A T cell antigen polypeptide and its application

Abstract

The present invention provides an antigenic polypeptide related to echinococcosis, a nucleic acid encoding the antigenic polypeptide and a vector containing the nucleic acid, and a cell containing the antigenic polypeptide or nucleic acid. At the same time, a echinococcosis vaccine is provided. This application uses the echinococcosis recombinant protein P29 as the research basis to screen and optimize peptide epitope molecules with immune effects, which is of great value in diagnosing echinococcosis, screening drugs for treating or preventing echinococcosis, and providing decision support for clinical treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunobiology, and in particular relates to a T cell antigen polypeptide and its application in biomedicine. Background Art

[0002] Hydatid disease is a chronic infectious disease caused by parasitic larvae that infect humans or livestock, severely endangering their health. It is widespread in areas with developed animal husbandry worldwide. Research on vaccines for hydatid disease started relatively late and remains relatively underdeveloped. To date, no vaccine for hydatid disease has been successfully commercialized, either domestically or internationally. Existing studies have shown that the recombinant protein P29 (rEg.P29), a potential vaccine, exhibits protective effects against Echinococcus granulosus in sheep. Immunization of sheep with rEg.P29 resulted in 94.5% of sheep developing protective immunity, meaning they were protected from infection. Immunization of mice with rEg.P29 and subsequent intraperitoneal injection of protoscoleci resulted in 96.6% protective immunity, suggesting that rEg.P29 has the potential for translational application as a hydatid disease vaccine.

[0003] Judging from the research progress of molecular vaccines in recent years, vaccines are becoming more and more epitope-based and molecules are miniaturized, which can make the immune effect of the vaccine more specific and have fewer side effects.

[0004] Historically, live, attenuated or inactivated forms of microbial pathogens (viruses, bacteria, etc.) have been used to induce antigen-specific responses and protect the host from infection. Depending on the pathogen used, such vaccine formulations may contain dozens to hundreds of proteins. However, protective immunity often relies on a few carefully selected proteins in these formulations, most of which are dispensable for inducing protective immunity. Furthermore, these additional proteins may induce allergic and / or reactive reactions, necessitating their elimination from the vaccine formulation. This rationale has led to interest in subunit vaccines, which use a single or select few microbial proteins in vaccine formulations to induce protective immunity. An extension of this logic is that even a single protein contains hundreds of antigenic epitopes, all of which are dispensable; some may even be detrimental to the induction of protective immunity. This has sparked interest in peptide vaccines, which contain only epitopes that are capable of inducing positive, desirable T- and B-cell-mediated immune responses. The peptides used in these vaccines are immunogenic peptide molecules synthesized from 20-30 amino acid sequences that represent specific epitopes of an antigen. On the one hand, since epitopes are antigenic determinants within a larger protein, these peptides are believed to be sufficient to activate appropriate cellular and humoral responses while eliminating allergic and / or reactive responses. In addition, peptide vaccines can be used to induce broad immunity against multiple serological variants (sera) or strains of a given pathogen by forming multiple non-contiguous immunodominant epitopes and / or epitopes that are conserved between different sera / strains of the pathogen.

[0005] The non-patent document Epitope specificities and antibody responses to the EG95 hydatid vaccine (DJ WOOLLARD et al., Parasite Immunology, 1998: 20: 535–540) discloses an EG95 hydatid antigen;

[0006] The non-patent document Immunoprotection of recombinant Eg.P29 against Echinococcus granulosus in sheep (Hao Wang et al., Vet Res Commun (2016) 40:73–79) discloses Eg.P29 recombinant protein;

[0007] Patent document CN105343873A discloses a gene rEg.P29 molecular engineered vaccine against sheep echinococcosis infection;

[0008] None of the above documents discloses the epitoped and miniaturized immunogenic antigenic polypeptide (epitope peptide) in this application. Even if a vaccine against rEg.P29 is disclosed, it is a vaccine that expresses the entire recombinant protein, and the full-length protein is likely to produce immune side effects.

[0009] Therefore, providing a miniaturized antigen polypeptide with a certain degree of immunogenicity is an important basis for developing small peptide vaccines and effectively treating echinococcosis. Summary of the Invention

[0010] This application uses the vaccine molecule rEg.P29 as the research basis to screen and optimize antigenic polypeptides with specific sequences that have immune effects. The antigenic polypeptides can induce the activation of lymphocytes in rEg.P29-immunized mice and cause them to proliferate. Moreover, the freeze-dried peptide is chemically relatively stable, and the vaccine prepared using the antigenic polypeptide remains stable under conventional cold chain storage and transportation. More importantly, under normal circumstances, the effects of polypeptides in activating specific cells and promoting cell proliferation are weaker than those of intact proteins, but the antigenic polypeptides provided in this application are basically close to the effects of intact proteins.

[0011] The first aspect of the present invention provides an antigenic polypeptide, wherein the antigenic polypeptide is derived from a recombinant echinococcosis protein or a fragment thereof.

[0012] Preferably, the hydatid disease is caused by infection with Echinococcus multilocularis or Echinococcus granulosus.

[0013] Preferably, the antigenic polypeptide comprises an epitope on the echinococcosis recombinant protein.

[0014] Preferably, the antigenic polypeptide is derived from an epitope on the recombinant protein rEg.P29 of Echinococcus granulosus.

[0015] Further preferably, the antigen polypeptide is derived from a T cell antigen epitope on the recombinant protein rEg.P29 of Echinococcus granulosus.

[0016] In a specific embodiment of the present invention, the antigen polypeptide is selected from one or more T cell antigen epitopes of SEQ ID NO: 47.

[0017] The T cell antigen epitope is a peptide segment that can bind to TCR or antibody.

[0018] Preferably, the T cell antigen epitope may be a continuous or discontinuous amino acid sequence of SEQ ID NO: 47.

[0019] Preferably, the T cell antigen epitope may be composed of spatially adjacent amino acid residues on the recombinant protein consisting of SEQ ID NO: 47.

[0020] Preferably, the antigenic polypeptide contains at least one antigenic epitope.

[0021] Preferably, the antigen polypeptide comprises at least 8 consecutive amino acids in SEQ ID NO: 47. Further preferably, the antigen polypeptide is less than 238 aa in length.

[0022] In a specific embodiment of the present invention, the antigen polypeptide comprises 8-20 consecutive amino acids in SEQ ID NO: 47. Further preferably, the antigen polypeptide comprises 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 consecutive amino acids in SEQ ID NO: 47.

[0023] Preferably, the antigenic polypeptide comprises:

[0024] A) one or more amino acid sequences of SEQ ID NOs: 1-46;

[0025] B) having an amino acid sequence identity of 80% or greater with one or more of SEQ ID NOs: 1-46;

[0026] C) differs from one or more of SEQ ID NOs: 1-46 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid sequence; or

[0027] D) an amino acid sequence represented by one or more of SEQ ID NOs: 1-46, comprising substitution, deletion and / or insertion of one or more amino acid residues.

[0028] In a specific embodiment of the present invention, the antigen polypeptide comprises a T cell antigen epitope, preferably any one of SEQ ID NOs: 1-46.

[0029] In a specific embodiment of the present invention, the antigen polypeptide comprises two or more T cell antigen epitopes, preferably two or more of SEQ ID NOs: 1 to 46, wherein the two or more T cell antigen epitopes are directly linked or connected via a linker.

[0030] Preferably, the antigen polypeptide induces a specific T cell immune response, and the cytokines secreted by the specific T cells include: interferon gamma (IFN-γ), interleukin 2 (IL-2), and tumor necrosis factor alpha (TNF-α).

[0031] Preferably, the antigen polypeptide can simultaneously stimulate the production of CD4 + IFN-γ + and CD8 +IFN-γ + T cells.

[0032] Preferably, the antigen polypeptide induces and assists B cells to produce specific antibodies against echinococcosis.

[0033] The second aspect of the present invention provides a nucleic acid encoding the above-mentioned antigen polypeptide.

[0034] The third aspect of the present invention provides an expression vector comprising a nucleic acid encoding the above-mentioned antigen polypeptide.

[0035] Preferably, the expression vector is capable of expression in vivo, in vitro, or ex vivo. Further preferably, the expression vector is capable of replication, transcription, and translation in a host cell. Therefore, it may further comprise other conventional expression elements, such as a promoter, terminator, or restriction enzyme cleavage sites.

[0036] Preferably, the expression vector can be a prokaryotic expression vector, a eukaryotic expression vector or a viral expression vector, preferably a prokaryotic expression vector, such as an Escherichia coli series.

[0037] Preferably, the expression vector can be a plasmid, cosmid, phage or virus.

[0038] The fourth aspect of the present invention provides a cell comprising the above-mentioned antigen polypeptide, the above-mentioned nucleic acid or the above-mentioned expression vector.

[0039] In a fifth aspect, the present invention provides a detection kit for echinococcosis, which includes reagents for detecting the above-mentioned antigen polypeptide, the above-mentioned nucleic acid, the above-mentioned expression vector or the above-mentioned cell.

[0040] The sixth aspect of the present invention provides a vaccine, which comprises the above-mentioned antigen polypeptide, the above-mentioned nucleic acid, the above-mentioned expression vector or the above-mentioned cell, and an immunologically acceptable carrier.

[0041] Preferably, the immunologically acceptable carrier includes adjuvants, diluents, solubilizers, lubricants, suspending agents, transfection accelerators, excipients, fillers, adhesives, absorption promoters and / or synergists, etc.

[0042] Preferably, the adjuvant includes but is not limited to surfactants such as immunostimulatory complexes, Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FIA), CpG, aluminum salt adjuvants (aluminum hydroxide or aluminum phosphate), LPS analogs (such as monophosphoryl ester A), cell wall peptides, benzoquinone analogs, squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, cations, polycations (such as poly-L-glutamic acid (LGS)) or nanoparticles, GM-CSF, IL-17, IFNg, IL-15, IL-21, anti-PD1 / 2, lactoferrin, protamine, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-α, INF-γ, Lymphotoxin-α, hGH, MCP-1, MIP-1a, MIP-1p, IL-8, RANT ES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, CD40, CD40L, vascular growth factor, fibroblast growth factor, nerve growth factor, vascular endothelial growth factor, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP 1, TAP2 and functional fragments thereof.

[0043] In a specific embodiment of the present invention, the immunologically acceptable carrier includes an adjuvant, and the adjuvant is FCA or CpG adjuvant.

[0044] Preferably, the vaccines include peptide vaccines, protein vaccines, mRNA vaccines and DNA vaccines.

[0045] The seventh aspect of the present invention provides a method for preparing the above-mentioned vaccine, which comprises mixing the above-mentioned antigen polypeptide with an immunologically acceptable carrier.

[0046] Preferably, the immunologically acceptable carrier includes adjuvants, diluents, solubilizers, lubricants, suspending agents, transfection accelerators, excipients, fillers, adhesives, absorption promoters and / or synergists, etc.

[0047] Preferably, the immunologically acceptable carrier includes an adjuvant. Further preferably, the adjuvant includes but is not limited to surfactants such as immunostimulatory complexes, Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FIA), CpG, aluminum salt adjuvants (aluminum hydroxide or aluminum phosphate), LPS analogs (such as monophosphoryl ester A), cell wall peptides, benzoquinone analogs, squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, cations, polycations (such as poly-L-glutamic acid (LGS)) or nanoparticles, GM-CSF, IL-17, IFNg, IL-15, IL-21, anti-PD1 / 2, lactoferrin, protamine, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-α, INF-γ, Lymphotoxin-α, hGH, MCP-1, MIP-1a, MIP- 1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF,, CD40, CD40L, vascular growth factor, fibroblast growth factor, nerve growth factor, vascular endothelial growth factor, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof.

[0048] Preferably, the adjuvant is FCA or CpG adjuvant.

[0049] In an eighth aspect, the present invention provides a method for screening anti-echinococcosis antibodies, the screening method comprising mixing the object to be screened with the above-mentioned antigen polypeptide.

[0050] The antibody screening method is not a treatment method. It is used to screen neutralizing antibodies and test and compare the efficacy of antibodies to determine which antibodies are suitable as drugs and which are not, or to compare the sensitivity of different drugs to their efficacy. In other words, a therapeutic effect is not guaranteed, but only a possibility.

[0051] The ninth aspect of the present invention provides a method for inducing a specific immune response, comprising administering the aforementioned antigen polypeptide, the aforementioned nucleic acid, the aforementioned expression vector or the aforementioned vaccine to an individual.

[0052] Preferably, the specific immune response includes a T cell response and / or a B cell response.

[0053] Preferably, the above immune response is an immune response against echinococcosis, and further preferably, the above immune response is an immune response against the echinococcosis recombinant antigen P29.

[0054] Preferably, the cytokines that the T cell response prompts T cells to secrete include: interferon gamma (IFN-γ), interleukin 2 (IL-2), and tumor necrosis factor alpha (TNF-α).

[0055] Preferably, the specific immune response can simultaneously produce CD4 + IFN-γ + and CD8 + IFN-γ + T cells.

[0056] The tenth aspect of the present invention provides a pMHC molecule, wherein the pMHC molecule comprises MHC and the above-mentioned antigen polypeptide.

[0057] The eleventh aspect of the present invention provides the use of the above-mentioned antigen polypeptide, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned cell, the above-mentioned vaccine, the above-mentioned pMHC molecule or the above-mentioned method in the preparation of products for preventing and / or treating echinococcosis.

[0058] Preferably, the products include but are not limited to vaccines, antibodies, drugs, and kits; preferably, the products are vaccines and / or antibodies.

[0059] The twelfth aspect of the present invention provides the use of the above-mentioned antigen polypeptide, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned cell, the above-mentioned kit, the above-mentioned pMHC molecule or the above-mentioned vaccine in preventing and / or treating echinococcosis.

[0060] The thirteenth aspect of the present invention provides an antibody, which can bind to the antigen polypeptide of the present invention.

[0061] The antigen polypeptides of the present invention can also be used for antibody identification using immunological methods, such as microcolumn gel assays, precipitation reactions, agglutination tests, complement fixation tests, labeled immunoassays (e.g., enzyme-linked immunosorbent assays, radioimmunoassays, fluorescence immunoassays, luminescence immunoassays, etc.), immunoblotting, and rapid assays (e.g., rapid dot immunobinding assays, liquid phase chip assays, etc.).

[0062] The "plurality" mentioned in the present invention means more than 2, including but not limited to 2-50, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.

[0063] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-associated signs, symptoms, conditions, or disorders.

[0064] The term "prevention" used in the present invention refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in the body.

[0065] The "nucleic acid" of the present invention includes natural or modified ribonucleotide sequences and deoxyribonucleotide sequences, preferably DNA, cDNA, pre-mRNA, mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, and tRNA.

[0066] The "individual" described in the present invention can be a human or a non-human animal, and the non-human animal can be a non-human mammal such as a mouse, cow, sheep, rabbit, pig, monkey, etc.

[0067] The term "and / or" as used herein includes all combinations of the items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A," "A and B," and "B." For another example, "A, B, and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0068] The terms "comprising" or "including" as used in the present invention are open-ended. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0069] The "homology" mentioned in the present invention refers to the fact that when using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the sequences used have (but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 112%, %, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% homology.

[0070] Beneficial effects:

[0071] The shortest and most effective rEg.P29 T cell epitope is provided as the core sequence of the rEg.P29 antigen polypeptide, which contains only 15aa of amino acids and can effectively stimulate cytokine production, promote T cell activation and proliferation, and simultaneously stimulate the production of CD4 + IFN-γ + and CD8 + IFN-γ + T cells.

[0072] The above merely summarizes some aspects of the present invention and is not and should not be considered to limit the present invention in any aspect.

[0073] All patents and publications mentioned in this specification are incorporated herein by reference as a whole. Those skilled in the art will recognize that certain changes can be made to the present invention without departing from the concept or scope of the present invention.

[0074] The following examples further illustrate the present invention and are not to be construed as limiting the scope of the invention or the specific methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0076] Figure 1 : SDS-PAGE analysis of rEg.P29 expression and purification, where M is a marker, lane 1 is E. coli containing pET28a before IPTG induction; lane 2 is E. coli containing pET28a-P29 after IPTG induction for 6 h; lane 3 is rEg.P29 purified by his affinity chromatography;

[0077] Figure 2 : Mouse immunization experimental plan, each group of mice was subcutaneously injected with rEg.P29+FCS, rEg.P29+CpG, or PBS, where W represents week, -2 and -1 represent three weeks and two weeks before sample collection, respectively;

[0078] Figure 3 : Specific cytokine detection results in spleen lymphocytes of mice immunized with rEg.P29, where * represents significant difference and the number represents the degree of significant difference. Splenic lymphocytes from mice in the PBS, rEg.P29+FCA and rEg.P29+CpG groups were stimulated with rEg.P29 for three days and the culture supernatants were collected. Cytokine release results;

[0079] Figure 4 : Schematic diagram of the design of the T cell antigen peptide of rEg.P29;

[0080] Figure 5 : Screening results of 45 T cell epitope mixed peptides. Spleen lymphocytes from mice in the PBS, rEg.P29+FCA, and rEg.P29+CpG groups were stimulated with T cell epitope mixed peptides or rEg.P29 for three days, and the culture supernatants were collected. Cytokine release results;

[0081] Figure 6 : The cytokine release results of spleen lymphocytes from mice in the rEg.P29+FCA immunization, rEg.P29+CpG immunization, or PBS groups were stimulated with ID15, ID16, ID17, ID18, ID19, and rEg.P29, respectively. ns represents non-significant difference, * represents significant difference, and the number represents the degree of significant difference;

[0082] Figure 7 : T cell antigen peptide and rEg.P29 stimulated the release of cytokines from spleen lymphocytes of mice in rEg.P29+FCA immunization, rEg.P29+CpG immunization or PBS groups, where ns represents non-significant difference, * represents significant difference, and the number represents the degree of significant difference;

[0083] Figure 8: T cell antigen peptide or rEg.P29 stimulated splenic lymphocytes of mice in the rEg.P29+FCA immunization, rEg.P29+CpG immunization, or PBS groups, and ELISPOT assayed the number of cells producing specific IFN-γ and IL-4. A and C are the results of cells producing specific IFN-γ or specific IL-4, respectively, and B and D correspond to the specific cell number statistics in A and C, respectively. ns represents non-significant difference, * represents significant difference, and the number represents the degree of significant difference.

[0084] Figure 9 : Schematic diagram of flow cytometry detection;

[0085] Figure 10 : The proportion of IFN-γ-producing spleen lymphocytes in mice in the rEg.P29+FCA, rEg.P29+CpG, or PBS groups stimulated with T cell antigen peptide or rEg.P29, where ns represents non-significant difference, * represents significant difference, and the number represents the degree of significant difference;

[0086] Figure 11 : Results of spleen lymphocyte activation induced by rEg.P29 stimulation in mice immunized with rEg.P29+FCA, mice immunized with rEg.P29+CpG or mice in the PBS group, where * represents significant difference and the number represents the degree of significant difference;

[0087] Figure 12 :rEg.P29 86-100 Stimulation-induced spleen lymphocyte activation results of rEg.P29+FCA immunized mice, rEg.P29+CpG immunized mice or PBS group mice, where * represents significant difference, and the number represents the degree of significant difference;

[0088] Figure 13 : The results of rEg.P29-induced spleen lymphocyte proliferation in rEg.P29+FCA-immunized mice, rEg.P29+CpG-immunized mice or PBS-treated mice, where * represents a significant difference and the number represents the degree of significant difference;

[0089] Figure 14 :rEg.P29 86-100 The results of stimulation-induced spleen lymphocyte proliferation in rEg.P29+FCA-immunized mice, rEg.P29+CpG-immunized mice, or PBS-group mice, where * represents significant difference, and the number represents the degree of significant difference;

[0090] Figure 15 : Schematic diagram of reagent dilution gradient. DETAILED DESCRIPTION

[0091] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0092] Example 1 Expression and purification of rEg.P29

[0093] rEg.P29 open reading frame amino acid sequence (SEQ ID NO: 47)

[0094] MSGFDVTKTFNRFTQRAGELVNKNEKTSYPTRTSDLIHEIDQMKAWISKIITATEEFVDINIASKVADAFQKNKEKITTTDKLGTALEQVASQSEKAAPQLSKMLTEASDVHQRMATAR KNFNSEVNTTFIEDLKNFLNTTLSEAQKAKTKLEEVRLDSDKTKLKNAKTAEQKAKWEAEVRKDESDFDRVHQESLTIFEKTCKEFDGLSVQLLDLIRAEKNYYEACAKECSMMLGE

[0095] Take out the BL21 strain containing the rEg.P29 / pET28a recombinant plasmid and the empty plasmid pET28a stored in this laboratory, pick the strain with an inoculation loop on the clean bench, and inoculate it into LB solid medium plate culture dishes containing kanamycin (Kana) at a final concentration of 50ug / mL. Place it in a 37℃ incubator overnight. Observe the growth of the colonies the next day, pick a single monoclonal colony, and inoculate the recombinant bacteria into LB liquid medium containing kanamycin (Kana) at a final concentration of 50ug / mL. Place it in a 37℃ shaker overnight at 200rpm until a distinct yellow color appears at OD600nm wavelength = 0.6. Then, inoculate it into LB liquid medium at a ratio of 1:100 to expand the culture. After culturing at 37℃ for 6h, OD = 0.6, add IPTG to a final concentration of 1mmol / L, and induce it in a 37℃ shaker for 6h. Collect the bacteria and detect the expression of the target protein by 12% SDS-PAGE. The induced expression bacterial solution was placed in an ultracentrifuge and centrifuged at 4°C, 8000 rpm / min for 10 mins to collect the bacterial cells for later use.

[0096] Purification of rEg.P29: After induction expression, the protein was found to be a fusion protein. After purification, the recombinant protein rEg.P29 with high purity was obtained. The target protein could be detected at around 31KD by SDS-PAGE ( Figure 1 The egg concentration was determined by BCA assay and adjusted to 1 mg / mL for storage. The endotoxin content was 0.356 EU / mL.

[0097] Example 2 Immunization of mice with rEg.P29 induces the production of specific cytokines

[0098] Purified rEg.P29 (1 mg / mL) was mixed with Freund's adjuvant (the volume ratio of rEg.P29 to Freund's adjuvant was 1:5) or CpG adjuvant (the volume ratio of rEg.P29 to CpG adjuvant was 1:2) to a total volume of 100 μl. The remainder was made up with PBS and immunized subcutaneously at three points in the abdomen. A booster was performed every 7 days (complete Freund's adjuvant was used for the first immunization and incomplete Freund's adjuvant was used for the booster immunization). The control group was injected with PBS 100 μL / head in the same way. The immunization schedule was as follows: Figure 2 shown.

[0099] (1) Isolation of mouse spleen lymphocytes

[0100] Excess tissue surrounding the spleen was separated, and the whole spleen was removed and placed in a 15ml centrifuge tube filled with Hank's solution on ice. Cells were first washed twice with Hank's solution, then gently ground through a 70μm filter using the plunger of an autoclaved 5ml syringe, while adding Hank's solution for rinsing. The filtrate was centrifuged and the supernatant discarded. The cells were resuspended in Hank's solution and plated on the surface of mouse spleen lymphocyte separation medium and centrifuged at 2200 rpm for 20 minutes at room temperature (7% increase, 0% decrease). After centrifugation, the buffy coat was removed, washed with Hank's solution, and centrifuged again. The cells were centrifuged at 1800 rpm for 8 minutes at room temperature, the supernatant discarded, and the cells were resuspended in complete 1640 culture medium and counted.

[0101] (2) Specific cytokine detection

[0102] Splenic lymphocytes from mice immunized with rEg.P29 were isolated and stimulated with rEg.P29+Anti-CD28 at 37°C and 5% CO2 for 72 h. Cytokines in the culture supernatant were detected by ELISA.

[0103] The detection steps are as follows:

[0104] 1. Coating with capture antibody: Dilute the capture antibody 1:250 with coating solution, mix well, add 100 μL / well to a 96-well plate, cover with tin foil, and store in a humidified box at 4°C overnight.

[0105] 2. Wash the plate: discard the liquid, wash with the washing solution 3 times, shake on a shaker, 1 minute each time, and pat dry the remaining liquid as much as possible on the last wash;

[0106] 3. Blocking: Reagent diluent, 200 μL / well, in a wet box, at room temperature for 1 hour;

[0107] 4. Preparation of standards and samples: (during the closed period)

[0108] ①Standard: Press with reagent diluent Figure 15 Serial dilution of standards;

[0109] ②Samples: Frozen samples should be thawed in advance to avoid repeated freezing and thawing;

[0110] Dilute the sample with reagent diluent at an appropriate ratio to ensure that the concentration of cytokines in the sample is within the detection range.

[0111] 5. Wash the plate: discard the liquid, wash with the washing solution 3 times, shake for 1 minute each time, and pat dry the residual liquid as much as possible for the last time;

[0112] 6. Add standards and samples: According to the 96-well plate layout, add standards and samples to the corresponding wells, 100 μL / well, mix on a shaker, and incubate in a wet box at room temperature for 2 hours;

[0113] Standard: 2 replicates per concentration, generally at position 1 B～H , 2 B～H

[0114] Sample: 3 replicate wells

[0115] 7. Wash the plate: discard the liquid, wash with the washing solution 5 times, shake on a shaker, 1 minute each time, and pat dry the remaining liquid as much as possible on the last wash;

[0116] 8. Add detection antibody and enzyme (Sav-HRP):

[0117] Dilute the detection antibody and HRP with reagent diluent at a ratio of 1:250; mix well, add 100 μL / well, and incubate at room temperature for 1 hour in a wet box;

[0118] 9. Wash the plate: discard the liquid, wash with the washing solution 7 times, shake for 1 minute each time, and pat dry the residual liquid as much as possible on the last wash;

[0119] 10. Color development: substrate TMB, 100 μL / well, in a humidified chamber at room temperature, protected from light, color development time no more than 30 minutes;

[0120] 11. Stop: Stop solution, 50 μL / well, mix on a shaker, and read the plate at a wavelength of λ = 450 nm.

[0121] 12. Data processing.

[0122] Splenic lymphocytes from mice in the rEg.P29+Freund's adjuvant, rEg.P29+CpG adjuvant, and PBS control groups were isolated 1, 2, 4, 8, and 16 weeks after the third immunization and added to microwells coated with rEg.P29. Cytokines in the culture supernatant were detected using ELISA. The levels of IFN-γ, IL-2, and TNF-α in the culture supernatant of spleen lymphocytes from mice immunized with rEg.P29 were significantly higher than those in the control group. These cytokines are important for combating parasitic infections ( Figure 3 ), the difference was statistically significant, while IL-4 and IL-10 were not produced.

[0123] Example 3 Screening, Optimization and Identification of T Cell Antigen Peptides of rEg.P29

[0124] (1) T cell epitope screening of rEg.P29

[0125] The 238 amino acids of rEg.P29 were synthesized into 45 overlapping peptides in a stepwise overlapping manner. Each peptide had 15 amino acids and each peptide overlapped by 10 amino acids. The 45 peptides covered the full length of rEg.P29. The schematic diagram of the T cell epitope design of rEg.P29 is shown in FIG. Figure 4 The amino acid sequence is shown in Table 1. As in Example 2, two weeks after the last immunization, spleen lymphocytes from mice immunized with rEg.P29 were isolated. The 45 synthesized peptides were divided into groups of three adjacent peptides (e.g., ID1-3; ID4-5, etc.) and anti-CD28 was added to stimulate spleen lymphocytes from mice immunized with rEg.P29 and the control group. The cells were cultured at 37°C, 5% CO2 for 72 h. The cytokines IFN-γ and IL-2 in the culture supernatant were detected by ELISA. Figure 5 As shown, ID16-18 can stimulate the spleen lymphocytes of mice immunized with rEg.P29 to produce IFN-γ and IL-2.

[0126] Next, we stimulated the spleen lymphocytes of mice immunized with rEg.P29 and the control group with ID15, ID16, ID17, ID18, ID19 and rEg.P29 plus Anti-CD28, and cultured them at 37°C, 5% CO2 for 72 h. The cytokines IFN-γ and IL-2 in the culture supernatant were detected by ELISA. Figure 6 As shown in the figure, ID18 and ID19 can stimulate the production of more cytokines IFN-γ and IL-2 than other peptides.

[0127] Table 1: T cell epitope sequences of rEg.P29

[0128]

[0129]

[0130] (2) Optimization and identification of T cell epitopes of rEg.P29

[0131] In order to further optimize the T cell epitopes of rEg.P29, rEg.P29 covering the full length of ID18 and ID19 was artificially synthesized. 86-105 (SEQ ID NO: 46) and their overlapping parts rEg.P29 91-100 , and then use rEg.P29 86-100 (ID18),rEg.P29 91-100 ,rEg.P29 86-105 , rEg.P29 91-105 The spleen lymphocytes of mice immunized with rEg.P29 and the control group were stimulated with rEg.P29 plus Anti-CD28 and cultured at 37°C, 5% CO2 for 72 h. The cytokines IFN-γ and IL-2 in the culture supernatant were detected by ELISA. Figure 7 As shown, rEg.P29 86-105 and rEg.P29 86-100 Compared with other peptides, it can stimulate the production of more cytokines IFN-γ and IL-2, and there is no statistical difference between them.

[0132] To verify the above results, we used ELISPOT and flow cytometry to analyze the above two peptides. 86-105 , rEg.P29 86-100 Splenic lymphocytes from mice immunized with rEg.P29 and the control group were stimulated with or without rEg.P29 plus Anti-CD28 and cultured at 37°C in 5% CO2 for 24 hours. The number of cells producing specific IFN-γ was determined by ELISPOT assay. The specific procedure was as follows:

[0133] Reagent preparation:

[0134] 1. Coating solution (1×PBS): 1L pH 7.2,

[0135] 8 g sodium chloride (NaCl), 0.2 g potassium chloride (KCl), 1.44 g disodium hydrogen phosphate (Na2HPO4), 0.24 g dipotassium hydrogen phosphate (KH2PO4), add 900 mL double distilled water (ddH2O); after complete dissolution, adjust the pH to 7.2, add double distilled water (ddH2O) to 1 L, autoclave or sterile filter, and store at 4°C.

[0136] 2. Blocking solution: cell culture medium (complete 1640)

[0137] 3. Wash solution I: 1×PBS + 0.05% Tween-20

[0138] 4. Washing solution II: 1× PBS

[0139] 5. Diluent: 1×PBS + 10% inactivated fetal bovine serum (FBS)

[0140] Sterile filter paper: high-pressure sterilized filter paper, used for washing plates.

[0141] 1. Aseptic operation (In ultra-clean bench)

[0142] 1. UV sterilization: irradiate the ELISPOT plate with UV light for 30 minutes before use;

[0143] 2. Capture antibody: Dilute the capture antibody 1:200 with coating solution, mix well, add 100 μL / well, and store in a humidified chamber at 4°C overnight.

[0144] 3. Wash the plate: discard the liquid and wash the plate twice with blocking solution, 200 μL / well, 3-5 minutes / time. After the last wash, try to pat dry any residual liquid on sterile filter paper.

[0145] 4. Blocking: Blocking solution, 200 μL / well, room temperature for 2 hours;

[0146] 5. Cell culture: Discard the solution and inoculate the cell suspension with stimulant into the plate at 200 μL / well incubate at 37°C in 5% CO2 for 2 to 24 hours (depending on the specific culture conditions).

[0147] 2. No need for sterile operation

[0148] 6. Wash the plate: discard the solution, wash the plate twice with deionized water, 200 μL / well, 3-5 minutes / time; wash the plate three times with Wash Solution I, 200 μL / well, 3-5 minutes / time, and pat dry any remaining liquid for the last wash;

[0149] 7. Add detection antibody: dilute the detection antibody 1:250 with diluent, mix well, filter through a 2μm filter, add 100μL / well, and incubate at room temperature for 2 hours;

[0150] 8. Wash the plate: discard the solution and wash the plate three times with Wash Solution I, 200 μL / well, 1-2 minutes / time. Pat dry any remaining liquid on the last wash.

[0151] 9. Add enzyme (Sav-HRP): dilute the enzyme 1:100 times with diluent, mix well, add 100 μL / well, and incubate at room temperature for 1 hour;

[0152] 10. Washing: Discard the solution, wash the plate 4 times with Washing Solution I, 200 μL / well, 1-2 minutes / time; wash the plate twice with Washing Solution II, 200 μL / well, 1-2 minutes / time, and discard the washing solution.

[0153] 11. Add substrate: substrate solution AEC ( BD Bioscience, Cat. No. 551951 ), 100 μL / well, and observe the generation of spots during the 5th to 60th minute to avoid excessive background.

[0154] 12. Termination: Wash the plate with deionized water to terminate the reaction.

[0155] 13. Drying: Leave the board at room temperature until it is completely dry.

[0156] 14. Data analysis: Read the plate using an ELISPOT reader and perform data analysis.

[0157] like Figure 8 As shown, rEg.P29 86-105 and rEg.P29 86-100 There was no statistical difference in the number of cells that could be stimulated to produce specific IFN-γ.

[0158] In addition, rEg.P29 86-105 and rEg.P29 86-100 None of them could stimulate the production of specific IL-4, corresponding to the above ELISA results.

[0159] rEg.P29 86-100 Splenic lymphocytes from mice immunized with rEg.P29 and control groups were cultured at 37°C in 5% CO2 for 24 h, and the proportion of cells producing specific IFN-γ was determined by flow cytometry as follows:

[0160] Reagent preparation:

[0161] 1. 1× phosphate buffered saline (1× PBS): 10 L pH 7.2–7.4

[0162] 80.0g sodium chloride (NaCl), 11.6g disodium hydrogen phosphate (Na2HPO4), 2.0g potassium dihydrogen phosphate (KH2PO4), 2.0g potassium chloride (KCl), add 9L double distilled water (ddH2O).

[0163] After complete dissolution, adjust the pH to 7.2-7.4, add double-distilled water to 1 L, and store at 2-8°C.

[0164] 2. Buffer II: 1× PBS + 0.1% bovine serum albumin (BSA) + 0.05% sodium azide (NaN3), store at 2-8°C;

[0165] 3. Fixative: 4% paraformaldehyde (PFA), store at 2-8°C.

[0166] 4. Buffer III: 1×PBS + 0.1% bovine serum albumin (BSA) + 0.05% sodium azide (NaN3) + 0.1% saponin, store at 2-8°C.

[0167] Experimental operation:

[0168] Centrifuge refrigerated at 4°C

[0169] The cells to be tested were unstimulated and / or stimulated according to the experimental design, and the concentration was adjusted to 1×10 6 / mL of cell suspension to be tested.

[0170] 1. Surface Molecular Staining

[0171] 1. Centrifugation: 4°C, 1800 rpm, 8 min;

[0172] 2. Resuspend: Discard the supernatant, add 2 mL of washing solution to each tube, centrifuge (4°C, 1800 rpm, 8 min), wash twice, and resuspend the cells with washing solution (100 μL / tube);

[0173] 3. Surface staining: Add corresponding surface molecule staining antibodies according to the experimental design;

[0174] 4. Incubation: Incubate at 4°C in the dark for 30 minutes;

[0175] 5. Wash: Add 2 mL of washing solution to each tube, centrifuge (4°C, 1800 rpm, 8 min), wash twice, discard the supernatant, add 150-200 μL of washing solution to each tube and resuspend, store at 4°C in the dark, and wait for loading;

[0176] 6. Test on the computer (Calibur or BD AriaⅡ).

[0177] 2. Intracellular Factor Staining

[0178] 1. Centrifugation: 4°C, 1800 rpm, 8 min;

[0179] 2. Resuspend: Discard the supernatant, add 2 mL of washing solution to each tube, centrifuge at 4°C (4°C, 1800 rpm, 8 min), wash twice, and resuspend the cells in 100 μL of washing solution to each tube;

[0180] 3. Surface staining: Add corresponding surface molecule staining antibodies according to the experimental design;

[0181] 4. Incubation: Incubate at 4°C in the dark for 30 minutes;

[0182] 5. Wash: Add washing solution, 2 mL / tube, 4°C, 1800 rpm, 8 min, twice;

[0183] 6. Fixation: Discard the supernatant and add 4% paraformaldehyde (PFA), 500 μL / tube, mix well, and incubate at room temperature in the dark for 8 minutes;

[0184] 7. Wash: Add 2 mL of washing solution to each tube and centrifuge (4°C, 2200 rpm, 8 min) once. Discard the supernatant and add 2 mL of membrane permeabilization solution to each tube and centrifuge (4°C, 2200 rpm, 8 min) once.

[0185] 8. Permeabilization: Discard the supernatant and resuspend the cells in permeabilization buffer (200 μL / tube) at 4°C for at least 2 hours or overnight.

[0186] 9. Wash: Add permeabilization buffer, 2 mL / tube, centrifuge (4°C, 2200 rpm, 8 min), and wash once;

[0187] 10. Resuspend: Resuspend cells with permeabilization buffer, 100 μL / tube;

[0188] 11. Intracellular staining: Add the corresponding staining antibody according to the experimental design, mix well, and incubate at 4°C in the dark for 30 minutes;

[0189] 12. Wash: Add 2 mL of washing solution to each tube and centrifuge (4°C, 2200 rpm, 8 min) twice;

[0190] Discard the supernatant, resuspend with 150-200 μL of washing solution, store at 4°C away from light, and wait for loading;

[0191] 13. Test on the computer (Calibur or BD AriaⅡ).

[0192] 3. Data Analysis

[0193] The data were analyzed and processed using Flowjo 7.6.1 software.

[0194] like Figure 9 and 10 As shown, rEg.P29 86-105 , rEg.P29 86-100 Both rEg.P29 and rEg.P29 can stimulate the production of specific IFN-γ, and there is no statistical difference in the proportion of stimulating the production of specific IFN-γ.

[0195] Example 4 rEg.P29 and rEg.P29 86-100 Can induce activation of spleen lymphocytes in mice immunized with rEg.P29

[0196] Since the epitope is the smallest immune functional unit in an antigen, we finally selected the shortest and most effective rEg.P29 86-100(15aa) as the T cell antigen polypeptide of rEg.P29. Isolate mouse spleen lymphocytes, rEg.P29 86-100 Splenic lymphocytes from mice immunized with rEg.P29 and control groups were cultured at 37°C, 5% CO2 for 24 h and 72 h, respectively, and their activation markers CD25 and CD69 were detected by flow cytometry. The specific operation was the same as the surface staining in Example 3.

[0197] like Figure 11 , 12, compared with the PBS group, rEg.P29 86-100 Or after rEg.P29 stimulation, the expression of CD69 increased on the first day, and the expression of CD25 increased significantly on the third day, indicating that rEg.P29 86-100 Or rEg.P29 stimulation can induce activation of spleen lymphocytes in rEg.P29 immunized mice.

[0198] Example 5 rEg.P29 and rEg.P29 86-100 Can induce the proliferation of spleen lymphocytes in mice immunized with rEg.P29

[0199] The cells were washed twice with preheated PBS, and then CFSE (Invitrogen, Carlsbad, USA, final concentration 2.5 mol / L) was added and incubated at 37°C in the dark for 15 min. The reaction was terminated with pre-cooled RPMI1640 (containing 10% fetal bovine serum), incubated at 4°C for 5 minutes, and washed twice with pre-cooled RPMI1640 (containing 10% fetal bovine serum). CFSE-labeled cells were then incubated with or without rEg.P29. 86-100 The cells were cultured in 37°C, 5% CO2, or rEg.P29. Cell samples were collected on days 3 and 5, and phenotypes were analyzed by staining with fluorescently labeled monoclonal antibodies at 4°C in the dark.

[0200] like Figure 13 , 14, compared with the PBS group, rEg.P29 86-100 After stimulation with rEg.P29 or rEg.P29, the CFSE fluorescence intensity decreased on the third day and continued to decrease on the fifth day, indicating that rEg.P29 86-100 Or rEg.P29 stimulation can induce the proliferation of spleen lymphocytes in rEg.P29-immunized mice, and there is no statistical difference between the two.

[0201] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0202] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0203] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention. Sequence Listing <110> Ningxia Medical University <120> A T cell antigen polypeptide and its application <130> 1 <160> 47 <170> SIPOSequenceListing 1.0 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <400> 1 Met Ser Gly Phe Asp Val Thr Lys Thr Phe Asn Arg Phe Thr Gln 1 5 10 15 <210> 2 <211> 15 <212> PRT <213> Artificial Sequence <400> 2 Val Thr Lys Thr Phe Asn Arg Phe Thr Gln Arg Ala Gly Glu Leu 1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <400> 3 Asn Arg Phe Thr Gln Arg Ala Gly Glu Leu Val Asn Lys Asn Glu 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <400> 4 Arg Ala Gly Glu Leu Val Asn Lys Asn Glu Lys Thr Ser Tyr Pro 1 5 10 15 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <400> 5 Val Asn Lys Asn Glu Lys Thr Ser Tyr Pro Thr Arg Thr Ser Asp 1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <400> 6 Lys Thr Ser Tyr Pro Thr Arg Thr Ser Asp Leu Ile His Glu Ile 1 5 10 15 <210> 7 <211> 15 <212> PRT <213> Artificial Sequence <400> 7 Thr Arg Thr Ser Asp Leu Ile His Glu Ile Asp Gln Met Lys Ala 1 5 10 15 <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <400> 8 Leu Ile His Glu Ile Asp Gln Met Lys Ala Trp Ile Ser Lys Ile 1 5 10 15 <210> 9 <211> 15 <212> PRT <213> Artificial Sequence <400> 9 Asp Gln Met Lys Ala Trp Ile Ser Lys Ile Ile Thr Ala Thr Glu 1 5 10 15 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <400> 10 Trp Ile Ser Lys Ile Ile Thr Ala Thr Glu Glu Phe Val Asp Ile 1 5 10 15 <210> 11 <211> 15 <212> PRT <213> Artificial Sequence <400> 11 Ile Thr Ala Thr Glu Glu Phe Val Asp Ile Asn Ile Ala Ser Lys 1 5 10 15 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <400> 12 Glu Phe Val Asp Ile Asn Ile Ala Ser Lys Val Ala Asp Ala Phe 1 5 10 15 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <400> 13 Asn Ile Ala Ser Lys Val Ala Asp Ala Phe Gln Lys Asn Lys Glu 1 5 10 15 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <400> 14 Val Ala Asp Ala Phe Gln Lys Asn Lys Glu Lys Ile Thr Thr Thr 1 5 10 15 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <400> 15 Gln Lys Asn Lys Glu Lys Ile Thr Thr Thr Asp Lys Leu Gly Thr 1 5 10 15 <210> 16 <211> 15 <212> PRT <213> Artificial Sequence <400> 16 Lys Ile Thr Thr Thr Asp Lys Leu Gly Thr Ala Leu Glu Gln Val 1 5 10 15 <210> 17 <211> 15 <212> PRT <213> Artificial Sequence <400> 17 Asp Lys Leu Gly Thr Ala Leu Glu Gln Val Ala Ser Gln Ser Glu 1 5 10 15 <210> 18 <211> 15 <212> PRT <213> Artificial Sequence <400> 18 Ala Leu Glu Gln Val Ala Ser Gln Ser Glu Lys Ala Ala Pro Gln 1 5 10 15 <210> 19 <211> 15 <212> PRT <213> Artificial Sequence <400> 19 Ala Ser Gln Ser Glu Lys Ala Ala Pro Gln Leu Ser Lys Met Leu 1 5 10 15 <210> 20 <211> 15 <212> PRT <213> Artificial Sequence <400> 20 Lys Ala Ala Pro Gln Leu Ser Lys Met Leu Thr Glu Ala Ser Asp 1 5 10 15 <210> 21 <211> 15 <212> PRT <213> Artificial Sequence <400> 21 Leu Ser Lys Met Leu Thr Glu Ala Ser Asp Val His Gln Arg Met 1 5 10 15 <210> twenty two <211> 15 <212> PRT <213> Artificial Sequence <400> twenty two Thr Glu Ala Ser Asp Val His Gln Arg Met Ala Thr Ala Arg Lys 1 5 10 15 <210> twenty three <211> 15 <212> PRT <213> Artificial Sequence <400> twenty three Val His Gln Arg Met Ala Thr Ala Arg Lys Asn Phe Asn Ser Glu 1 5 10 15 <210> twenty four <211> 15 <212> PRT <213> Artificial Sequence <400> twenty four Ala Thr Ala Arg Lys Asn Phe Asn Ser Glu Val Asn Thr Thr Phe 1 5 10 15 <210> 25 <211> 15 <212> PRT <213> Artificial Sequence <400> 25 Asn Phe Asn Ser Glu Val Asn Thr Thr Phe Ile Glu Asp Leu Lys 1 5 10 15 <210> 26 <211> 15 <212> PRT <213> Artificial Sequence <400> 26 Val Asn Thr Thr Phe Ile Glu Asp Leu Lys Asn Phe Leu Asn Thr 1 5 10 15 <210> 27 <211> 15 <212> PRT <213> Artificial Sequence <400> 27 Ile Glu Asp Leu Lys Asn Phe Leu Asn Thr Thr Leu Ser Glu Ala 1 5 10 15 <210> 28 <211> 15 <212> PRT <213> Artificial Sequence <400> 28 Asn Phe Leu Asn Thr Thr Leu Ser Glu Ala Gln Lys Ala Lys Thr 1 5 10 15 <210> 29 <211> 15 <212> PRT <213> Artificial Sequence <400> 29 Thr Leu Ser Glu Ala Gln Lys Ala Lys Thr Lys Leu Glu Glu Val 1 5 10 15 <210> 30 <211> 15 <212> PRT <213> Artificial Sequence <400> 30 Gln Lys Ala Lys Thr Lys Leu Glu Glu Val Arg Leu Asp Leu Asp 1 5 10 15 <210> 31 <211> 15 <212> PRT <213> Artificial Sequence <400> 31 Lys Leu Glu Glu Val Arg Leu Asp Leu Asp Ser Asp Lys Thr Lys 1 5 10 15 <210> 32 <211> 15 <212> PRT <213> Artificial Sequence <400> 32 Arg Leu Asp Leu Asp Ser Asp Lys Thr Lys Leu Lys Asn Ala Lys 1 5 10 15 <210> 33 <211> 15 <212> PRT <213> Artificial Sequence <400> 33 Ser Asp Lys Thr Lys Leu Lys Asn Ala Lys Thr Ala Glu Gln Lys 1 5 10 15 <210> 34 <211> 15 <212> PRT <213> Artificial Sequence <400> 34 Leu Lys Asn Ala Lys Thr Ala Glu Gln Lys Ala Lys Trp Glu Ala 1 5 10 15 <210> 35 <211> 15 <212> PRT <213> Artificial Sequence <400> 35 Thr Ala Glu Gln Lys Ala Lys Trp Glu Ala Glu Val Arg Lys Asp 1 5 10 15 <210> 36 <211> 15 <212> PRT <213> Artificial Sequence <400> 36 Ala Lys Trp Glu Ala Glu Val Arg Lys Asp Glu Ser Asp Phe Asp 1 5 10 15 <210> 37 <211> 15 <212> PRT <213> Artificial Sequence <400> 37 Glu Val Arg Lys Asp Glu Ser Asp Phe Asp Arg Val His Gln Glu 1 5 10 15 <210> 38 <211> 15 <212> PRT <213> Artificial Sequence <400> 38 Glu Ser Asp Phe Asp Arg Val His Gln Glu Ser Leu Thr Ile Phe 1 5 10 15 <210> 39 <211> 15 <212> PRT <213> Artificial Sequence <400> 39 Arg Val His Gln Glu Ser Leu Thr Ile Phe Glu Lys Thr Cys Lys 1 5 10 15 <210> 40 <211> 15 <212> PRT <213> Artificial Sequence <400> 40 Ser Leu Thr Ile Phe Glu Lys Thr Cys Lys Glu Phe Asp Gly Leu 1 5 10 15 <210> 41 <211> 15 <212> PRT <213> Artificial Sequence <400> 41 Glu Lys Thr Cys Lys Glu Phe Asp Gly Leu Ser Val Gln Leu Leu 1 5 10 15 <210> 42 <211> 15 <212> PRT <213> Artificial Sequence <400> 42 Glu Phe Asp Gly Leu Ser Val Gln Leu Leu Asp Leu Ile Arg Ala 1 5 10 15 <210> 43 <211> 15 <212> PRT <213> Artificial Sequence <400> 43 Ser Val Gln Leu Leu Asp Leu Ile Arg Ala Glu Lys Asn Tyr Tyr 1 5 10 15 <210> 44 <211> 15 <212> PRT <213> Artificial Sequence <400> 44 Asp Leu Ile Arg Ala Glu Lys Asn Tyr Tyr Glu Ala Cys Ala Lys 1 5 10 15 <210> 45 <211> 15 <212> PRT <213> Artificial Sequence <400> 45 Glu Lys Asn Tyr Tyr Glu Ala Cys Ala Lys Glu Cys Ser Met Met 1 5 10 15 <210> 46 <211> 20 <212> PRT <213> Artificial Sequence <400> 46 Ala Leu Glu Gln Val Ala Ser Gln Ser Glu Lys Ala Ala Pro Gln Leu 1 5 10 15 Ser Lys Met Leu 20 <210> 47 <211> 238 <212> PRT <213> Echinococcus granulosus <400> 47 Met Ser Gly Phe Asp Val Thr Lys Thr Phe Asn Arg Phe Thr Gln Arg 1 5 10 15 Ala Gly Glu Leu Val Asn Lys Asn Glu Lys Thr Ser Tyr Pro Thr Arg 20 25 30 Thr Ser Asp Leu Ile His Glu Ile Asp Gln Met Lys Ala Trp Ile Ser 35 40 45 Lys Ile Ile Thr Ala Thr Glu Glu Phe Val Asp Ile Asn Ile Ala Ser 50 55 60 Lys Val Ala Asp Ala Phe Gln Lys Asn Lys Glu Lys Ile Thr Thr Thr 65 70 75 80 Asp Lys Leu Gly Thr Ala Leu Glu Gln Val Ala Ser Gln Ser Glu Lys 85 90 95 Ala Ala Pro Gln Leu Ser Lys Met Leu Thr Glu Ala Ser Asp Val His 100 105 110 Gln Arg Met Ala Thr Ala Arg Lys Asn Phe Asn Ser Glu Val Asn Thr 115 120 125 Thr Phe Ile Glu Asp Leu Lys Asn Phe Leu Asn Thr Thr Leu Ser Glu 130 135 140 Ala Gln Lys Ala Lys Thr Lys Leu Glu Glu Val Arg Leu Asp Leu Asp 145 150 155 160 Ser Asp Lys Thr Lys Leu Lys Asn Ala Lys Thr Ala Glu Gln Lys Ala 165 170 175 Lys Trp Glu Ala Glu Val Arg Lys Asp Glu Ser Asp Phe Asp Arg Val 180 185 190 His Gln Glu Ser Leu Thr Ile Phe Glu Lys Thr Cys Lys Glu Phe Asp 195 200 205 Gly Leu Ser Val Gln Leu Leu Asp Leu Ile Arg Ala Glu Lys Asn Tyr 210 215 220 Tyr Glu Ala Cys Ala Lys Glu Cys Ser Met Met Leu Gly Glu 225 230 235

Claims

1. An antigen polypeptide, characterized in that The antigen polypeptide is selected from the amino acid sequence shown in SEQ ID NO: 18, 19 or 46.

2. A nucleic acid encoding the antigenic polypeptide according to claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A cell comprising the antigenic polypeptide of claim 1, the nucleic acid of claim 2 or the expression vector of claim 3.

5. A vaccine, characterized in that The vaccine comprises the antigen polypeptide according to claim 1, the nucleic acid according to claim 2, the expression vector according to claim 3 or the cell according to claim 4, and an immunologically acceptable carrier.

6. The vaccine according to claim 5, characterized in that The immunologically acceptable carrier includes an adjuvant, a diluent, a solubilizer, a lubricant, a suspending agent, a transfection accelerator, an excipient, a filler, a binder, an absorption promoter and / or a synergist.

7. The vaccine according to claim 6, characterized in that The immunologically acceptable carrier includes an adjuvant, and the adjuvant is FCA or CpG.

8. A method for preparing the vaccine according to any one of claims 5 to 7, characterized in that: The preparation method comprises mixing the antigen polypeptide according to claim 1 with an immunologically acceptable carrier.

9. A method for screening anti-echinococcosis antibodies, characterized in that: The screening method comprises mixing the object to be screened with the antigen polypeptide according to claim 1.

10. Use of the antigen polypeptide according to claim 1, the nucleic acid according to claim 2, the expression vector according to claim 3, or the cell according to claim 4 in the preparation of a product for preventing and / or treating echinococcosis.

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