Antigen polypeptide and its application

Abstract

The present invention provides an antigenic polypeptide, a nucleic acid encoding the antigenic polypeptide, an expression vector comprising the nucleic acid, a cell comprising the antigenic polypeptide, the nucleic acid, or the expression vector, and a vaccine for echinococcosis and a method for preparing the same. The antigenic polypeptide described herein can induce an immune response and induce the proliferation of specific B cells.

Description

Technical field:

[0001] The present invention belongs to the technical field of immunobiology, and in particular relates to an anti-echinococcosis recombinant protein P29 antigen polypeptide and an application thereof. Background technology:

[0002] Hydatid disease is a chronic infectious disease caused by parasitic larvae infecting humans or livestock, seriously endangering their health. The disease is widely prevalent in areas with developed animal husbandry around the world. Research on vaccines for hydatid disease started relatively late and the level of research is relatively low. To date, no vaccine for hydatid disease has been successfully commercialized and marketed domestically or internationally. Existing studies have shown that the recombinant protein P29 (rEg.P29) of Echinococcus granulosus is antigenic and can induce an immune response dominated by Th1 cells in the animal immune system in anti-hydatid disease experiments using mice and sheep as infection models. After rEg.P29 was used to immunize sheep, the proportion of sheep that developed protective immunity, that is, those that were protected from infection, was 94.5%. After rEg.P29 was used to immunize mice, and then the mice were challenged with protoscolecus via intraperitoneal injection, a protective immunity of 96.6% was obtained. rEg.P29 has the potential for translational application as a hydatid disease vaccine.

[0003] Currently, a variety of proteins or related vaccines related to the prevention and treatment of echinococcosis have been disclosed. For example, patents CN113214374A and CN113214373A disclose two new echinococcosis antigens, Cystatin protein and Murinoglobulin-2 protein; patent CN105343873A discloses a gene rEg.P29 molecular engineering vaccine for sheep echinococcosis infection, its preparation method and application; patent CN106397610A discloses a method for preparing a multi-epitope fusion diagnostic antigen protein of Echinococcus granulosus and its application, which is to prepare a diagnostic antigen by concatenating antigen epitopes in a certain order. Although most veterinary-approved vaccines use whole attenuated or inactivated pathogens, in fact, most pathogen proteins are unnecessary for achieving a complete protective response, and some of these proteins may also cause unnecessary side effects, such as allergies, autoimmunity and off-target reactions. Therefore, it is very necessary to design and produce epitope-based vaccines. Summary of the invention:

[0004] To address the above-mentioned issues, the present invention provides an antigenic polypeptide that has the effect of inducing lymphocyte activation and proliferation. Furthermore, the lyophilized peptide is chemically stable, and vaccines prepared using this antigenic polypeptide remain stable even under conventional cold chain storage and transportation. More importantly, while polypeptides are typically less effective than intact proteins in activating specific cells and promoting cell proliferation, the antigenic polypeptides provided herein are substantially similar in effectiveness to intact proteins.

[0005] 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.

[0006] Preferably, the hydatid disease is caused by infection with Echinococcus multilocularis or Echinococcus granulosus.

[0007] Preferably, the antigenic polypeptide comprises an epitope on the echinococcosis recombinant protein.

[0008] Preferably, the antigenic polypeptide is derived from an epitope on the recombinant protein rEg.P29 of Echinococcus granulosus.

[0009] Further preferably, the antigen polypeptide is derived from a B cell antigen epitope on the recombinant protein rEg.P29 of Echinococcus granulosus.

[0010] In a specific embodiment of the present invention, the antigen polypeptide is selected from one or more B cell antigen epitopes of SEQ ID NO: 2.

[0011] The B cell antigen epitope is a peptide segment that can bind to BCR or antibody.

[0012] Preferably, the B cell antigen epitope may be a continuous or discontinuous amino acid sequence in SEQ ID NO:2.

[0013] Preferably, the B cell antigen epitope may be composed of spatially adjacent amino acid residues on the recombinant protein consisting of SEQ ID NO: 2.

[0014] Preferably, the antigenic polypeptide contains at least one antigenic epitope.

[0015] Preferably, the antigen polypeptide comprises at least 8 consecutive amino acids in SEQ ID NO: 2. Further preferably, the antigen polypeptide is less than 238 aa in length.

[0016] Preferably, the antigen polypeptide comprises 8-20 consecutive amino acids in SEQ ID NO: 2, specifically 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acids.

[0017] Preferably, the antigenic polypeptide comprises:

[0018] A) one or more amino acid sequences of SEQ ID NO: 3 to SEQ ID NO: 26;

[0019] B) having an amino acid sequence identity of 80% or greater with one or more of SEQ ID NO: 3 to SEQ ID NO: 26;

[0020] C) differs from one or more of SEQ ID NO:3 to SEQ ID NO:26 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid sequence; or

[0021] D) an amino acid sequence represented by one or both of SEQ ID NO: 3 to SEQ ID NO: 26, comprising substitution, deletion and / or insertion of one or more amino acid residues.

[0022] Preferably, the antigen polypeptide comprises a B cell antigen epitope, preferably represented by any one of SEQ ID NO: 3 to SEQ ID NO: 26.

[0023] Preferably, the antigen polypeptide comprises two or more B cell antigen epitopes, preferably two or more of SEQ ID NO: 3 to SEQ ID NO: 26. The two or more B cell antigen epitopes are directly linked or connected via a linker.

[0024] More preferably, the antigen polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 22.

[0025] More preferably, the amino acid sequence of the antigenic polypeptide comprises the sequence shown in SEQ ID NO: 22.

[0026] The second aspect of the present invention provides a nucleic acid encoding the above-mentioned antigen polypeptide.

[0027] The third aspect of the present invention provides an expression vector comprising the above nucleic acid.

[0028] 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.

[0029] 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.

[0030] Preferably, the expression vector can be a plasmid, cosmid, phage or virus.

[0031] 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.

[0032] The fifth aspect of the present invention provides a use of the above-mentioned antigen polypeptide, the above-mentioned nucleic acid, the above-mentioned expression vector or the above-mentioned cell in the preparation of a product for treating or preventing echinococcosis.

[0033] Preferably, the "product" includes but is not limited to vaccines, antibodies, drugs, and kits; preferably, the product is a vaccine and / or an antibody.

[0034] In a sixth 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.

[0035] The seventh aspect of the present invention provides a vaccine for echinococcosis, 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.

[0036] Preferably, the immunologically acceptable carrier includes adjuvants, diluents, solubilizers, lubricants, suspending agents, transfection accelerators, excipients, fillers, adhesives, absorption promoters and / or synergists, etc.

[0037] 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, TAP1, TAP2 and functional fragments thereof.

[0038] Further preferably, the adjuvant is selected from FCA, CpG, aluminum adjuvant and / or peanut oil emulsified adjuvant.

[0039] In a specific embodiment of the present invention, the adjuvant is FCA and / or CpG.

[0040] Preferably, the vaccines include peptide vaccines, protein vaccines, mRNA vaccines and DNA vaccines.

[0041] The eighth aspect of the present invention provides a method for preparing the above-mentioned vaccine, which comprises mixing the above-mentioned antigen polypeptide with the above-mentioned immunologically acceptable carrier.

[0042] The ninth aspect of the present invention provides a method for screening anti-echinococcosis antibodies, which comprises mixing the object to be screened with the above-mentioned antigen polypeptide.

[0043] 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.

[0044] The tenth 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, the aforementioned cell or the aforementioned vaccine to an individual.

[0045] Preferably, the specific immune response includes a T cell response and / or a B cell response.

[0046] 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.

[0047] In an eleventh aspect, the present invention provides an antibody that can bind to the antigen polypeptide of the present invention.

[0048] 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.).

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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."

[0055] 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.

[0056] 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. Description of the drawings:

[0057] Figure 1 : SDS-PAGE analysis of rEg.P29 expression and purification, wherein, M: marker, lane 1: E. coli containing pET28a before IPTG induction, lane 2: E. coli containing pET28a-P29 after IPTG induction for 6 h, lane 3: rEg.P29 purified by his affinity chromatography.

[0058] Figure 2 : Specific antibodies in the serum of mice immunized with rEg.P29 bind to the rEg.P29 protein, where M is marker. Lane 1: The primary antibody is the serum of mice immunized with anti-His tag antibody. Lane 2: The primary antibody is the serum of mice in the PBS group. Lane 3: The primary antibody is the serum of mice in the rEg.P29+FCA group. Lane 4: The primary antibody is the serum of mice in the rEg.P29+CpG group.

[0059] Figure 3 : Immunization of mice with rEg.P29 can induce the production of anti-rEg.P29 specific antibodies.

[0060] Figure 4 : Screening of B cell dominant epitope peptides. Sera from mice immunized with rEg.P29+FCA, mice immunized with rEg.P29+CpG or mice in the PBS group recognized the B1-24 epitope peptide or rEg.P29 coated on the ELISA plate, respectively.

[0061] Figure 5 : B cell dominant epitope peptides react with specific antibodies, that is, the sera of rEg.P29+FCA immunized mice, rEg.P29+CpG immunized mice or PBS group mice respectively recognize B4, B6, B7, B8, B11, B12, B15, B16, B17, B20 or rEg.P29 coated on the ELISA plate.

[0062] Figure 6 : B cell dominant epitope binding to specific IgG antibody subtype detection, that is, the sera of rEg.P29+FCA immunized mice, rEg.P29+CpG immunized mice or PBS group mice respectively recognized B4, B6, B7, B8, B11, B12, B15, B16, B17, B20 or rEg.P29 coated on ELISA plates.

[0063] Figure 7 : B cell dominant epitope-specific antibody IgG titer, that is, the sera of rEg.P29+FCA-immunized mice, rEg.P29+CpG-immunized mice or PBS group mice respectively recognized B4, B6, B7, B8, B11, B12, B15, B16, B17, B20 or rEg.P29 coated on ELISA plates.

[0064] Figure 8 : The results of spleen lymphocyte activation stimulated by B20 and rEg.P29 in rEg.P29+FCA immunized mice, rEg.P29+CpG immunized mice or PBS group mice, respectively, where unsti represents the control group, i.e. the spleen lymphocyte group without B20 or rEg.P29 stimulation.

[0065] Figure 9 : The results of B20 and rEg.P29 stimulating the proliferation of splenic B lymphocytes in rEg.P29+FCA immunized mice, rEg.P29+CpG immunized mice or PBS group mice, respectively. Among them, Medium represents the splenic lymphocyte group without B20 or rEg.P29 stimulation.

[0066] Figure 10 : Histogram of spleen B lymphocyte proliferation in rEg.P29+FCA immunized mice, rEg.P29+CpG immunized mice or PBS group mice stimulated by B20 and rEg.P29, respectively. Among them, Medium represents the spleen lymphocyte group without B20 or rEg.P29 stimulation. Specific implementation method:

[0067] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0069] The ICR mice used in the examples of the present invention were bred by Hauschka using a Swiss mouse population for high fertility. They were then distributed by the Institute of Cancer Research in the United States to various countries for breeding experiments. These mice are called ICR mice in these countries.

[0070] Example 1 Expression and purification of rEg.P29 recombinant protein

[0071] rEg.P29 open reading frame gene sequence (GenBank sequence number: AF078931): (SEQ ID NO: 1)

[0072] ATGTCCGGATTTGACGTTACTAAGACTTTCAATAGATTTACCCAGCGGGCTGGTGAGCTTGTAAATAAGAATGAAAAGACCTCATATCCTACCCGAACCTCAGATCTTATCCATGAGATCGACCAAATGAAAGCATGGATCAGCAAGATCATCACCGCTACTGAGGAATTCGTAGACATCAACATTGCATCTAAAGTCGCGGATGCTTTCCAGAAGAATAAGGAGAAGATTACTACTACCGACAAACTGGGTACTGCTCTCGAGCAGGTTGCTTCCCAATCAGAAAAGGCAGCTCCCCAACTTTCTAAAATGCTGACGGAAGCTTCTGATGTCCATCAGCGTATGGCCACTGCCAGAAAGAATTTCAATAGTGAGGTTAATACCACCTTCATTGAAGATTTGAAAAACTTCTTGAACACCACGCTTAGCGAGGCCCAGAAAGCAAAGACCAAGCTGGAGGAGGTTCGACTAGATTTGGACTCTGACAAGACTAAATTGAAGAATGCTAAGACTGCGGAACAGAAGGCCAAGTGGGAGGCCGAGGTGCGAAAAGACGAAAGTGACTTCGATCGAGTGCACCAAGAATCTCTTACTATCTTTGAGAAGACTTGCAAAGAATTCGATGGGTTGAGCGTTCAGCTGTTGGATCTGATCCGTGCAGAGAAGAATTACTACGAAGCCTGTGCCAAAGAGTGCAGTATGATGCTGGGCGAGTAG

[0073] Amino acid sequence of the rEg.P29 open reading frame (SEQ ID NO: 2)

[0074] MSGFDVTKTFNRFTQRAGELVNKNEKTSYPTRTSDLIHEIDQMKAWISKIITATEEFVDINIASKVADAFQKNKEKITTTDKLGTALEQVASQSEKAAPQLSKMLTEASDVHQRMATAR KNFNSEVNTTFIEDLKNFLNTTLSEAQKAKTKLEEVRLDSDKTKLKNAKTAEQKAKWEAEVRKDESDFDRVHQESLTIFEKTCKEFDGLSVQLLDLIRAEKNYYEACAKECSMMLGE

[0075] Take out the BL21 strain containing the rEg.P29 / pET28a recombinant plasmid and the empty plasmid pET28a stored in this room, pick the strain with an inoculation loop on the clean bench, and inoculate it into LB solid medium flat culture dishes with a final concentration of kanamycin (Kana) of 50 μg / mL, and place it in a 37°C incubator that has been opened in advance for overnight. Observe the growth of the colonies the next day, pick a single monoclonal colony, and inoculate the recombinant bacteria into LB liquid culture medium containing kanamycin (Kana) at a final concentration of 50 μg / mL. Place it in a 37°C shaker and shake overnight at 200 rpm until a clear yellow color appears at OD 600 When the OD was 0.6 at nm wavelength, the cells were inoculated into LB liquid culture medium at a ratio of 1:100 to expand the culture. After culturing at 37°C for 6 h, the OD was 0.6. IPTG was added to a final concentration of 1 mmol / L, and the cells were shaken and induced at 37°C for 6 h. The cells were collected and the expression of the target protein was detected by 12% SDS-PAGE.

[0076] 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 BCA assay detected an egg concentration of 1 mg / mL and an endotoxin content of 0.358 EU / mL.

[0077] Example 2 Binding of specific antibodies from serum of mice immunized with rEg.P29 to rEg.P29

[0078] After SDS-PAGE electrophoresis, the membrane was electroblotted onto a nitrocellulose membrane using conventional methods. The membrane was blocked with 5% skim milk powder at 37°C for 2 h and then washed three times with PBST (containing 0.5‰ Tween-20). The membrane was then soaked in a 1:100 dilution of immune serum at 4°C overnight. After washing, the membrane was incubated with a 1:100 dilution of goat anti-mouse IgG-horseradish peroxidase (HRP) at 37°C on a shaker at 60 rpm for 2 h. After washing four times with PBST, freshly prepared color development solution was added for 5 min, and the reaction was terminated by rinsing with distilled water.

[0079] Western blot results showed that rEg.P29 could not be recognized by the mouse serum samples in the PBS group, but could be recognized by the mouse monoclonal His tag antibody and the immunized mouse serum ( Figure 2 shown).

[0080] Example 3 Immune Effects of rEg.P29 Protein

[0081] 1. Animal immunization

[0082] Twenty-four ICR mice were divided into two groups. Group A was immunized with 10 μg / 100 μl of rEg.P29 emulsified in Freund's adjuvant; Group B was the control group, which received 100 μl of Freund's adjuvant emulsified in PBS. Immunizations were conducted once every two weeks for three times. Antigen was quantified using Bio-Rad's Gel Doc 1000 gel analysis software. By the end of the experiment, two mice in Group A and three mice in Group B had died.

[0083] 2. Attack infection

[0084] Establishment of experimental animal infection model: 8 weeks after immunization of mice, each mouse was intraperitoneally injected with 0.1 ml of protoscolex suspension, that is, each mouse was infected with approximately 1,500 live protoscolex.

[0085] 3. Observation of immune protection

[0086] (1) After 20 weeks of infection, mice were killed, their abdomens were opened, and their spleens were weighed. The spleen index was calculated using the formula:

[0087] Spleen index = spleen weight (mg) / mouse body weight (g);

[0088] (2) Count the cysts of echinococcosis in the liver and mesentery and calculate the immune protection. The formula is:

[0089] Immune protection (%) = (1-average number of cysts in the immunized group / average number of cysts in the control group) × 100.

[0090] 4. Results

[0091] rEg.P29 induces protective effect against echinococcosis infection in mice: the average echinococcosis cyst diameters of the rEg.P29 immunization group and the PBS control group were 0.9 mm and 8.1 mm, respectively; among them, ICR mice immunized with rEg.P29 recombinant antigen obtained 96.6% immune protection and increased spleen index, which was significantly different from that of the PBS control group (P<0.05) (Table 1).

[0092] Table 1 Number of echinococcosis cysts, immune protection and spleen index of mice immunized with rEg.P29 after challenge infection

[0093]

[0094] Note: Compared with the PBS control group, * P<0.05

[0095] Example 4 Immunization of mice with rEg.P29 induces the production of anti-rEg.P29 specific antibodies

[0096] Purified rEg.P29 was adjusted to a concentration of 1 mg / mL using enzyme-free aqueous buffer and mixed with Freund's adjuvant (rEg.P29 to Freund's adjuvant volume ratio of 1:5) or CpG adjuvant (rEg.P29 to CpG adjuvant volume ratio of 1:2). Immunization was performed subcutaneously in the abdomen at a dose of 100 μg / animal. Booster immunizations were performed every 7 days (complete Freund's adjuvant for the first immunization and incomplete Freund's adjuvant for the booster immunization). A control group received 100 μL of normal saline per animal in the same manner.

[0097] Peptide Identification and Preparation: Based on the rEg.P29 amino acid sequence available in our laboratory, we used bioinformatics software such as ABCPred to predict and analyze the B cell antigen epitopes of rEg.P29. After comparative screening, we selected several 16-amino acid peptides with good hydrophilicity and other properties. These peptides were synthesized at Shanghai Bioengineering Co., Ltd. with a purity of ≥95%. The peptide sequences are shown in Table 2:

[0098] Table 2 B cell epitope peptides of rEg.P29

[0099]

[0100] ELISA plates were coated with 24 synthetic B cell epitope peptides (Table 2) and rEg.P29. Serum from Eg.P29-immunized mice was used as the primary antibody. ELISA was used to detect the immunoreactivity of B cell epitope peptides (Table 2) with antibodies in serum from rEg.P29-immunized mice. The results were analyzed to identify the positively reactive peptides. ELISA test steps:

[0101] (1) Dilute the peptide to a concentration of 10 μg / mL and rEg.P29 to a concentration of 10 μg / mL in coating buffer. Add 100 μL per well of the ELISA plate for coating and incubate overnight at 4°C.

[0102] (2) The next day, the coating solution was poured off, and the cells were washed three times with PBST. After patting dry, PBS containing 10% FBS (feto-calf serum) was added, 200 μL per well, and incubated at 37°C for 2 h.

[0103] (3) Pour off the blocking solution, wash 5 times with PBST, then add rEg.P29 immune mouse serum (diluted with PBS containing 10% FBS at a ratio of 1:100), 100 μL per well, and incubate at 37°C for 2 h.

[0104] (4) Pour off the primary antibody incubation solution, wash five times with PBST, and then add Goat anti-mouse IgG (diluted 1:5000 with PBS containing 10% FBS), 100 μL per well, and incubate at 37°C for 1 h.

[0105] (5) Pour off the secondary antibody incubation solution, wash seven times with PBST, then add 100 μL of TMB colorimetric solution to each well. After developing for 8–10 minutes, add stop solution to terminate the reaction. Measure OD450 using an A450 nm microplate reader within 15 minutes.

[0106] (6) Analyze data

[0107] The serum of rEg.P29 immunized group and control group was separated at 7, 14, 21, 28 and 35 days after the third immunization, and added into micro-wells coated with rEg.P29. The serum anti-rEg.P29 specific antibodies were detected by ELISA. The IgG, IgM, IgA and IgE antibody levels in the serum of rEg.P29 immunized mice were significantly higher than those in the control group ( Figure 3 ), the difference is statistically significant.

[0108] Example 5 Identification of B cell dominant epitopes

[0109] (1) Screening of B cell dominant epitopes

[0110] ELISA plates were coated with 24 B cell epitope peptides predicted by bioinformatics (Table 2) and rEg.P29. Diluted serum from mice immunized with rEg.P29 was used as the primary antibody to detect specific antibodies IgG, IgM, IgE and IgA.

[0111] Test results such as Figure 4The results showed that in the rEg.P29+FCA group, the levels of specific IgG antibodies against B4, B6, B7, B8, B11, B12, B15, B16, B17, and B20 were significantly higher than those against other B cell epitope peptides. In the rEg.P29+CpG group, only the levels of specific IgG antibodies against B11 and B20 were significantly higher than those against other B cell epitope peptides.

[0112] (2) B cell dominant epitope peptide antibody detection

[0113] Specific antibodies to each B cell epitope peptide and rEg.P29 were tested, and the test results were as follows: Figure 5 As can be seen from the figure, all B cell epitope peptides bind to IgG, while B7, B8, B12, B15, B16, and B20 bind to IgM. IgE and IgA do not bind to the epitope peptides.

[0114] (3) Detection of IgG subtypes of B cell dominant epitope peptides

[0115] The 10 epitope peptides screened in step (1) and rEg.P29 were used to coat the ELISA plate, and the diluted serum of rEg.P29-immunized mice was used as the primary antibody to detect the specific IgG subtype of each B cell epitope peptide.

[0116] Test results such as Figure 6 It was found that in the rEg.P29+FCA group, none of the B cell epitope peptides bound to IgG3, while both IgG1 and IgG2b could bind to the epitope peptides. B4, B15, B16, and B20 could react with IgG2a, and B4, B6, B8, B11, B15, B16, and B20 could bind to IgG2c.

[0117] (4) Detection of B cell dominant epitope peptide antibody titer

[0118] The 10 epitope peptides (B4, B6, B7, B8, B11, B12, B15, B16, B17, B20) screened in step (1) and rEg.P29 were respectively coated on ELISA plates, and the serum of mice immunized with rEg.P29 at different dilutions was used as the primary antibody to detect specific antibodies.

[0119] Test results such as Figure 7 The results showed that the titers of B4 and B15 were 1:25,600, B6, B7, and B8 were 1:12,800, B11, B12, and B20 were 1:3,200, and B16 and B17 were 1:1,600. The titer of rEg.P29, used as a control, reached 1:64,000.

[0120] comprehensive Figure 4, 5, 6, 7. Based on these results, we know that different adjuvants may select different B cell epitope peptides due to their different mechanisms of action. CpG adjuvant is an oligodeoxynucleotide containing a CpG sequence that has strong immune activation properties and few side effects, making it an adjuvant that can be used in humans. The results show that both CpG and Freund's adjuvant can enhance humoral immune responses. Considering the translational application of this vaccine, we tend to use CpG adjuvant in subsequent experiments. Combined with the types of specific antibodies, a comprehensive evaluation of antibody titers showed that among the epitope peptides mentioned above, B20 was able to produce anti-B20-specific IgM, IgG, IgG2b, and IgG2c antibodies in the sera of both adjuvant-immunized groups, with an antibody titer of 1:3200. Based on this comprehensive evaluation, we believe that the effect of B20 may be closer to that of rEg.P29. To further verify this, we also tested its ability to induce specific B lymphocyte activation and proliferation.

[0121] B20 amino acid sequence: (SEQ ID NO: 22)

[0122] TKLKNAKTAEQKAKWE

[0123] Example 6 B20 and rEg.P29 induce specific B cell activation

[0124] Take the mouse spleen, chop it into small pieces and place it on a filter to grind gently. Add the sample diluent while grinding. Prepare the spleen into a cell suspension of about 5 ml. Slowly add it to the 5 ml mouse spleen lymphocyte separation solution. Centrifuge at 450g for 20 minutes at room temperature. Take the lymphocyte layer, add the cleaning solution to wash and discard the supernatant to obtain the mouse spleen lymphocytes. Add or do not add rEg.P29 or B20 stimulation, culture at 37 degrees, 5% CO2 for 1 day, and then use flow cytometry to detect the activation markers CD25 and CD69. Figure 8 As shown, one day after B20 and rEg.P29 stimulation and culture, the expression of CD69 and CD25 in the immunized group increased compared with the PBS group, indicating that both B20 and rEg.P29 can induce specific B cell activation in the rEg.P29 immunized group mice, but the ability of B20 to induce specific B cell activation is slightly weaker than that of rEg.P29.

[0125] Example 7 B20 and rEg.P29 induce specific B cell proliferation

[0126] As described in Example 6, spleen lymphocytes from mice immunized with rEg.P29 were obtained, washed twice with preheated PBS, and then CFSE (Invitrogen, Carlsbad, USA, final concentration of 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 medium (containing 10% fetal bovine serum), incubated at 4°C for 5 minutes, and washed twice with pre-cooled RPMI1640 medium (containing 10% fetal bovine serum). CFSE-labeled cells were then cultured with or without B20 or rEg.P29. The cells were cultured at 37°C with 5% CO2. Cell samples were collected on days 3 and 5, and phenotypic analysis was performed using fluorescently labeled monoclonal antibodies stained at 4°C in the dark.

[0127] like Figure 9 As shown in the figure, compared with the PBS group, the CFSE fluorescence intensity decreased on the third day after B20 or rEg.P29 stimulation and continued to decrease on the fifth day, indicating that B20 or rEg.P29 stimulation can induce the proliferation of spleen-specific B lymphocytes in rEg.P29-immunized mice, and there is no statistical difference between the two ( Figure 10 ).

[0128] 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.

[0129] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the claims and their equivalents. Sequence Listing <110> Ningxia Medical University <120> Antigen polypeptide and its application <130> P0102021100791 <160> 26 <170> SIPOSequenceListing 1.0 <210> 1 <211> 717 <212> DNA <213> Artificial Sequence <400> 1 atgtccggat ttgacgttac taagactttc aatagattta cccagcgggc tggtgagctt 60 gtaaataaga atgaaaagac ctcatatcct acccgaacct cagatcttat ccatgagatc 120 gaccaaatga aagcatggat cagcaagatc atcaccgcta ctgaggaatt cgtagacatc 180 aacattgcat ctaaagtcgc ggatgctttc cagaagaata aggagaagat tactactacc 240 gacaaactgg gtactgctct cgagcaggtt gcttcccaat cagaaaaggc agctccccaa 300 ctttctaaaa tgctgacgga agcttctgat gtccatcagc gtatggccac tgccagaaag 360 aatttcaata gtgaggttaa taccaccttc attgaagatt tgaaaaactt cttgaacacc 420 acgcttagcg aggcccagaa agcaaagacc aagctggagg aggttcgact agatttggac 480 tctgacaaga ctaaattgaa gaatgctaag actgcggaac agaaggccaa gtgggaggcc 540 gaggtgcgaa aagacgaaag tgacttcgat cgagtgcacc aagaatctct tactatcttt 600 gagaagactt gcaaagaatt cgatgggttg agcgttcagc tgttggatct gatccgtgca 660 gagaagaatt actacgaagc ctgtgccaaa gagtgcagta tgatgctggg cgagtag 717 <210> 2 <211> 238 <212> PRT <213> Artificial Sequence <400> 2 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 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <400> 3 Ser Gln Ser Glu Lys Ala Ala Pro Gln Leu Ser Lys Met Leu Thr Glu 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <400> 4 Gln Arg Met Ala Thr Ala Arg Lys Asn Phe Asn Ser Glu Val Asn Thr 1 5 10 15 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <400> 5 Ser Asp Leu Ile His Glu Ile Asp Gln Met Lys Ala Trp Ile Ser Lys 1 5 10 15 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <400> 6 Ala Glu Val Arg Lys Asp Glu Ser Asp Phe Asp Arg Val His Gln Glu 1 5 10 15 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <400> 7 Ile Ser Lys Ile Ile Thr Ala Thr Glu Glu Phe Val Asp Ile Asn Ile 1 5 10 15 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <400> 8 Leu Glu Glu Val Arg Leu Asp Leu Asp Ser Asp Lys Thr Lys Leu Lys 1 5 10 15 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <400> 9 Ile Arg Ala Glu Lys Asn Tyr Tyr Glu Ala Cys Ala Lys Glu Cys Ser 1 5 10 15 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <400> 10 Glu Lys Thr Ser Tyr Pro Thr Arg Thr Ser Asp Leu Ile His Glu Ile 1 5 10 15 <210> 11 <211> 16 <212> PRT <213> Artificial Sequence <400> 11 Arg Phe Thr Gln Arg Ala Gly Glu Leu Val Asn Lys Asn Glu Lys Thr 1 5 10 15 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <400> 12 Thr Ile Phe Glu Lys Thr Cys Lys Glu Phe Asp Gly Leu Ser Val Gln 1 5 10 15 <210> 13 <211> 16 <212> PRT <213> Artificial Sequence <400> 13 Ser Lys Val Ala Asp Ala Phe Gln Lys Asn Lys Glu Lys Ile Thr Thr 1 5 10 15 <210> 14 <211> 16 <212> PRT <213> Artificial Sequence <400> 14 Thr Ala Glu Gln Lys Ala Lys Trp Glu Ala Glu Val Arg Lys Asp Glu 1 5 10 15 <210> 15 <211> 16 <212> PRT <213> Artificial Sequence <400> 15 Arg Val His Gln Glu Ser Leu Thr Ile Phe Glu Lys Thr Cys Lys Glu 1 5 10 15 <210> 16 <211> 16 <212> PRT <213> Artificial Sequence <400> 16 Lys Met Leu Thr Glu Ala Ser Asp Val His Gln Arg Met Ala Thr Ala 1 5 10 15 <210> 17 <211> 16 <212> PRT <213> Artificial Sequence <400> 17 Lys Glu Lys Ile Thr Thr Thr Asp Lys Leu Gly Thr Ala Leu Glu Gln 1 5 10 15 <210> 18 <211> 16 <212> PRT <213> Artificial Sequence <400> 18 Thr Asp Lys Leu Gly Thr Ala Leu Glu Gln Val Ala Ser Gln Ser Glu 1 5 10 15 <210> 19 <211> 16 <212> PRT <213> Artificial Sequence <400> 19 Val Asp Ile Asn Ile Ala Ser Lys Val Ala Asp Ala Phe Gln Lys Asn 1 5 10 15 <210> 20 <211> 16 <212> PRT <213> Artificial Sequence <400> 20 Phe Asp Val Thr Lys Thr Phe Asn Arg Phe Thr Gln Arg Ala Gly Glu 1 5 10 15 <210> twenty one <211> 16 <212> PRT <213> Artificial Sequence <400> twenty one Gly Leu Ser Val Gln Leu Leu Asp Leu Ile Arg Ala Glu Lys Asn Tyr 1 5 10 15 <210> twenty two <211> 16 <212> PRT <213> Artificial Sequence <400> twenty two Thr Lys Leu Lys Asn Ala Lys Thr Ala Glu Gln Lys Ala Lys Trp Glu 1 5 10 15 <210> twenty three <211> 16 <212> PRT <213> Artificial Sequence <400> twenty three Asn Phe Leu Asn Thr Thr Leu Ser Glu Ala Gln Lys Ala Lys Thr Lys 1 5 10 15 <210> twenty four <211> 16 <212> PRT <213> Artificial Sequence <400> twenty four Asn Phe Asn Ser Glu Val Asn Thr Thr Phe Ile Glu Asp Leu Lys Asn 1 5 10 15 <210> 25 <211> 16 <212> PRT <213> Artificial Sequence <400> 25 Asp Leu Asp Ser Asp Lys Thr Lys Leu Lys Asn Ala Lys Thr Ala Glu 1 5 10 15 <210> 26 <211> 16 <212> PRT <213> Artificial Sequence <400> 26 Thr Thr Phe Ile Glu Asp Leu Lys Asn Phe Leu Asn Thr Thr Leu Ser 1 5 10 15

Claims

1. An antigen polypeptide, characterized in that The amino acid sequence of the antigen polypeptide is shown in SEQ ID NO: 6, 8-10, 13-14, 17-19 or 22.

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. 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 treating or preventing echinococcosis.

6. A vaccine for echinococcosis, 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.

7. The vaccine according to claim 6, 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.

8. The vaccine according to claim 7, characterized in that The adjuvant is selected from FCA or CpG.

9. A method for preparing the vaccine according to any one of claims 6 to 8, characterized in that: The preparation method comprises mixing the antigen polypeptide according to claim 1 with an immunologically acceptable carrier.

10. 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.

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