CN110862435B

CN110862435B - African swine fever CTL epitope polypeptide and application thereof

Info

Publication number: CN110862435B
Application number: CN201911237087.3A
Authority: CN
Inventors: 张念之; 魏孝辉; 何俊磊; 李忠华
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-12-05
Filing date: 2019-12-05
Publication date: 2021-09-24
Anticipated expiration: 2039-12-05
Also published as: CN110862435A

Abstract

The invention provides an African swine fever CTL epitope polypeptide and application thereof, belonging to the technical field of polypeptide vaccine preparation. The invention screens polypeptide which is derived from the stable combination of MHC I in African Swine Fever Virus (ASFV) through a bioinformatics method and a method for identifying MHC I binding motif, and the screened polypeptide has the characteristic of starting CD8⁺T cells generate immune response and stimulate the capability of an organism to specifically generate CTL, and the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO. 1-18. The polypeptide provided by the invention can be used for preparing an African swine fever specific epitope vaccine or polypeptide vaccine, has high safety and good specificity, and has potential application value in the prevention and control work of African swine fever.

Description

African swine fever CTL epitope polypeptide and application thereof

Technical Field

The invention belongs to the technical field of preparation of polypeptide vaccines, and particularly relates to African swine fever CTL epitope polypeptide and application thereof.

Background

African Swine Fever (ASF) is an acute, hemorrhagic, virulent infectious disease caused by African Swine Fever Virus (ASFV) infecting domestic pigs and various wild pigs (African wild pigs, European wild pigs, etc.). It is characterized by short onset course, generally 5-14 days, and the death rate of the most acute and acute infections is up to 100%. African swine fever is caused by double-stranded DNA viruses and is the only member of the African swine fever virus family. Serotypes are classified based on the erythrocyte adsorption inhibition assay (HAI), and 32 ASFV strains can be divided into 8 serogroups; the genotype is determined according to a single gene sequence of the encoding VP-72 protein, and is divided into 24 genotypes in total. The genome size varies among different strains, and is between 170-190kb, and the genome encodes about 150-167 proteins in total, including proteins required for virus replication, structural proteins and related proteins for inhibiting and escaping the host defense system.

At present, effective prevention and control and treatment measures aiming at African swine fever are still lacked, and the main means is to strengthen the isolation prevention and control of a pig farm, and to adopt the tooth extraction type treatment and thorough disinfection for the sick pigs. Attempts have been made in many countries to develop inactivated ASFV vaccines, recombinant protein vaccines, DNA + protein vaccines, live vector vaccines and attenuated live vaccines, but because of their unsatisfactory safety and protective efficacy, they have failed to achieve the desired safety and protective efficacy.

Major histocompatibility complex I (MHC I) molecule-mediated Cytotoxic T Lymphocyte (CTL) responses are an important tool for the body to clear diseased cells from the body and to protect against viral infection. The MHC I molecules are expressed on the surfaces of all nucleated cells and platelets, bind to and present intracellular antigen polypeptides (such as viral polypeptides degraded by proteasomes), are specifically recognized by T Cell Receptors (TCR) on the surfaces of CD8+ T cells, and activate CD8+ T cells to specifically kill target cells under the synergistic action of co-receptor molecules (CD8 molecules and the like). This process is called a CTL response, and polypeptides presented by MHC I molecules that induce a CTL response are called CTL epitopes. For many years, studies have demonstrated that ASF cannot achieve effective control against viruses by humoral immunity, and CTL has been reported as an essential means for eliminating ASFV. The combination of MHC I molecules and polypeptide epitopes is a prerequisite for starting CTL response, so that the MHC I molecules and the polypeptide epitopes also become the basis for researching cellular antiviral immune response and provide conditions for developing novel polypeptide epitope vaccines.

Disclosure of Invention

At present, an effective vaccine aiming at ASF is lacked, and the invention aims to provide an African swine fever CTL epitope polypeptide with the potential of being developed into an African swine fever polypeptide vaccine and application thereof.

In order to achieve the purposes, the invention screens the polypeptide which is derived from the stable combination of MHC I in ASFV through a bioinformatics method and a method for identifying MHC I combination motif, and develops and identifies a novel polypeptide vaccine based on the potential polypeptide epitope, and the invention has the greatest advantages that the polypeptide prediction accuracy is greatly improved, and the developed novel polypeptide vaccine has high safety. In order to achieve the purpose, the invention adopts the following technical scheme to realize the screening and identification of the African swine fever CTL epitope. The screening method of the African swine fever CTL epitope mainly comprises the following steps:

(1) aiming at the existing research, the protein difference among different genotype strains is analyzed and compared, and the protein with larger variation and the structural protein are selected;

(2) performing epitope prediction statistics on the protein in the step (1) through a plurality of porcine major histocompatibility complex class I molecules (SLA I) by a bioinformatics method, and screening a polypeptide with strong binding capacity;

(3) performing frequency calculation on the polypeptides screened in the step (2) by a bioinformatics method, and screening the polypeptides with the occurrence frequency higher than 50%;

(4) synthesizing a random peptide library in vitro, and identifying and analyzing the amino acid distribution of the random peptide library by utilizing a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology and protein De Novo Sequencing from head;

(5) identifying SLA I molecules mainly expressed by a plurality of swinery groups, performing in-vitro expression, performing renaturation purification on the SLA I through a random peptide library, and separating polypeptides through thermal denaturation;

(6) identifying in vitro binding motifs of SLA I molecules by LC-MS/MS and De Novo, further screening the polypeptides in step (3) by the motifs;

(7) in vitro synthesizing the polypeptide screened in the step (6) and purifying; namely screening to obtain the polypeptide with biological activity.

Then, researching a polypeptide vaccine, and selecting a corresponding adjuvant to emulsify the quantitative polypeptide; selecting a reasonable number of experimental animals for programmed immunization; collecting blood at appropriate time before and after immunization, separating peripheral blood lymphocytes, and detecting CD8 induced by polypeptide by flow cytometry⁺Changes in T cell numbers and detection of polypeptide-specific CTL by the ELISPOT method.

In the step (1) of the screening process, strains with different genotypes are selected by the strains, and the Chinese II type strength is combined; most preferred are type I and type II virulent strains. Both type I and type II virulent and avirulent genomic sequences have been disclosed in GenBank.

In the step (1), the non-homotypic difference protein is preferably a protein with larger difference; most preferably P-Value < 0.01.

In the step (2), ASF polypeptide prediction is carried out through the archived SLA-I in a plurality of bioinformatics websites; for example, NetMHCpan 4.0(http:// www.cbs.dtu.dk/services/NetMHCpan /).

In the step (2), screening binding polypeptides, including strong binding and weak binding; most preferred are Affinity Threshold >500 or Rank Threshold > 0.5.

In the step (3), merging statistics is carried out on the polypeptides with walking, and polypeptide screening is carried out according to the polypeptide with the largest span.

In the step (4), the length of the random polypeptide is preferably 8-10; most preferably 9.

In the step (4), the synthesis method of the random polypeptide can adopt a conventional method; preferably Fmoc method.

In the step (4) and the step (6), the FDR takes 1% value, and the De Novo analysis ALC (%) is more than or equal to 50 minutes.

In the step (5), an alpha chain with higher SLA-I homology is screened and identified for expression through clone expression analysis.

In the step (5), the protein renaturation purification method comprises the following steps: the molar ratio of the heavy chain to the light chain to the random polypeptide is 1:1: 5; conventional protein renaturation solution (100mM Tris, 400mM L-arginine, 2mM EDTA, 1.5306g/L reduced glutathione and 0.3064g/L glutathione, pH 8.0); conventional separation and purification methods (molecular sieve chromatography, ion exchange chromatography).

In the step (5), the polypeptide may be isolated and purified by a method commonly used in the art; such as membrane dialysis, solid phase extraction and desalination. The filter preferably has a molecular weight cut-off of 3 kDa.

In the step (6), the method for judging the main anchor position and the amino acid preference thereof comprises the following steps: judgment is directly carried out by De Novo analysis by using Weblogo or icelogo mapping. The number of primary anchor locations is 3-4.

In the step (6), the FDR value is 1%, De Novo ALC (%) >50, the Weblogo and/or icelogo mapping motif analysis is carried out, and the polypeptides are screened by scoring the anchoring sites.

In the step (7), the Fmoc method is selected to synthesize the polypeptide, and the polypeptide is purified by HPLC, so that the purity reaches 99%.

Through the screening of the method, 18 polypeptides with higher fractions are obtained, and the amino acid sequences of the African swine fever CTL epitope polypeptides are shown in any one of SEQ ID No.1-18, or the amino acid sequences of the SEQ ID No.1-18 are subjected to substitution and/or deletion of one or more amino acid residues and/or addition of amino acid sequences which are derived from the SEQ ID No.1-18 and maintain the functions of the proteins shown in the SEQ ID No. 1-18.

The encoding gene of the African swine fever CTL epitope polypeptide belongs to the protection scope of the invention.

The invention provides a biological material containing the African swine fever CTL epitope polypeptide, and the biological material is a recombinant expression vector, an expression cassette, a recombinant bacterium or a host cell.

6 pigs of 2 months of age are randomly selected for testing, wherein 3 pigs are used as an experimental group, and 3 pigs are used as a control group. The immunization program is a conventional immunization program, all immunization programs are cancelled before immunization, and boosting is carried out 2 weeks after first immunization. Detection by CD3, CD4 and CD8 specific monoclonal antibody by flow cytometry, and attention is paid to CD4⁺、CD8⁺Number of positive T lymphocytes and change of the corresponding ratio. Selecting an ELISPOT kit for detecting IFN-gamma, and detecting the cell number of the IFN-gamma generated by the stimulation of the polypeptide. The African swine fever CTL epitope polypeptide obtained by screening or the coding gene thereof or the biological material containing the same is found to be capable of improving the CD8 of the organism⁺T cell number, and promoting the secretion of IFN-gamma.

Based on the findings, the African swine fever CTL epitope polypeptides have potential capability of preparing polypeptide vaccines. The invention further provides a medicine containing the African swine fever CTL epitope polypeptide.

Preferably, the medicament is a polypeptide vaccine.

Furthermore, the polypeptide vaccine also contains an adjuvant, wherein the adjuvant is chitosan and carrier protein; preferably MONTANIDE ISA 50V2, MONTANIDE ISA 61VG, and/or MONTANIDE ISA 201 VG.

The invention provides an application of the African swine fever CTL epitope polypeptide or the coding gene thereof or the biological material containing the African swine fever CTL epitope polypeptide in preparation of a vaccine for preventing infection of African swine fever virus.

The invention provides application of the African swine fever CTL epitope polypeptide or the coding gene thereof or the biological material containing the African swine fever CTL epitope polypeptide in preparation of a medicine for treating African swine fever virus infection.

The invention provides application of the African swine fever CTL epitope polypeptide or the coding gene thereof or a biological material containing the African swine fever CTL epitope polypeptide in preparation of an African swine fever detection reagent or kit.

The invention predicts the corresponding ASFV epitope peptide based on the vaccine safety by combining the existing SLA I molecular bioinformatics and a method for identifying a specific SLA I motif by random peptide, and develops the polypeptide vaccine aiming at the ASFV specific epitope. The random peptide is used for identifying the pig herd mainly expressing SLA I alleles and corresponding binding motifs thereof at one time, so that the identification of the pig herd mainly expressing multiple SLA I allele binding polypeptide motifs is completed, the research cost and the time cost are greatly reduced, the prediction accuracy of the virus potential epitope peptide is improved, and the obtained result can be used for screening specific T cell epitopes of the species for a long time, particularly T cell epitopes of African swine fever. The screened polypeptide has the capability of stimulating organism specificity to generate CTL and can start CD8⁺T cells generate immune response and promote the organism to secrete IFN-gamma, and based on corresponding animal experiments, the safety of the polypeptide vaccine and the reliability of CTL immune response activation are verified. The polypeptide provided by the invention can be used for preparing an African swine fever specific epitope vaccine or polypeptide vaccine, has high safety and good specificity, and has potential application value in the prevention and control work of African swine fever.

Drawings

FIG. 1 is a protein scanning screen of Chinese strains of African swine fever by comparison proteins.

Fig. 2 is a total ion flux (TIC) chromatogram (left panel) and basepeel plot (right panel) of the bound 9 peptide.

FIG. 3 is a weblogo map showing the results of motif identification of SLA-1. multidot. 0401 and SLA-1. multidot. 1301.

FIG. 4, FIG. 5A, FIG. 5B, FIG. 6A and FIG. 6B are the detection of CD4 by flow cytometry at different times before and after immunization, respectively⁺CD8^-And CD4^-CD8⁺T cell identification results.

FIG. 7 shows CD4 before and after immunization of control group and immune group^-CD8⁺T cell change comparison graph.

FIG. 8 is a graph of the results of polypeptide-specific ELISPOT assay at day 3 post-immunization, which detects IFN γ secretion in PBMCs before and after immunization.

FIG. 9 is a graph of the results of polypeptide-specific ELISPOT assay at day 14 after immunization, which detects IFN γ secretion in PBMCs before and after immunization.

FIG. 10 is a graph of the results of polypeptide-specific ELISPOT assay at day 28 post-immunization, which detects IFN γ secretion in PBMCs before and after immunization.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The reagents used in the examples are commercially available unless otherwise specified.

Example 1 screening and application of potential T cell epitope based on African swine fever

1. Preliminary screening for viral polypeptide epitopes

1.1 selection of viral proteins

Based on the existing research, after ASFV is attenuated, the ASFV can resist the homotypic intensity infection but cannot resist the different homotypic intensity infection, so in order to find out the specific CTL epitope aiming at Chinese strain ASFV China/2018/AnhuiXCGQ (GenBank: MK128995.1), the protein difference analysis among I/II different genotype strains is carried out, and the protein and the structural protein with larger variation are screened, and the specific steps are as follows:

(1) based on the studies of scientists such as Linda Dixon, the following four different genotypic strains were selected for analytical comparison, as detailed in FIG. 1:

ASFV China/2018/AnhuiXCGQ (GenBank: MK128995.1) type II virulent strain, ASFV OURT 88/3 (avimulant field isolate) (GenBank: AM712240.1) type I attenuated strain, ASFV NHV (GenBank: KM262845.1) type II attenuated strain, and ASFV strain L60(GenBank: KM262844.1) type I virulent strain.

(2) And (2) selecting proteins of MGF families (MFG-110, MGF-360 and MGF-505), structural proteins (CD2V, P30, P54 and P72) and the like for potential polypeptide epitope prediction according to the comparison result in the step (1) by combining the existing domestic and foreign researches.

1.2 preliminary prediction of viral epitopes by bioinformatics

39 registered SLAs I (shown in Table 1) (comprising SLA-1, SLA-2 and SLA-3) are analyzed and predicted through bioinformatics websites such as NetMHCpan 4.0Server (http:// www.cbs.dtu.dk/services/NetMHCpan /), and polypeptides with statistical frequency higher than 50% are screened, which is detailed in Table 2.

TABLE 1 SLA-I for bioinformatics Web site login

TABLE 2 bioinformatics-based epitope screening

2. Pig SLAI polypeptide motif identification

2.1 Synthesis and identification of random peptide library the Fmoc method is adopted to synthesize the sequence random nonapeptide library, and the specific steps are as follows:

(1) polypeptide-solid phase carrier crosslinking: preparing 19 amino acids (except cysteine) with alpha-amino protecting groups by Fmoc (9-fluorenylmethyloxycarbonyl), and reacting with an alkoxybenzyl alcohol type resin in DMSO at the temperature of 20-25 ℃;

(2) merging and deprotection: mixing 19 crosslinked polypeptide-solid phase carriers, and deprotecting amino group with TFA (trifluoroacetic acid);

(3) neutralizing and washing: neutralizing the free amino terminus with triethylamine and washing well;

(4) grouping and condensing: equally dividing the washed mixture into 19 parts, activating carboxyl of a new round of amino acid by DCC, and respectively adding 19 kinds of amino acid into the 19 equally divided mixtures in excess for condensation reaction;

(5) repeating the processes (2) to (4) for 7 times circularly, mixing uniformly and fully washing;

(6) solid phase carrier excision to obtain cross-linked random 9 peptide-solid phase carrier, and solid phase carrier excision by 90% TFA;

(7) and (3) purification: purification using reverse-phase column (C18) to remove short peptides and residual TFA;

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

Random 9 peptide quality testing procedure was as follows: the random 9 peptide was re-dissolved in 20. mu.L of 0.1% formic acid/water solution, 10. mu.L of sample was injected, and the mixture was purified by column chromatography (C18,3 μm, 100 μm,

75 μm by 15cm) were separated: mobile phase: a: 0.1% aqueous formic acid solution, B: 0.1% formic acid in acetonitrile

Chromatographic gradient:

q active ultra-high resolution mass spectrometer (Thermo Scientific Q active) detection conditions: spraying voltage: 2.0 kV, capillary temperature: 320 ℃, S-lens RF Level: 55, resolution setting: primary 70000@ m/z 200, secondary 17500@ m/z 200, parent ion scan range: m/z 200-1800; ion scanning range: automatic, MS1 AGC: 3e6, ion implantation time: 60MS, MS2 AGC: 5e4, ion implantation time: 50ms, ion screening window: 2.2 m/z, fragmentation pattern: HCD, energy NCE 27, Data-dependent MS/MS: top10, dynamic exclusion time: for 10 s.

Data De Novo parsing: the collected raw data were first analyzed by De Novo sequencing using De Novo software with the following software parameters:

the TIC spectrum and Base peak of the random nonapeptide library are shown in FIG. 2, and the distribution of the proportion of 19 amino acids at each site in the random 9 peptides is obtained.

2.2 identification and expression of SLA I alpha and beta chains

(1) Peripheral blood lymphocytes are isolated using conventional methods.

(2) Lymphocyte RNA is extracted by a conventional TRIZOL method.

(3) Cloning and identification of SLA I:

mu.g of the sample was subjected to reverse transcription using a kit, according to the instructions (Takara Prime Script II 1)^stStrand cDNA Synthesis Kit, Code No. 6210A). The cDNA after completion of the reverse transcription was subjected to PCR amplification with primer sequences shown in Table 3 to obtain two amplification bands of 1100bp (α strand of SLA-1. multidot. 0401 and SLA 1. multidot. 1301) and 300bp (β strand of SLA-1. multidot. 0401 and SLA-1. multidot. 1301), the amplification products were recovered by agarose gel electrophoresis (Axygen) and the recovered products were ligated to pMD-18T cloning vector (Takara) by solution I ligase. The ligation products were transformed to Top10 clonotype competence and sequenced according to the conventional transformation methods of molecular clonology.

TABLE 3 SLA-I alpha and beta primers

(4) Expression purification of SLA I Inclusion bodies

Introducing enzyme cutting sites (Nde I and Xho I) into extracellular regions of SLA1 0401 and SLA-1 1301 and beta 2m subjected to sequencing analysis in a PCR mode, performing double enzyme cutting recovery on a PCR product and pET-21a stored in a laboratory, connecting overnight at 16 ℃, and performing transformation sequencing;

the correctly sequenced plasmid was transformed into BL21(DE3) expression competence, mass-induced expression was performed by adding IPTG, the collected cells were sonicated at low temperature, centrifuged at 8000g for 10min, washed with 20mL washing buffer (0.5% Triton-100, 50mM Tris pH8.0, 300mM NaCl, 10mM EDTA, 10mM DTT), centrifuged at 8000g for 10min, and repeated once. 20mL of resuspension buffer (50mM Tris pH8.0, 100mM NaCl, 10mM EDTA, 10mM DTT) was used for washing, and 20. mu.L of the washed solution was used for SDS-PAGE identification, centrifuged at 8000g for 10min, weighed, and the pellet was dissolved by guanidine hydrochloride denaturing solution (6M guanidine hydrochloride, 10% glycerol, 50mM Tris pH8.0, 100mM NaCl, 10mM EDTA, 10mM DTT) at a concentration of 30 mg/mL.

2.3 identification of the SLA I motif

(1) Renaturation of SLA I-random nonapeptide library

An MHC I renaturation solution is prepared, and comprises 100Mm Tris pH8.0, 400mM L-arginine, 2mM EDTA, 0.7653g/500mL of reduced glutathione and 0.1532g/500mL of glutathione. 1mL of beta 2m inclusion body is dropwise added into a renaturation solution, 10mg of random nonapeptide library dissolved by DMSO is added after 8 hours of renaturation, the mixture is fully and uniformly mixed, 3mL of SLA-I alpha chain is dropwise added, concentration is carried out after 12 hours of renaturation, the concentration is carried out until the concentration is less than 50mL, solution exchange is carried out by using a molecular sieve (20mM Tris-HCl pH8.0, 50mM NaCl), and the concentration is highly reduced until the concentration is less than 2 mL. Further purification was carried out by molecular sieve chromatography (Superdex 200 Incase 10/300GL, GE), ion exchange chromatography (Resource Q, GE) and sampling for SDS-PAGE purity identification.

(2) Washing peptide

The purified identified SLA I-random nonapeptide complexes were highly concentrated to 200. mu.L and the MHC-polypeptide complexes were incubated at 65 ℃ for 1 hour to allow for sufficient dissociation of the polypeptides. Denatured SLA-I alpha and beta chains were retained by a 3KD filter, and the filtrate was collected several times and subjected to desalting purification by a Stagetip C18 column.

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

The same as the random nonapeptide quality detection method in step 2.1. Partial De Novo resolution of SLA-I binding 9 peptide, webblog mapping. According to De Novo resolution of two SLA-I binding 9 peptides, the following operations were performed: performing statistics on the distribution of each amino acid for more than 50 scores of polypeptide data, performing histogram logo mapping (http:// webbloo. bergelley. edu/logo. cgi) on the Weblogo exceeding the theoretical distribution ratio of each amino acid (the amino acid distribution obtained in step 2.1), and visually displaying the distribution percentage of each amino acid, wherein the result is shown in fig. 3: where each column from left to right represents each site from N-terminus to C-terminus in the binding 9 peptide, the ordinate corresponds to the maximum entropy for a given sequence type (log 220-4.3 bits).

3. Epitope screening and Synthesis based on motifs

The polypeptides screened in step 1.2 were scored and screened through multiple SLA I motifs, and 18 polypeptides with higher scores were synthesized by Fmoc method as shown in table 4, the method was the same as step 2.1, and purified by HPLC, with a purity of 95%.

TABLE 4 SLA I-based potential epitopes of African swine fever

4. Selection of adjuvants and emulsification of vaccines

Refer to the montainide ISA 61VG adjuvant specification, configured at an adjuvant to vaccine mass ratio of 3: 2. Dissolving and mixing 18 polypeptides shown in Table 4 in 200 μ g/peptide per head, diluting to 70mL with PBS, preparing vaccine with 130mL MONTANIDE ISA 61VG (density of 0.83g/mL) according to the mass ratio of 4:6, performing high-speed splicing to prepare polypeptide vaccine, and injecting 2mL per head.

5. Programmed immunization of laboratory animals

6 pigs of 2 months of age with good mental status were randomly selected for testing, 3 of which were experimental groups (ear tags: 3-1,3-2,3-3) and 3 of which were control groups (1-1,1-2, 1-3). To avoid interference from other vaccines, all conventional vaccine immunization programs were cancelled prior to immunization. The total groups are as follows: experimental group and control group. The experiment group is immunized with 2 mL/head part on day 0 and injected into neck muscle; on day 14, boost immunization was performed at 2 mL/head, neck intramuscular injection. The control group was injected with an equal amount of physiological saline at the corresponding time.

6. Detection of polypeptide vaccine induced CD4 by flow cytometry⁺、CD8⁺Alteration of Iso T cells

(1) Peripheral blood lymphocyte isolation

Blood is collected once three days before immunization, and blood is collected 1 week after primary immunization and boosting immunization of the polypeptide vaccine. 10 mL/head of peripheral blood was taken each time for lymphocyte separation in the same manner as in step 2.2(1), diluted and counted using DMEM containing 10% fetal bovine serum, and the number of cells was adjusted to 1X 10⁶/mL。

(2) Flow cytometry detection

Taking 100 mu L of the cell suspension with the adjusted concentration in the step (1) to a flow detection tube, adding each labeled antibody into each detection sample according to the table 5, uniformly mixing, setting a negative control group, and incubating for 30 minutes at room temperature in a dark place;

adding 2mL of PBS washing liquor to resuspend the cells, centrifuging for 5 minutes at 600g, discarding the supernatant, and repeating for 2 times;

and thirdly, adding 0.5mL of PBS washing solution to resuspend the cells, and detecting on a flow-type machine.

TABLE 5 antibody added to each test sample by flow

The flow results are shown in fig. 4, fig. 5A, fig. 5B, fig. 6A and fig. 6B, respectively, and the flow test results of blood collection from three columns to left to right are shown at-3 (third day before immunization), 14 and 28 days, respectively, and each row represents the test result of the sample of each pig. In the single flow chart, the ordinate represents Anti-CD4 mAb-Alexa Flour 647, the abscissa represents Anti-CD8 mAb-PE, and Q1/Q2/Q3/Q4 represents CD4⁺CD8^-T lymphocyte/CD 4⁺CD8⁺T lymphocyte/CD 4⁰CD8⁺T lymphocyte/CD 4^-CD8^-T lymphocytes.

The proportion of T cells in each pig was classified and counted, and as shown in Table 6 and FIG. 7, the experimental group showed significant CD4^-CD8⁺Proliferation phenomenon, CD4⁺CD8^-No obvious proliferation phenomenon.

TABLE 6 proportion of T cell subsets

7、

Detection of polypeptide specificity CTL by ELISPOT method

(1) Peripheral blood lymphocyte isolation

Blood is collected once three days before immunization, and the blood is collected 1 week after the primary immunization and the boosting immunization of the polypeptide vaccine. 10 mL/head of peripheral blood was taken each time for lymphocyte separation in the same manner as in step 2.2(1), diluted and counted using DMEM containing 10% fetal bovine serum, and the number of cells was adjusted to 1X 10⁷/mL。

(2) ELISPOT detection

Adding 15 mu L of 35% ethanol into each hole for pre-wetting for 1 minute;

washing: adding deionized water into 200 mu L/hole, and washing for 5 times;

coating: pIFN γ -I coating antibody was diluted by PBS to a final concentration of 10 μ g/mL. Adding 100 μ L/well, and coating overnight at 4 deg.C;

sealing: the following day the coating solution was poured and washed 5 times with sterile 1 XPBS 200. mu.l. And finally, drying the sterilized absorbent paper in a buckling manner. Adding diluted sealing solution (10% fetal calf serum culture medium) into 200 μ L/hole, sealing at 37 deg.C for 1 hr;

fifthly, pouring the confining liquid without washing, adding 100 mu L of the counted cells in the step (1) into each well, diluting the polypeptide to the final concentration of 100 mu g/mL by DMEM of 10% fetal calf serum, and adding 100 mu L into each well. Setting negative control, positive control and blank control at the same time, incubating at 37 ℃ for 12-48h, referring to Table 7, polypeptide is abbreviated as head and tail letters plus length, for example QVVFHAGSLYNWFSV is abbreviated as QV-15;

table 7 test sample set-up reference

Sixthly, removing cells, washing 5 times by 200 mu L/hole PBS, and diluting P2C11-biotin to 0.5 mu g/mL by 0.5 percent of PBS solution of fetal calf serum. Adding 100 mu L/hole and incubating for 2h at room temperature;

seventhly, PBS is washed for five times, and HRP is diluted to the PBS with the final concentration of 0.5 percent fetal bovine serum. For AEC stain, HRP was diluted 1:100, 100ul was added to each well, and allowed to stand at room temperature for 1 h. Washing for 5 times;

adding 100 mul AEC until some AEC appears, stopping the reaction by water, naturally drying in the air, and storing in the dark.

The ELISPOT results are counted and counted to obtain a more obvious phenomenon, as shown in fig. 8, 9 and 10, which are ELISPOT detection results at-3 days, 14 days and 28 days respectively, the result at-3 days is almost non-reactive, and a negative-positive control is established, which indicates that no non-specific reaction interference exists in the experimental process. Of the 18 polypeptides that caused significant secretion of IFN γ interferon at both

days

14 and 28, the best effect of TY21 stimulation of IFN γ was followed by YF19, DF13, II 14.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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<213> Artificial Sequence (Artificial Sequence)

<400> 10

His Tyr Met Asn Cys Ser Leu Pro Thr Tyr Phe

1 5 10

<210> 11

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Ser Ala Met Leu Ala Cys Val Arg Phe Tyr Asn Met Asp Asn Leu Phe

1 5 10 15

Phe Cys Ile

<210> 12

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Glu Ala Ile Ser Tyr Val Tyr Gln His Phe Lys Tyr Leu Asn Thr Trp

1 5 10 15

Trp Leu Ile

<210> 13

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Ile Gln Asp Tyr Ser Tyr Ser Ala Ile Tyr Tyr Cys Phe Ile

1 5 10

<210> 14

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

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

20

<210> 15

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Ile Leu Asp Asp Ile Ser Phe Ser Glu Met Leu Thr Arg Tyr Trp Tyr

1 5 10 15

Ser Met

<210> 16

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Tyr Asn Leu Thr Glu Ala Ile Gln Tyr Phe Tyr Gln Arg Tyr

1 5 10

<210> 17

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Asp Leu Thr Met Tyr Ser Leu Gly Tyr Ile Phe Leu Phe

1 5 10

<210> 18

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Ile Asn Met Arg His His Thr Ser Tyr Thr Glu Asn Ser Val Leu Thr

1 5 10 15

Tyr

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

atggggcctg gagccctctt cc 22

<210> 20

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tcacactcta ggatcctggg tgagggacac 30

<210> 21

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gtcgcgcgtc ccccgaaggt tcagg 25

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ttagtggtct cgatcccact taact 25

Claims

1. The African swine fever CTL epitope polypeptide is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID No. 3.

2. The African swine fever CTL epitope polypeptide coding gene of claim 1.

3. The biomaterial containing the African swine fever CTL epitope polypeptide of claim 1, wherein the biomaterial is a recombinant expression vector, an expression cassette, a recombinant bacterium or a host cell.

4. A medicament comprising the African swine fever CTL epitope polypeptide of claim 1.

5. The medicament according to claim 4, characterized in that it is a polypeptide vaccine.

6. The medicament of claim 5, wherein the polypeptide vaccine further comprises an adjuvant.

7. The medicament of claim 6, wherein the adjuvant is MONTANIDE ISA 50V2, MONTANIDE ISA 61VG, and/or MONTANIDE ISA 201 VG.

8. Use of the African swine fever CTL epitope polypeptide or its coding gene of claim 1 or the biomaterial of claim 3 in preparation of a medicament for improving CD8 production in vivo⁺T cells or drugs for increasing the number of cells secreting IFN-gamma.

9. Use of the African swine fever CTL epitope polypeptide of claim 1 or encoding gene thereof or the biomaterial of claim 3 in the preparation of a medicament for preventing or treating African swine fever virus infection.

10. Use of the African swine fever CTL epitope polypeptide or the coding gene thereof of claim 1 or the biomaterial of claim 3 in preparation of a reagent or a kit for detecting African swine fever.