

[55] (2) According to the comparison results in step (1) and the existing domestic and foreign research, the proteins of MGF family (MFG-110, MGF-360, MGF-505) and structural proteins (CD2V, P30, P54, P72) were selected to predict the potential epitopes.

[56] 1.2 Preliminary prediction of virus epitopes by bioinformatics method

[57] Through bioinformatics websites, such as NetMHCpan 4.0 Server (http://www.cbs.dtu.dk/services/NetMHCpan/), 39 registered SLA I (as shown in Table 1) (including SLA-1, SLA-2 and SLA-3) were analyzed and predicted, and peptides with statistical frequency higher than 50% were screened, see Table 2 for details.

[58] Table 1 Registered SLA-I in bioinformatics website

SLA+1 OO SLA2 ~~ SLA3 "SLA-1¥0101 SLA-2*0101 SLA-3*0101

SLA-1*0201 SLA-2*0102 SLA-3*0301

SLA-1*0202 SLA-2*0201 SLA-3*0302

SLA-1*0401 SLA-2*0202 SLA-3*0303

SLA-1*0501 SLA-2*0301 SLA-3*0304

SLA-1*0601 SLA-2*0302 SLA-3*0401

SLA-1*0701 SLA-2*0401 SLA-3*0501

SLA-1*0702 SLA-2*0402 SLA-3*0502

SLA-1*1101 SLA-2*0501 SLA-3*0503

SLA-1*1201 SLA-2*0502 SLA-3*0601

SLA-1*1301 SLA-2*0601 SLA-3*0602

SLA-2*0701 SLA-3*0701

SLA-2*1001

SLA-2*1002

SLA-2*1101

SLA-2*1201

[59] Table 2 Epitopes screening based on bioinformatics

Peptide sequence Peptide sequence

ASFV G | QLMGCTATY MGF 110 | TYQSPTTPWC

ACD 0021 -12L FYEI 0

ASFV G | MIQATMITMYNSIVIFFF HYMNCSLPTY

ACD 0036 MGF 110 |F 0 -13L

ETNECTSSFETLF VLNRPLSIFY

KTVQHIEQY QLSIKQYCLYF

P30 RAHNFIQTIYGTPL KMIKNTYVLK

MGF 360 | FWFKI

QVVFHAGSLYNWFSV -6L GNVDEIHHAY

F

P54 CLSPVTTPSFFSTHMYTILI STYEYTETFHS

TL ew

TQNTASQTMSAIENLRQRN EAISYVYQHF

INVSRAREFYISWDTDYV RGLMEITFML

DHDDSFSTVL

TKYWY

P72 VVSASAINFLLL MGF 360 | SAMLACVRFY

SSISDISPVTYPITLPI NNVFDLHELY

HGINLIDKFPSKFCSSYIPFH ALAEQRNYYL

Y ISHHLSL

HKPHQSKPILTDENDTQRT IQDYSYSAIYY

MGF 505- | LLAWEGNLYY ALAEQKENYL om

MGF 505- | MRAYLHETLFELACLWQR SINMGIFLDY 8

DLTMYSLGYIFLF VKTDLLNNEF

SLSTLLLKYW

MGF 360 |Y

MGF 505- | YTDYLDRWEYCSQMLF -12L DHNLSTMYY wT

A238L NLSTCISLFTSWMF NQAMLSSIQY mm

KTLNLTKTYNHESNYWVN YVSKNMMIFL

WVGYNNVCYY MGF 360 | NQAMLSSIQY

I il

CP2475L | INMRHHTSYTENSVLTY MGF 360 | HNFTKAIHYF

A

ALQNDIEAMMTMVINPHP AYMYNLSNIF

A a

DP63R KNVSTVFTYY VRNKAIELYW ee

YTNESILEYNWNNSNINNF | MGF 360 | YEEPDFAELA

CD2V Ke -18R FICAAYF

CTYLTLSSNYFYTFFKLYYI YFGEPQVMYL

NINDTFVKYTNESILEY ILDDISFSEML

MGF 360 | TRYWYSM -21R YNLTEAIQYF

EMMKLTCSTY

DGNYSTIYYC

FML

[60] 2 Identification of swine SLA I peptide motif

[61] 2.1 Synthesis and identification of random peptide library

[62] The Fmoc method was used to synthesize the sequence random nonapeptide library. The specific steps were as follows:

[63] (1) Peptide-solid phase carrier cross-linking: 19 kinds of a-amino protecting group amino acids (except cysteine) were prepared by Fmoc(9- fluorenylmethoxycarbonyl) method, and reacted with alkoxybenzyl alcohol resin in

DMSO at 20-25°C;

[64] (2) Mix and deprotection: 19 kinds of cross-linked peptide-solid phase carrier were evenly mixed and deprotected by TFA (trifluoroacetic acid);

[65] (3) Neutralization and washing: the free amino terminal with triethylamine was neutralized and fully washed,

[66] (4) Grouping and condensation: the washed mixture was equally divided into 19 parts, the carboxyl groups of a new round of amino acids were activated by DCC, and 19 kinds of amino acids were respectively added into 19 equal parts of the mixture in excess for condensation reaction;

[67] (5) The processes (2)-(4) were circularly repeated for 7 times, mixing evenly and washing thoroughly;

[68] (6) The cross-linked random nonapeptide-solid phase carrier was cut off by 90%

TFA to obtain a cross-linked random nonapeptide library;

[69] (7) Purification: reverse column (C18) was used for purification to remove short peptide and residual TFA,

[70] (8) Mass spectrometry sequencing analysis: the synthesized random nonapeptide library was analyzed by LC-MS/MS and De Novo Sequencing, and the distribution of each amino acid was analyzed and quality checked .

[71] The quality inspection steps of random nonapeptide were as follows: the obtained random nonapeptide was redissolved with 20 uL of 0.1% formic acid/water solution, injected with 10 pL, and separated by chromatographic column (C18, 3pm, 100um, 100A, 75um* 15cm). Mobile phase: A: 0.1% formic acid water solution, B: 0.1% formic acid acetonitrile solution.

[72] Chromatographic gradient: /mL*min? 0 99% 1% 400 1 95% 5% 400 90 70% 30% 400 92 60% 40% 400 95 10% 90% 400 105 10% 90% 400 106 99% 1% 400 125 99% 1% 400

Ce

[73] Detecting parameters of the Q Exactive ultra-high resolution mass spectrometer (Thermo Scientific Q Exactive):

[74] spray voltage: 2.0 kV; capillary temperature: 320°C; S-lens RF Level: 55; resolution setting: Level 1-70000@m/z 200, Level2-17500@m/z 200; parent ion scanning range: m/z 200-1800, daughter ion: automatic; MS1 AGC: 3e6, ion implantation time: 60ms; MS2 AGC: Se4, ion implantation time: S0ms; ion screening window: 2.2 m/z; fragmentation mode: HCD, energy NCE 27; Data-dependent MS/MS:

top 10; dynamic exclusion time: 10s.

[75] Data De Novo analysis: firstly, the collected original data were analyzed by De

Novo software Peaks studio, and the software parameters were set as follows:

Item ~~ valwe

Enzyme ~~ No-Specific

Variable modifications Oxidation (M),Deamidated(N,Q)

Peptide Mass Tolerance + 10 ppm

Fragment Mass Tolerance 0.02 Da

[76] The TIC spectrum and Base peak diagram of the random nonapeptide library were shown in Fig. 2A and Fig. 2B, and the distribution of the ratio of 19 amino acids at each locus in the random nonapeptide library was obtained.

[77] 2.2 Identification and expression of SLA I a chain and B chain

[78] (1) The peripheral blood lymphocytes were isolated by conventional methods.

[79] (2) The lymphocyte RNA was extracted by conventional TRIZOL method.

[80] (3) The cloning and identification of SLA I were performed:

[81] 1pg cDNA was taken for reverse transcription with the kit and the method refers to the manual (Takara Prime Script IT 1% Strand cDNA Syntheis Kit, Code No. 6210A).

The cDNA after reverse transcription was conducted with PCR amplification. The primer sequences were shown in Table 3, and two amplified bands were obtained, which were 1100 bp (achain of SLA-1*0401 and SLA-1*1301) and 300 bp (B chain of SLA- 10401 and SLA-1*1301), respectively. The amplified products were recovered by agarose gel electrophoresis (Axygen) and ligated to the pMD-18T vector (Takara) through solution I ligase. According to the conventional methods, the ligation products were transformed into Top10 clonal chemically competent cells cultured and sequenced.

[82] Table 3 Primers of SLA-I a chain and B chain

Primer Primer sequence ~~ Fowad 5’-ATGGGGCCTGGAGCCCTCTTCC-3’ primer

SLA-I-a 5-

Reverse

TCACACTCTAGGATCCTGGGTGAGGGACAC- primer 3

~~ Forward ~~ 5’- GTCGCGCGTCCCCCGAAGGTTCAGG-3°

SLA-LB primer

Reverse / 5’- TTAGTGGTCTCGATCCCACTTAACT-3’ primer

[83] (4) Expression and purification of SLA I inclusion body

[84] The extracellular regions of SLA-1*0401, SLA-1*1301 and B2m after sequencing analysis were introduced into enzyme digestion sites (Nde I and Xho I) by

PCR method. The PCR products and stored pET-21a vector digested with double enzymes and recovered by gel. the digested product and ligase were mixed overnight at 16°C, transformed and sequenced.

[85] The correctly sequenced plasmids were transformed into BL2 1(DE3) expressing chemically competent cells. IPTG was added to induce a massive expression and the collected thallus was performed with low-temperature ultrasonic crushing. The inclusion body was washed with 20 ml washing buffer (0.5% Triton-100, 50mM Tris pHS8.0, 300mM NaCl, 10mM EDTA, 10mM DTT), centrifuged at 8000g for 10min, and repeated once. The resulting precipitate was washed with 20ml resuspension buffer (50mM Tris pH8.0, 100mM NaCl, 10 mM EDTA, 10 mM DTT), 20uL washing liquid was taken for SDS-PAGE identification, centrifuged at 8000g for 10min, weighed, and dissolved with guanidine hydrochloride denaturing solution (6M guanidine hydrochloride, 10% glycerol, 50mM Tris with pH 8.0, 100mM EDTA, 100mMDTT) with a concentration of 30mg/mL.

[86] 2.3 Identification of SLA I motif

[87] (1) Renaturation of SLA I-random nonapeptide library

[88] The MHC I renaturation solution was prepared, which contained 100Mm Tris with pH8.0, 400mM L-arginine, 2mM EDTA, reducing glutathione 0.7653g/500mL and glutathione 0.1532g/500ml. ImL B2m inclusion body was added dropwise into the renaturation solution. after renaturating for 8h, 10 mg random nonapeptide library dissolved in DMSO was added. 3mL SLA-I a chain was added dropwise after full mixing. After renaturating for 12h, the renaturation solution was concentrated to less than S0mL, fluid change was performed with molecular sieve (20mM Tris-HCI with pH 8.0, 50 mM NaCl) and highly concentrated to less than 2mL. Further purification was carried out by molecular sieve chromatography (Superdex 200 Increase 10/300 GL, GE)

and ion exchanGE chromatography (Resource Q, GE), and samples were taken for SDS-

PAGE purity identification.

[89] (2) Peptide elution

[90] The purified SLA I-random nonapeptide complex was highly concentrated to 200uL, and the MHC-peptide complex was incubated at 65°C for 1 hour to fully dissociate the peptide. The a chain and B chain of denatured SLA-I were intercepted by 3KD filter membrane, and the filtrate was collected several times and desalted and purified by Stagetip C18 column.

[91] (3)LC-MS/MS mass spectrometry sequencing and analysis

[92] Same as the random nonapeptide quality inspection method in step 2.1. The part of SLA-I binding nonapeptide was analyzed by De Novo analysis and plotted by weblogo. According to the De Novo analysis of two SLA-I-binding nonapeptides, the following operations were performed: the distribution statistics of each amino acid was carried out on peptide data with a score of more than 50, and the amino acid distribution which exceeds the theoretical distribution ratio of each amino acid (the amino acid distribution obtained in step 2.1) was plotted through Weblogo (http://weblogo.berkeley.edu/logo.cgi). The distribution percentage of each amino acid was displayed visually in Fig. 3, wherein each column from left to right represents each locus from N to C in the binding nonapeptide, and the ordinate corresponds to the maximum entropy of a given sequence type (logz 20 = 4.3 bits).

[93] 3 Screening and synthesizing epitopes based on motifs

[94] The peptides screened in step 1.2 were scored and screened by multiple SLA I motifs, and 18 peptides with higher scores were synthesized by Fmoc method as shown in Table 4. The method was the same as that in step 2.1, and the synthesized peptides were purified to 95% by HPLC.

[95] Table 4 Potential epitopes of ASFV based on SLA I

Protein

MGF 110-

QVVFHAGSLYNWFSV u HYMNCSLPTYF 11L NLFFCI

TONTASQTMSAIENLRORN EAISYVYQHFKYLN

12L LLLKYWY br HKPHQSKPILTDENDTQRTC ILDDISFSEMLTRYW nn

NINDTFVKYTNESILEY CP2475L

LTY

[96] 4 Selection of adjuvant and emulsification of vaccine

[97] According to the instruction manual of MONTANIDE ISA 61 VG adjuvant, it was prepared referring to the mass ratio of adjuvant to vaccine of 3:2. 18 peptides shown in Table 4 were dissolved with 200 pg/ peptide per head, and the solution was adjusted to 70mL with PBS. At the same time, 130mL of MONTANIDE ISA 61 VG (density: 0.83g/mL) was used to prepare the vaccine according to the mass ratio of adjuvant to vaccine of 4:6, and the peptide vaccine was prepared by high-speed splicing, and each head was injected with 2mL.

[98] 5 Programmed immunization of experimental animals

[99] Six 2-month-old pigs in good mental state were randomly selected for the experiment, wherein three were experimental group (ear tag: 3-1,3-2,3-3) and three were control group (1-1,1-2,1-3). In order to avoid interference from other vaccines, all routine vaccine immunization procedures were cancelled before immunization. They were divided into two groups: experimental group and control group. The experimental group was immunized with 2mL/ head on day 0, neck intramuscular injection and on the 14th day, booster immunization was given, 2mL/ head, neck intramuscular injection.

The control group was injected with the same amount of normal saline at corresponding time.

[100] 6 The changes of CD4* and CD8" T cells induced by peptide vaccine detected by flow cytometry

[101] (1) Separation of peripheral blood lymphocytes

[102] Blood samples were collected three days before immunization, and one week after the first immunization and booster immunization for the peptide vaccine. Every time, 10mL/ head of peripheral blood was taken for lymphocyte separation. The method was the same as step 2.2 (1), the DMEM containing 10% fetal bovine serum was diluted for counting, and the number of cells was adjusted to 1x10° /mL.

[103] (2) Detection by flow cytometry

[104] (1) 100pL of the cell suspension that the concentration has been adjusted in step (1) was added into a flow detection tube, and each labeled antibody was added to each detection sample according to Table 5. The contents in the tube were mixed well, and a negative control group was set, and the tube was incubated for 30 minutes at room temperature in the dark;

[105] (2) 2mL PBS solution was added to resuspend the cells. The tube was centrifuged at 600g for 5min. The supernatant was discarded and the process was repeated twice;

[106] (3) The cells were resuspended by adding 0.5mL PBS solution, and were detected by flow cytometry.

[107] Table 5 Antibodies needed for flow cytometry

Negative Experimental

No. 1 2 3 control group

EE CDp3-FITC

Types of CD3. DP CD8- CD4-Alexa antibodies FITC Alexa PE None Fluor

Fluor

CD8-PE

Amount of 978 antibody added 0.2ug 0.1pg O.lug None 0.1ug 0.lug

[108] The flow cytometry results were shown in Fig. 4, Fig. 5A, Fig. 5B, Fig. 6A and

Fig. 6B. The flow cytometry results of blood samples collected from the -3 day (the 3™ day before immunization), the 14! day, and the 28! day were shown in the three columns from left to right, and each row represents the test results of each pig. In a single flow cytometer, the ordinate represents anti-CD4 mAb-Alexa Flour 647, the abscissa represents Anti-CD8 mAb-PE, and Q1/Q2/Q3/Q4 respectively represent CD4*CD8T lymphocytes /CD4*CD8* T lymphocytes /CD4CD8* T lymphocytes /CD4CD8T lymphocytes.

[109] According to the classified statistics of the proportion of T cell subsets in each pig, as shown in Table 6 and Fig. 7, the experimental group showed obvious CD4CD8* proliferation, while CD4"CD8 had no obvious proliferation.

[110] Table 6 Proportion of T cell subsets

No. CDS ND CDS” CDS CoE CDE | CDE | ODT | CDR

[111] Peptide specific CTL detected by ELISPOT method

[112] (1) Separation of peripheral blood lymphocytes

[113] Blood was collected three days before immunization, and one week after primary immunization and booster immunization. Each time, 10 mL/ head of peripheral blood was used for lymphocyte separation. The method was the same as step 2.2 (1), the

DMEM containing 10% fetal bovine serum was used to dilute cells for counting, and the number of cells was adjusted to 1x10” /mL.

[114] (2) ELISPOT detection

[115] (1) 15 ul of 35% ethanol was added to each well for pre-wetting for 1 minute;

[116] (2) Washing: 200uL deionized water was added into each well, and washing for 5 times;

[117] (3) Coating: the antibody coated with pIFNy-I was diluted by PBS to a final concentration of 10ug/mL. Add 100uL to each well and wrap at 4°C overnight;

[118] (4) Sealing: The coating solution was poured and the plate was washed with 200ul sterile 1<PBS for 5 times. For the last time, the plate was buckle dried on sterilized absorbent paper. The diluted sealing solution (10% fetal bovine serum medium) was added in 200uL/ well, and blocked at 37°C for 1 hour;

[119] (5) The sealing solution was poured without washing, 100uL of cells counted in step (1) was added to each well, the peptide was diluted with DMEM containing 10% fetal bovine serum to a final concentration of 100ug/mL, and 100uL of the resulting solution was added to each well. At the same time, negative control, positive control and blank control were set up and incubated at 37°C for 12-48 hours, referring to Table 7.

The peptides were abbreviated as first and last letter + length, such as

QVVFHAGSLYNWFSV abbreviated as QV-15;

[120] Table 7 References of test sample setting.

Liles les leas lew | ow

EMS | Ii IVI? | WE Poaitive contrel | contre

YEG | HERS WY17 | EFI Megafive contrel | control control | comrel

Fp contra! | comrel contral | control

F [VLIZ | YFIS | HPI: | SHS | VYI4 | KYIV | EF13 | SHY | TEL] | Positive | Negative venkel | contd conto! | zende control | comrel

[121] (©) The cells were removed, the plate was washed with 200uL/well PBS for 5 times, and the P2C11-biotin was diluted to 0.5pug/mL with 0.5% fetal bovine serum PBS solution. 100uL/well resulting solution was added and incubated at room temperature for 2h;

[122] (7) The plate was washed with PBS for 5 times, and HRP was diluted into PBS with a final concentration of 0.5% fetal bovine serum. For AEC stain, HRP was diluted in a ration of 1:100 and 100ul dilution was added to each well, and stood at room temperature for 1h. Washing for 5 times;

[123] 100uL AEC was added until spots appear and the reaction was terminated with water. The solution was air dried naturally and stored in the dark.

[124] By performing counting statistics to the ELISPOT results, obvious phenomena were obtained, as shown in Fig. 8, Fig. 9 and Fig. 10, which were the ELISPOT detection results on the -3, 14 and 28 days respectively. The results on the -3 days are almost non- reactive, and the positive and negative control is established, indicating that there is no non-specific reaction interference during the experiment. On the 14th and 28th days, different peptides cause the secretion of IFN-y to varying degrees. Among the 18 peptides, TY21 has the best effect in stimulating IFN-y, followed by YF19, DF13 and 14.

[125] Although the present disclosure has been described in detail above with general descriptions and specific embodiments, it was obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present disclosure.

Therefore, these modifications or improvements made without deviating from the spirit of the disclosure belong to the scope of the disclosure.

SEQLTXT

SEQUENCE LISTING

<110> China Agricultural University <120> CTL EPITOPE PEPTIDE OF AFRICAN SWINE FEVER AND USE THEREOF <130> SWP202106464 <160> 58 <170> PatentIn version 3.5 <21e> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of SLA-I alpha chain <400> 1 atggggcctg gagccctctt cc 22 <2105 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of SLA-I alpha chain <400> 2 tcacactcta ggatcctggg tgagggacac 30 <2105 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of SLA-I bata chain <400> 3 gtcgcgcgtc ccccgaaggt tcagg 25 <2105 4 <211> 25 <212> DNA <213> Artificial Sequence

Pagina 1

SEQLTXT

<220> <223> Reverse primer of SLA-I bata chain <400> 4 ttagtggtct cgatcccact taact 25 <216> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein ASFV G ACD 00210 <400> 5

Gln Leu Met Gly Cys Thr Ala Thr Tyr 1 5 <210> 6 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein ASFV G ACD 00360 <400> 6

Met Ile Gln Ala Thr Met Ile Thr Met Tyr Asn Ser Ile Val Ile Phe 1 5 10 15

Phe Phe <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein P30(1) <400> 7

Glu Thr Asn Glu Cys Thr Ser Ser Phe Glu Thr Leu Phe 1 5 10

Pagina 2

SEQLTXT

<210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein P30(2) <400> 8

Lys Thr Val Gln His Ile Glu Gln Tyr 1 5 <210> 9 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein P30(3) <400> 9

Arg Ala His Asn Phe Ile Gln Thr Ile Tyr Gly Thr Pro Leu 1 5 10 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein P30(4) <400> 10

Gln Val Val Phe His Ala Gly Ser Leu Tyr Asn Trp Phe Ser Val 1 5 10 15 <210> 11 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein P54(1)

Pagina 3

SEQLTXT

<400> 11

Cys Leu Ser Pro Val Thr Thr Pro Ser Phe Phe Ser Thr His Met Tyr 1 5 10 15

Thr Ile Leu Ile <210> 12 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein P54(2) <400> 12

Thr Gln Asn Thr Ala Ser Gln Thr Met Ser Ala Ile Glu Asn Leu Arg 1 5 10 15

Gln Arg Asn Thr Tyr 20 <210> 13 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein P72(1) <400> 13

Ile Asn Val Ser Arg Ala Arg Glu Phe Tyr Ile Ser Trp Asp Thr Asp 1 5 10 15

Tyr Val <210> 14 <211> 12 <212> PRT <213> Artificial Sequence <220>

Pagina 4

SEQLTXT

<223> Peptide of protein P72(2) <400> 14

Val Val Ser Ala Ser Ala Ile Asn Phe Leu Leu Leu 1 5 10 <210> 15 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein P72(3) <400> 15

Ser Ser Ile Ser Asp Ile Ser Pro Val Thr Tyr Pro Ile Thr Leu Pro 1 5 10 15

Tle <210> 16 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein P72(4) <400> 16

His Gly Ile Asn Leu Ile Asp Lys Phe Pro Ser Lys Phe Cys Ser Ser 1 5 10 15

Tyr Ile Pro Phe His Tyr <210> 17 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein P72(5)

Pagina 5

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<400> 17

His Lys Pro His Gln Ser Lys Pro Ile Leu Thr Asp Glu Asn Asp Thr 1 5 10 15

Gln Arg Thr Cys Ser His Thr Asn Pro <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_505-1R <400> 18

Leu Leu Ala Trp Glu Gly Asn Leu Tyr Tyr 1 5 10 <210> 19 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF _5985-2R(1) <400> 19

Met Arg Ala Tyr Leu His Glu Thr Leu Phe Glu Leu Ala Cys Leu Trp 1 5 10 15

Gln Arg Tyr <210> 20 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF _5985-2R(2) <400> 20

Pagina 6

SEQLTXT

Asp Leu Thr Met Tyr Ser Leu Gly Tyr Ile Phe Leu Phe 1 5 10 <210> 21 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_505-3R <400> 21

Tyr Thr Asp Tyr Leu Asp Arg Trp Glu Tyr Cys Ser Gln Met Leu Phe 1 5 10 15 <210> 22 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein A238L <400> 22

Asn Leu Ser Thr Cys Ile Ser Leu Phe Thr Ser Trp Met Phe 1 5 10 <210> 23 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein EP153R(1) <400> 23

Lys Thr Leu Asn Leu Thr Lys Thr Tyr Asn His Glu Ser Asn Tyr Trp 1 5 10 15

Val Asn Tyr Ser Leu <210> 24 <211> 10

Pagina 7

SEQLTXT

<212> PRT <213> Artificial Sequence <220> <223> Peptide of protein EP153R(2) <400> 24

Trp Val Gly Tyr Asn Asn Val Cys Tyr Tyr 1 5 10 <210> 25 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein C2475L(1) <400> 25

Ile Asn Met Arg His His Thr Ser Tyr Thr Glu Asn Ser Val Leu Thr 1 5 10 15

Tyr <210> 26 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein C2475L(2) <400> 26

Ala Leu Gln Asn Asp Ile Glu Ala Met Met Thr Met Val Ile Asn Pro 1 5 10 15

His Pro Pro Val <210> 27 <211> 10 <212> PRT <213> Artificial Sequence

Pagina 8

SEQLTXT

<220> <223> Peptide of protein D63R <400> 27

Lys Asn Val Ser Thr Val Phe Thr Tyr Tyr 1 5 10 <210> 28 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein CD2V(1) <400> 28

Tyr Thr Asn Glu Ser Ile Leu Glu Tyr Asn Trp Asn Asn Ser Asn Ile 1 5 10 15

Asn Asn Phe <210> 29 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein CD2V(2) <400> 29

Cys Thr Tyr Leu Thr Leu Ser Ser Asn Tyr Phe Tyr Thr Phe Phe Lys 1 5 10 15

Leu Tyr Tyr Ile <210> 30 <211> 17 <212> PRT <213> Artificial Sequence <220>

Pagina 9

SEQLTXT

<223> Peptide of protein CD2V(3) <400> 30

Asn Ile Asn Asp Thr Phe Val Lys Tyr Thr Asn Glu Ser Ile Leu Glu 1 5 10 15

Tyr <210> 31 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_110-12L <400> 31

Thr Tyr Gln Ser Pro Thr Thr Pro Trp Cys Phe Tyr Glu Ile 1 5 10 <210> 32 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_110-13L(1) <400> 32

His Tyr Met Asn Cys Ser Leu Pro Thr Tyr Phe 1 5 10 <210> 33 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_110-13L(2) <400> 33

Val Leu Asn Arg Pro Leu Ser Ile Phe Tyr 1 5 10

Pagina 10

SEQLTXT

<210> 34 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_110-13L(3) <400> 34

Gln Leu Ser Ile Lys Gln Tyr Cys Leu Tyr Phe 1 5 10 <210> 35 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-6L(1) <400> 35

Lys Met Ile Lys Asn Thr Tyr Val Leu Lys Phe Trp Phe Lys Ile 1 5 10 15 <210> 36 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-6L(2) <400> 36

Gly Asn Val Asp Glu Ile His His Ala Tyr Phe 1 5 10 <210> 37 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-6L(3)

Pagina 11

SEQLTXT

<400> 37

Ser Thr Tyr Glu Tyr Thr Glu Thr Phe His Ser Phe Ser Ser Leu Arg 1 5 10 15 <210> 38 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-11L(1) <400> 38

Glu Ala Ile Ser Tyr Val Tyr Gln His Phe Lys Tyr Leu Asn Thr Trp 1 5 10 15

Trp Leu Ile <210> 39 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-11L(2) <400> 39

Arg Gly Leu Met Glu Ile Thr Phe Met Leu Asp His Asp Asp Ser Phe 1 5 10 15

Ser Thr Val Leu Thr Lys Tyr Trp Tyr <210> 40 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-11L(3) <400> 40

Pagina 12

SEQLTXT

Ser Ala Met Leu Ala Cys Val Arg Phe Tyr Asn Met Asp Asn Leu Phe 1 5 10 15

Phe Cys Ile <210> 41 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-11L(4) <400> 41

Asn Asn Val Phe Asp Leu His Glu Leu Tyr 1 5 10 <210> 42 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-11L(5) <400> 42

Ala Leu Ala Glu Gln Arg Asn Tyr Tyr Leu Ile Ser His His Leu Ser 1 5 10 15

Leu <210> 43 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-11L(6) <400> 43

Ile Gln Asp Tyr Ser Tyr Ser Ala Ile Tyr Tyr Cys Phe Ile 1 5 10

Pagina 13

SEQLTXT

<210> 44 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-11L(7) <400> 44

Ala Leu Ala Glu Gln Lys Glu Asn Tyr Leu Ile Ala His Ala Leu Ser 1 5 10 15

Leu <210> 45 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-11L(8) <400> 45

Ser Ile Asn Met Gly Ile Phe Leu Asp Tyr 1 5 10 <210> 46 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-12L(1) <400> 46

Val Lys Thr Asp Leu Leu Asn Asn Glu Phe Ser Leu Ser Thr Leu Leu 1 5 10 15

Leu Lys Tyr Trp Tyr

Pagina 14

SEQLTXT

<210> 47 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-12L(2) <400> 47

Asp His Asn Leu Ser Thr Met Tyr Tyr Cys Tyr Val Leu 1 5 10 <210> 48 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-12L(3) <400> 48

Asn Gln Ala Met Leu Ser Ser Ile Gln Tyr Tyr 1 5 10 <210> 49 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-12L(4) <400> 49

Tyr Val Ser Lys Asn Met Met Ile Phe Leu Thr Tyr Asp Leu Arg 1 5 10 15 <210> 50 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-13L <400> 50

Pagina 15

SEQLTXT

Asn Gln Ala Met Leu Ser Ser Ile Gln Tyr Tyr 1 5 10 <210> 51 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-14L(1) <400> 51

His Asn Phe Thr Lys Ala Ile His Tyr Phe Tyr Lys Arg 1 5 10 <210> 52 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-14L(2) <400> 52

Ala Tyr Met Tyr Asn Leu Ser Asn Ile Phe Leu Val Lys Gln Leu Phe 1 5 10 15 <210> 53 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-18R(1) <400> 53

Val Arg Asn Lys Ala Ile Glu Leu Tyr Trp Val Phe 1 5 10 <210> 54 <211> 17 <212> PRT <213> Artificial Sequence <220>

Pagina 16

SEQLTXT

<223> Peptide of protein MGF_360-18R(2) <400> 54

Tyr Glu Glu Pro Asp Phe Ala Glu Leu Ala Phe Ile Cys Ala Ala Tyr 1 5 10 15

Phe <210> 55 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-18R(3) <400> 55

Tyr Phe Gly Glu Pro Gln Val Met Tyr Leu Leu Tyr Lys Tyr 1 5 10 <210> 56 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-21R(1) <400> 56

Ile Leu Asp Asp Ile Ser Phe Ser Glu Met Leu Thr Arg Tyr Trp Tyr 1 5 10 15

Ser Met <210> 57 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-21R(2)

Pagina 17

SEQLTXT

<400> 57

Tyr Asn Leu Thr Glu Ala Ile Gln Tyr Phe Tyr Gln Arg Tyr 1 5 10 <210> 58 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Peptide of protein MGF_360-21R(3) <400> 58

Glu Met Met Lys Leu Thr Cys Ser Thr Tyr Asp Gly Asn Tyr Ser Thr 1 5 10 15

Ile Tyr Tyr Cys Phe Met Leu

Pagina 18

Claims

Conclusions

A CTL epitopes of African swine fever ("African swine fever"), comprising an amino acid sequence set forth in any one of SEQ ID No.: 1 - 18; or the amino acid sequence set forth in any of SEQ ID No.: 1 to 18 which is substituted and/or deleted with one or more amino acid residues; and/or the amino acid sequence is joined to an amino acid sequence derived from SEQ ID No.:1 - 18 and the function of the protein set out in SEQ ID

No.: 1 — 18 Holds.

Encoding genes of the ASF-CTL epitopes according to claim 1.

A biomaterial containing the AfSF CTL epitopes according to claim 1, wherein the biomaterial is a recombinant expression vector, an expression cassette, a recombinant bacterium or a host cell.

A medicament containing the CTL epitopes of ASF according to claim 1.

A medicament according to claim 4, wherein the medicament is a peptide vaccine.

A medicament according to claim 5, wherein the peptide vaccine further contains: adjuvants, and wherein the adjuvants are chitosan and carrier protein; preferably MONTANIDE ISA 50 V2, MONTANIDE ISA 61 VG and/or MONTANIDE ISA 201

VG.

Use of the African swine fever CTL epitopes of claim 1 or the coding gene of claim 2 or the biomaterial of claim 3 in increasing the number of CD8+ T cells produced by somatic cells or IFN-γ secreting cells .

Use of the CTL epitopes of ASF according to claim 1 or the coding gene according to claim 2 or the biomaterial according to claim 3 in the preparation of a vaccine for the prevention of ASFV infection.

Use of the CTL epitopes of ASF according to claim 1 or the coding gene according to claim 2 or the biomaterial according to claim 3 in the manufacture of a medicament for treatment of ASFV infection.

Use of the CTL epitopes of ASF according to claim 1 or the coding gene according to claim 2 or the biomaterial according to claim 3 in the preparation of a reagent or a kit for detecting ASFV.