CN1634978A - SARS virus HLA-A2 restricted epitope polypeptide and its application - Google Patents

Abstract

The invention discloses a restrictive epitope polypeptide Ssp-1 of a SARS virus HLA-A2, a complex containing said epitope, antigen presenting cells and application method of the specific immunity effector cells corresponding to said epitope in SARS treatment and prevention.

Description

SARS virus HLA-A2 restricted epitope polypeptide and its application

technical field

The present invention relates to the fields of biology and medicine, more specifically to a SARS virus HLA-A2 restricted epitope polypeptide Ssp-1, a complex containing the epitope, an antigen-presenting cell and specificity for the epitope The application method of immune effector cells in the treatment and prevention of SARS.

Background technique

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease. Since mid-November 2002, it first appeared in some parts of Guangdong Province, China, and then quickly spread to 33 countries and regions on five continents, including Hong Kong Special Administrative Region and Taiwan, Vietnam, Singapore, Canada, and Germany. Local transmission occurred in 6 countries. Due to the rapid spread of the disease, the general susceptibility of the population and the high mortality rate, it has become a global epidemic that seriously endangers human health and life safety. The clinical features of the disease are: fever, dry cough, dyspnea, lymphopenia, and rapidly progressive changes in lung imaging. Some patients may die due to respiratory failure caused by severe respiratory distress, and the upper respiratory symptoms are not obvious. Have diarrhea symptoms. Antibiotics used for traditional treatment are ineffective.

Under the coordination of the World Health Organization (WHO), 13 laboratories in 9 countries around the world worked together to isolate the SARS virus and complete the whole genome sequencing in only 3 weeks. On April 16, 2003, on the basis of the isolation of the pathogen and the completion of genome sequencing, the WHO officially announced a new coronavirus that had not been reported before as the pathogen that caused SARS, and named it SARS coronavirus (SARS Coronavirus, SARS-CoV) ).

SARS coronavirus is a positive-strand RNA virus with a genome length of about 30kb and 11 open reading frames (ORFs), in which the genes for non-structural proteins are located at the 5' end and the structural proteins are located at the 3' end, including the spine protein (spike), surface protein (E), membrane protein (membrane) and nucleocapid (nucleocapid). Among them, the S protein plays a key role in the process of virus binding to host cell surface receptors and mediating membrane fusion into cells, and is also the main antigenic protein of coronaviruses.

The diameter of SARS-CoV is 80-220nm, and it has an envelope. There are 2-3 kinds of glycoproteins on the envelope, and one of the long envelope glycoproteins has a protrusion length of 20nm. Each protrusion is composed of three S proteins connected by non-covalent bonds. The S protein trimeric protrusion has various functions, which are responsible for binding sensitive cell receptors, inducing fusion between the viral envelope and cell membrane and cells , stimulate the body to produce neutralizing antibodies and mediate cellular immune responses. The S glycoprotein has four domains: a short C-terminus constituting the inner or cytoplasmic region of the envelope: a transmembrane region and two outer domains (referred to as S1 and S2, respectively). Its cytoplasmic domain is rich in conserved cysteine residues and is involved in the formation of complex tertiary structures with the S protein, as well as the assembly of protrusions or interactions with other viral proteins. The transmembrane region is connected with S2 protein and contains a highly conserved sequence KWPWYVWL. Usually, the cytoplasmic domain or the inner region of the envelope and the transmembrane region account for a small proportion in the S protein, and most of them are the extracellular domain composed of S1 and S2. There is a group of basic amino acid residues between S1 and S2, which constitute the sensitive motif of trypsin cleavage. The cleavage of S protein is necessary to induce membrane fusion activity, but S2 and S1 produced by protease cleavage do not dissociate, while Being held together by non-covalent bonds, virions that remove S1 tend to lose their infectivity, but there are exceptions.

Therefore, if the extracellular region of the S protein is used as a target to immunize the body, the body will produce a specific immune response against the virus S protein, so that the body can effectively clear the virus and provide a new source of protection for the prevention and treatment of SARS-CoV. measure.

Synthetic peptide vaccine is a new vaccine developed with the progress of molecular biology and immunology in recent years. It can induce the body to produce a specific immune response, but its side effects are mild and its safety is good. It is the current vaccine research. A new direction, widely used with anti-tumor and anti-viral immunotherapy. At present, a variety of vaccines based on epitope peptides have entered clinical research or entered the market.

Cellular immunity plays a key role in antitumor and antiviral immunity. The antigen recognized by T cells is a peptide bound to MHC class I or class II molecules on the cell surface, and its length is about 8-12 amino acids. As the main effector cell CTLs that kill tumor cells, they recognize endogenous peptides combined with MHC class I molecules. Evidence has shown that synthetic peptides can directly bind to MHC class I molecules without processing and processing by APCs, and they have the same efficacy as natural endogenous peptides in activating the immune system. Peptide vaccine has become a new strategy against malignant tumors, and it is also the most studied tumor therapeutic vaccine. The following five types have been studied more:

(1) gp 100: Peptides G9154 (KTWGQYWQV) and G9209 (ITDQVPPFSV) derived from gp 100 can induce antigen-specific CTLs. Using it to sensitize peripheral blood lymphocytes (PBL) from HLA-A2+ melanoma patients can significantly enhance the ability to induce specific CTLs: the induced CTLs can recognize unmodified Gg100.

(2) CEA: Zaremba et al. reported that the CEA peptide CAP1-6D can not only sensitize CEA-specific CTLs in vitro, but its potency is 100-1000 times that of CAPI, and can induce CEA-specific CTLs in vivo (CAP1 cannot); And the sensitized CTLS can also recognize CAP1. More importantly, these CTLs were able to lyse cognate CEA-expressing human tumors.

(3) MAGE-2: MAGE-2 is widely present in various tumors such as melanoma, laryngeal cancer, lung cancer, and sarcoma; it does not exist in normal tissues except testis. In MAGE-2, three polypeptides have been found to form stable complexes with MHC class I molecules at 37°C, and at least two of them can be processed and presented by HLA-A 0201, making them new candidates for polypeptide vaccine research.

(4) P21 ^WAFI : Fusion of P21 ^WAFI peptide with endogenous peptide can inhibit tumor growth.

(5) HER2/neu: Peptides derived from HER2/neu can sensitize specific CTLs in vitro, induce specific CTLs in mice, and inhibit tumor growth. In addition, HPV peptide vaccines have also entered clinical research.

HLA-A2 is a kind of MHC-I molecule with a relatively high distribution in the Chinese population, with a positive rate of 40-60%, accounting for the first place among all subgroups of MHC-I molecules, and is the first choice in vaccine design related molecules.

With the in-depth understanding of the interaction between MHC class I molecules and peptide epitope molecules, scientists have established a relatively mature epitope identification technology route, the main steps are as follows: ① According to the interaction between epitopes and MHC class I molecules Features, predict MHC class I molecule-restricted CTL epitopes, and use computer molecular simulation technology to further screen the predicted epitopes; ②Determine the binding force between epitopes and MHC class I molecules; ③Use in vitro cytotoxicity analysis or immune tumor-bearing transgenic small Identify whether the peptide epitope can induce CTL in mice, and seek the best dominant epitope. Based on this technical route, a variety of HLA-A2.1 restricted CTL epitopes have been identified, some of which have shown good clinical efficacy. T2 cells are one of the tool cells used to determine the binding ability of epitopes to HLA-A2.1 molecules. It is an HLA-A2.1 cell line with defects in antigen presentation transporters. The HLA-A2.1 molecule of the endogenous antigen molecule can be used to determine the affinity between the HLA-A2.1 molecule and the target epitope by using its binding degree with the target peptide.

Since there is currently no effective method for preventing and treating SARS, there is an urgent need in this field to develop new antigens that can be used for preventing and treating SARS, especially synthetic peptide vaccines with high safety.

Contents of the invention

The purpose of the present invention is to provide a synthetic peptide with high safety and high immunogenicity and its application.

In the first aspect of the present invention, an HLA-A2 restricted epitope polypeptide is provided, and the polypeptide is selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of SEQ ID NO: 13;

(b) The amino acid sequence of SEQ ID NO: 13 is formed by substitution, deletion or addition of one or more amino acid residues, and a polypeptide derived from (a) having the activity of inducing cytotoxic T lymphocyte killing.

In another preferred embodiment, the length of the polypeptide is 8-20 amino acids, more preferably, the length is 8-12 amino acids.

In another preferred embodiment, the polypeptide has the amino acid sequence of SEQ ID NO: 13.

The present invention also provides a recombinant protein (such as a fusion protein, etc.), which contains the above-mentioned HLA-A2 restricted epitope polypeptide of the present invention.

In the second aspect of the present invention, an isolated nucleic acid is provided, which encodes the above-mentioned HLA-A2 restricted epitope polypeptide of the present invention.

In the third aspect of the present invention, an antigen-presenting cell is provided, which is sensitized by the above-mentioned HLA-A2 restricted epitope polypeptide of the present invention.

In another preferred example, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, B cells, fibroblasts, and endothelial cells.

In the fourth aspect of the present invention, a pharmaceutical composition is provided, which contains:

(a) a safe and effective amount of the above-mentioned HLA-A2 restricted epitope polypeptide of the present invention or the above-mentioned sensitized antigen-presenting cells of the present invention; and

(b) A pharmaceutically acceptable carrier or excipient.

In the fifth aspect of the present invention, the use of the above-mentioned HLA-A2 restricted epitope polypeptide or sensitized antigen-presenting cells of the present invention is provided, and they are used to prepare drugs for preventing and treating SARS.

In the sixth aspect of the present invention, specific antibodies against the above-mentioned epitope polypeptides of the present invention are also provided.

Description of drawings

Figure 1 shows that Ssp-1-sensitized dendritic cells induce the killing activity of cytotoxic T lymphocytes in HLA-A2.1/K ^b transgenic mice in vivo.

Figure 2 shows the killing activity of normal human peripheral blood cytotoxic T lymphocytes induced by dendritic cells sensitized by SARS HLA-A2-restricted polypeptide Ssp-1 in vitro.

Detailed ways

After in-depth and extensive research, the inventors selectively synthesized a number of SARS antigen peptides that may combine with HLA-A*0201 and induce the body to produce CTL on the basis of computer simulation analysis of the SARS coronavirus S protein. Through the T2 peptide binding experiment, the epitope peptide Ssp-1 with strong affinity to HLA-A*0201 was screened out, and its ^{immunogenicity} was evaluated. Specific, HLA-A*0201-restricted cytotoxic T lymphocytes were induced in the peripheral blood of mice and healthy humans, and it is an immunogenic polypeptide naturally processed and presented by cells. The present invention has been accomplished on this basis.

As used herein, "polypeptide of the present invention", "SARS-specific polypeptide", and "HLA-A2 restricted epitope polypeptide" can be used interchangeably, referring to the polypeptide Ssp-1 shown in the amino acid sequence SEQ ID NO: 13. In addition, variant forms of the sequence of SEQ ID NO: 13 having the same function as Ssp-1 are also included. These variations include (but are not limited to): one or more (usually 1-20, preferably 1-10, more preferably 1-5, and most preferably 1-3) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active derivatives of Ssp-1.

The polypeptide of the present invention can be artificially synthesized by conventional methods, and can also be produced by recombinant methods.

The polypeptide of the present invention can be directly used to detect anti-SARS antibodies, and can also be used to couple with proteins with molecular weights such as BSA to form polypeptide conjugates. Generally, the conjugate is composed of a polypeptide, a cross-linking agent, and BSA, wherein the cross-linking agent is preferably glutaraldehyde (when coupled to the N-terminus of the polypeptide of the present invention), EDAC (when combined with the present invention) when the C-terminus of the inventive polypeptide is coupled).

The conjugate of the present invention can be used to immunize animals such as rabbits and mice, so as to obtain antibodies against the polypeptide of the present invention ("the antibody of the present invention"). The antibodies of the present invention include specific polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies. Here, "specificity" means that the antibody can bind to SARS virus, gene product or fragment.

The present invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments (such as Fab' or (Fab) ₂ fragments), antibody heavy chains, antibody light chains, chimeric antibodies, humanized antibodies wait.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified polypeptides or conjugates of the invention can be administered to animals to induce polyclonal antibody production. For monoclonal antibodies, hybridoma technology can be used to prepare (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J.Immunol.6: 511, 1976; Kohler et al., Eur.J.Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).

The polypeptide, conjugate and antibody of the present invention can be used to detect whether there is SARS virus or anti-SARS virus antibody in a sample, so as to be one of the effective indicators for early detection of SARS.

In addition to being used for detection, the SARS-specific polypeptide of the present invention can also compete with the virus for receptors in vivo, prevent the fusion of the virus and the membrane, and inhibit the virus from infecting normal cells. In addition, the polypeptides and conjugates of the present invention can also be used to prepare therapeutic pharmaceutical compositions or preventive vaccine compositions.

Therefore, in another aspect, the present invention also provides a composition, which contains (a) a safe and effective amount of the polypeptide of the present invention, a conjugate or a composition thereof; and (b) a pharmaceutically acceptable carrier or excipient agent. The amount of the polypeptide of the present invention is usually 10 μg-100 mg/dose, preferably 100-1000 μg/dose.

As used herein, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or exhibits a detectable therapeutic or preventive effect. The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agents and/or combination of therapeutic agents chosen for administration. Therefore, it is not useful to prespecify an exact effective amount. However, the effective amount can be determined by routine experimentation, within the judgment of the clinician, for a given situation.

For the purposes of the present invention, an effective dosage is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg body weight of the polypeptide of the present invention administered to an individual. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. The term refers to pharmaceutical carriers which do not, by themselves, induce the production of antibodies deleterious to the individual receiving the composition and which are not unduly toxic upon administration. These vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

Pharmaceutically acceptable carriers in therapeutic compositions can contain liquids, such as water, saline, glycerol and ethanol. In addition, there may also be auxiliary substances in these carriers, such as wetting agents or emulsifying agents, pH buffering substances and the like. In addition, immune adjuvants may also be included in the immune composition.

Typically, therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution, or suspension, in liquid carriers prior to injection can also be prepared.

Once formulated, the compositions of the invention can be administered directly to a subject. The subject to be prevented or treated can be an animal; especially a human.

The therapeutic or preventive pharmaceutical composition (including vaccines) containing the polypeptide of the present invention can be applied orally, subcutaneously, intradermally, or intravenously. The therapeutic dosage regimen can be a single dose regimen or a multiple dose regimen.

The main advantages of the present invention are: the Ssp-1 epitope peptide is a cytotoxic T lymphocyte epitope peptide derived from the SARS coronavirus S protein and HLA-A2 restricted, which can safely and effectively cause an immune response against SARS, and not only The research on the pathogenesis of SARS, and the development of its vaccine and therapeutic preparations are of great significance.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in the manufacture conditions recommended by the manufacturer.

Example 1, Screening of HLA-A*0201 High Affinity Peptides

First, on the basis of computer simulation analysis of the S protein of SARS coronavirus, a number of SARS antigen peptides that may bind to HLA-A*0201 and induce the body to produce CTL were prepared by using the conventional peptide total artificial synthesis method (Table 1 ).

Next, peptide binding assays were used to screen for epitope peptides with high affinity to HLA-A*0201. The method is as follows: first collect T2 cells, wash three times with serum-free 1640, adjust the cell concentration to 2×10 ⁵ /ml, spread in 24-well plate, 0.5ml/well. Then co-incubate with 50uM candidate polypeptide and 2.5μg/ml β2 microglobulin at 37°C, 5% CO ₂ incubator for 18h. The incubated cells were washed three times with ice-cold PBS, FITC-labeled HLA-A2-specific mAb BB7.2 (Sterotec Ltd, Oxford, UK) was added, kept in ice bath for 45 min, and the average fluorescence intensity was detected by flow cytometry after washing with PBS . The positive known peptide CEA HLA-A2 restricted epitope polypeptide CAP-1 was used as a positive control, and the simple T2 cells stimulated without peptide were used as a background control.

Result judgment: Immunofluorescence method to detect the combination of peptides and HLA-A*0201 molecules is based on the fact that the combination of exogenous peptides and MHC-I molecules on the surface of T2 cells can increase the expression of MHC-I molecules on the surface , the more stable the combination of the two is, the more the expression of MHC-I molecules can be detected, and the average fluorescence intensity is used as the detection index. The results were measured by fluorescence coefficient (FI). A polypeptide with FI > 1 is considered a high affinity epitope.

The results are shown in Table 1.

Table 1. T2-HLA-A*0201-binding affinity for SARS-spike protein-derived peptides SEQ ID NO: starting point sequence score* FI ^ 1 1042 VVFLHVTYV 484.238 0.5 2 982 RLQSLQTYV 382.536 0.3 3 958 VLNDIL SRL 342.461 0.6 4 897 VLYENQKQI 232.693 0.4 5 1174 NLNES LIDL 201.447 1.1 6 734 LLLQYGSFC 171.868 0.4 7 411 KLPDDFMGC 135.453 0.4 8 787 ILPDPLKPT 119.463 0.3 9 151 MIFDNAFNC 102.176 0.4 10 2 FIFLLFLTL 94.987 -0.1 11 596 VLYQDVNCT 93.239 0.4 12 1096 IITTDNTFV 89.418 0.3 13 1167 RLNEVAKNL 87.586 1.7 14 846 LLTDDMIAA 79.642 0.4 15 735 LLQYGSFCT 73.477 0.2 16 1214 ILLCCMTSC 71.872 0.3 17 803 LLFNKVTLA 71.872 0.3 18 106 TMNNKSQSV 50.232 0.2 19 940 ALNTLVKQL 49.134 0.3 20 131 ELCDNPFFA 48.732 0.4

An HLA-A2 high-affinity epitope Ssp-1 was screened out from the spike protein S2 of SARS coronavirus, and its sequence is RLNEVAKNL (SEQ ID NO: 13).

Example 2, Induction of SARS HLA-A2-restricted polypeptide-specific cytotoxic T lymphocytes in HLA-A2.1/K ^b transgenic mice

Dendritic cells (DC) derived from bone marrow of HLA-A2.1/K ^b transgenic mice were prepared according to conventional methods. Collect DCs cultured to day 7, adjust the cell concentration to 1×106/ml, add Ssp-1 (final concentration 10 μg/ml) and β2 microglobulin (final concentration 3 μg/ml), 37°C, 5% CO ₂ Incubate for 3 h in the incubator. The peptide-sensitized DCs were collected, washed three times with PBS, and the cell concentration was adjusted to 1×10 ⁶ /0.4ml to immunize mice. Each mouse was intraperitoneally injected with 1×10 ⁶ /0.4ml, and immunized three times with an interval of one week. Seven days after the last immunization, the mouse spleen was removed aseptically, and the red blood cells were dissolved to make a single cell suspension. Splenocyte suspension (5×10 ⁶ /ml) and peptide-sensitized irradiated autologous dendritic cells were cultured in RPMI 1640 complete medium at a ratio of 10:1. After 6 days of culture, the effector cells were collected, and their specific killing activity was detected by a standard 4-hour ⁵¹ Cr release assay. Use Ssp-1, CAP-1 loaded T2 cells and unloaded T2 cells as target cells respectively, add ⁵¹ Cr (100 μCi/10 ⁶ cells), place in a water bath at 37 degrees Celsius for 90 minutes, and mix gently every 15 minutes Homogenize once, wash 3 times, thoroughly wash away residual Na ₂ ⁵¹ CrO ₄ , use complete medium to adjust the cell concentration of marked target cells to 1×10 ⁵ /ml, add to 96-well round bottom plate, 100 μl per well. Add effector cells according to three different effector-target ratios of 50:1, 25:1, and 12.5:1, incubate at 37°C for 4 hours, collect 100 μl of supernatant from each well, and detect the cpm value with a gamma counter. For each well of the maximum release group, individual target cells were added with 100 μl of 1% SDS; for the spontaneous release wells, individual target cells were added with 100 μl of complete medium.

The specific lysis rate or killing rate was calculated according to the following formula: specific lysis rate or killing rate (%)=(cpm of experimental group-cpm of spontaneous release group)/(cpm of maximum release group-cpm of spontaneous release group).

The results are shown in Figure 1 , indicating that Ssp-1-sensitized dendritic cells can significantly induce the killing activity of cytotoxic T lymphocytes in HLA-A2.1/K ^b transgenic mice in vivo.

Example 3. Induction of Ssp-1-specific cytotoxic T lymphocytes from healthy human peripheral blood in vitro

Peripheral blood mononuclear cells from healthy people were isolated and cultured at 37 degrees Celsius for 2 hours. The adherent cells were used to cultivate dendritic cells, and the non-adherent cells were suspended in RPMI 1640 medium with 5% fetal bovine serum and passed through a nylon hair column ( Incubate for 1 hour at 37°C). Purified T lymphocytes (2×10 ⁶ ) were co-cultured with Ssp-1-sensitized dendritic cells (2×10 ⁵ ) in a 24-well plate, and recombinant human IL-7 (10ng/ml; Peprotech Inc ), 24h later, recombinant human IL-10 (10ng/ml; Peprotech Inc) was added. Cultured T cells were stimulated once a week with Ssp-1-sensitized irradiated autologous dendritic cells for a costimulation of four times. 24 and 48 hours after each stimulation, recombinant human IL-10 (10 ng/ml) and recombinant human IL-2 (20 IU/ml) were added respectively. Seven days after the last stimulation, the effector cells were collected, and their specific killing activity was detected by 51Cr release assay as above. Target cells were divided into two groups, one group was T2 cells loaded with Ssp-1 and CAP-1 and T2 cells without loading (as shown in Figure 2A); the other group was SW480 (HLA- A2.1 ⁺ ) and HT29 (HLA-A2.1 ⁻ ) cells ( FIG. 2B ).

The results are shown in Figure 2, indicating that dendritic cells sensitized by the SARS HLA-A2-restricted polypeptide Ssp-1 induce the killing activity of normal human peripheral blood cytotoxic T lymphocytes in vitro.

Example 4

Antigen peptide immunization animals to prepare antibodies:

Take 1mg of Ssp-1 prepared in Example 1, and use a conventional method to couple with BSA at a ratio of 1:15, then take about 0.5ml of the conjugate (the content of the conjugate is 0.1mg and 0.5mg respectively), and after adding 1.5ml of PBS, Mixed with CFA (Complete Freund's Adjuvant). Rabbits and mice were immunized with antigen emulsified by adjuvant; each rabbit was injected with about 3ml of antigen, and mice were injected with 0.8ml/monkey. Three weeks after the initial immunization, take the same dose of polypeptide antigen and PBS, mix with 2ml IFA (incomplete Freund's adjuvant), emulsify the animals for booster immunization, and the injection dose remains unchanged. Animals were bled three weeks later and antiserum was collected. Use a BSA-coated column to remove antibodies against BSA. Select the antigen polypeptide with higher antibody titer to prepare monoclonal antibody.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Sequence Listing

<110> Institute of Immunology, Second Military Medical University

  Shanghai Haixin Biotechnology Co., Ltd.

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Claims (10)

1. A HLA-A2 restricted epitope polypeptide, characterized in that the polypeptide is selected from the group consisting of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 13; (b) The amino acid sequence of SEQ ID NO: 13 is formed by substitution, deletion or addition of one or more amino acid residues, and the polypeptide derived from (a) has the activity of inducing cytotoxic T lymphocyte killing. 2. The polypeptide according to claim 1, wherein the length of the polypeptide is 8-20 amino acids. 3. The polypeptide according to claim 1, wherein the length of the polypeptide is 8-12 amino acids. 4. The polypeptide of claim 1, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 13. 5. A recombinant protein, characterized in that it contains the HLA-A2 restricted epitope polypeptide according to claim 1. 6. An isolated nucleic acid, characterized in that it encodes the HLA-A2 restricted epitope polypeptide of claim 1. 7. An antigen-presenting cell, characterized in that the antigen-presenting cell is sensitized by the HLA-A2 restricted epitope polypeptide according to claim 1. 8. The antigen-presenting cell according to claim 7, wherein the antigen-presenting cell is selected from the group consisting of dendritic cells, macrophages, B cells, fibroblasts, and endothelial cells. 9. A pharmaceutical composition, characterized in that it contains: (a) a safe and effective amount of the HLA-A2 restricted epitope polypeptide of claim 1 or the antigen-presenting cell of claim 7; and (b) A pharmaceutically acceptable carrier or excipient. 10. The use of the HLA-A2 restricted epitope polypeptide according to claim 1 or the antigen-presenting cell according to claim 7, characterized in that it is used for the preparation of medicaments for the prevention and treatment of SARS.

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