CN111434680B

CN111434680B - Epitope peptide of IL 33-related diseases and application thereof

Info

Publication number: CN111434680B
Application number: CN201910036482.9A
Authority: CN
Inventors: 钱峰; 王军
Original assignee: Nanjing Saixin Biotechnology Co ltd
Current assignee: Nanjing Saixin Biotechnology Co ltd
Priority date: 2019-01-15
Filing date: 2019-01-15
Publication date: 2021-11-23
Anticipated expiration: 2039-01-15
Also published as: CN111434680A

Abstract

The invention relates to a novel epitope peptide of IL33 related diseases and application thereof, wherein the epitope peptide is as follows: (1) is positioned in SEQ ID NO. 1; or, (2) an amino acid sequence that is at least 80% identical to the sequence of seq id No.1 in (1). And the above-mentioned SEQ ID NO.1 contains one or more antigen epitopes, and the amino acid sequence length of the antigen epitope peptide is 2-70% of the full length of SEQ ID NO. 1. The application of the epitope peptide can comprise the preparation of vaccines or antibodies or antibody drugs and the like. The epitope peptide in the present application has immunogenicity, which can induce immune response. The epitope peptide or gene molecule, fusion protein, vector or host cell containing the epitope peptide can be used for preparing biological medicines, vaccines and the like, and has important application significance.

Description

Epitope peptide of IL 33-related diseases and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an epitope peptide of IL 33-related diseases and application thereof.

Background

IL33 is a new member of the IL1 family discovered in 2005. The human IL33 gene is located on chromosome 9p24.1 and encodes 270 amino acids. The full length IL33 has a molecular weight of about 30kDa and is cleaved into a mature protein of about 18kDa in size. IL33 is constitutively expressed in smooth muscle cells of various tissues in humans, as well as in tracheal and bronchial epithelial cells. In lung tissue fibroblasts and dermal keratinocytes, IL33 was induced by TNF-. alpha.stimulation.

IL33 has dual biological functions, under normal physiological condition, IL33 is positioned in a full-length form in a cell nucleus to play a role of a transcription factor and regulate the expression of other genes. When a cell is stimulated or damaged, IL33 is transferred from the nucleus to the cytoplasm and is cleaved into a mature protein, which acts as a cytokine. The mature IL33 is combined with ST2 molecules of a receptor thereof, activates an NF-kB pathway, and stimulates the production of proinflammatory factors such as IL5, IL13 and the like. Mature IL33, like an "alarm," is released upon injury to the body's receptors, recruits and activates a variety of immune cells such as neutrophils, eosinophils, mast cells, Th2 cells, etc., to release cytokines to elicit an inflammatory response.

The IL33/ST2 signaling pathway has been implicated in a variety of human diseases, such as asthma, rheumatoid arthritis, sepsis, heart disease, atherosclerosis, malignancies, and the like. Therefore, the research of the epitope related to IL33 has great significance for the research and development of antibody drugs or vaccines and the like, and the research and development of new drugs with even very good effects for treating asthma, rheumatoid arthritis, septicemia, heart diseases, atherosclerosis, malignant tumors and the like can be directly determined, so the research and development of a new epitope is very important for the medical field, and the research and development work is carried out.

Disclosure of Invention

In view of the above, the present invention aims to provide an epitope peptide of IL 33-related diseases and its application, which solves the deficiencies in the prior art.

The purpose of the invention is realized by the following technical scheme:

an object of the present invention is to provide an epitope peptide, which is: (1) is positioned in SEQ ID NO. 1; or, (2) an amino acid sequence that is at least 80% identical to the sequence of seq id No.1 in (1).

And the above-mentioned SEQ ID NO.1 contains one or more antigen epitopes, and the amino acid sequence length of the antigen epitope peptide is 2-70% of the full length of SEQ ID NO. 1.

Furthermore, the epitope peptide is located in a continuous amino acid sequence in the amino acid sequence from 65 th position to 160 th position in SEQ ID NO. 1.

Further, the epitope peptide is a continuous amino acid sequence in the amino acid sequence from 80 th position to 117 th position in SEQ ID NO. 1.

Furthermore, the epitope peptide is located in a continuous amino acid sequence in the amino acid sequence from the 100 th position to the 115 th position in SEQ ID NO. 1.

Furthermore, the epitope peptide is a continuous amino acid sequence located in the 108 th-112 th amino acid sequence in SEQ ID NO. 1.

Furthermore, the epitope peptide is an amino acid sequence which has more than 80% of overlapping degree with the continuous amino acids from 108 th position to 112 th position in SEQ ID NO. 1.

Further, SEQ ID NO.1 is a sequence located in IL 33.

Another objective of the invention is to provide a fusion protein formed by fusing the epitope peptide of any one of the above with a carrier protein.

It is still another object of the present invention to provide a gene molecule capable of encoding the epitope peptide, wherein the gene molecule is a gene sequence or a gene fragment capable of encoding the epitope peptide.

It is still another object of the present invention to provide an expression vector containing the gene molecule described above.

It is a further object of the present invention to provide a host cell comprising an expression vector as described above or having integrated into its genome a nucleic acid molecule as described above.

An object of the present invention is to provide an antibody capable of binding to the epitope peptide described in any one of the above.

It is still another object of the present invention to provide an isolated antigen-antibody complex, wherein the antigen of the complex comprises the epitope peptide described in any one of the above or the fusion protein described above, the epitope peptide forms an epitope, and the antibody specifically binds to the epitope.

It is a final object of the present invention to provide a vaccine comprising the epitope peptide of any one of the above, the fusion protein of the above, the nucleic acid molecule of the above, the expression vector of the above, or the host cell of the above.

The invention provides an epitope peptide of IL33 related diseases and application thereof, which at least have the following beneficial effects: the epitope peptide has immunogenicity, and can induce and generate immune response reaction. The epitope peptide or gene molecule, fusion protein, vector or host cell containing the epitope peptide can be used for preparing biological medicines, vaccines and the like, and has important application significance.

The epitope peptide has a particularly strong binding effect with the antibody in the application, the affinity is very good, the concentration of the antibody can be as low as pmol level, the affinity can be comparable with that of herceptin, the method is very surprising, and the binding force is stable. This has an extremely important role for an antigen or antibody to exert a medicinal effect. Because the interaction effect among the antigen antibodies is good, the antibody has great potential medicinal value for preparing medicaments, and the medicaments containing the antibodies can treat, prevent or relieve IL-33 related diseases, such as asthma, rheumatoid arthritis, septicemia, heart diseases, atherosclerosis, malignant tumors and the like.

In addition, the antibody has excellent neutralizing activity, can neutralize IL33 to stimulate macrophage (PM) to induce the bioactivity of IL6 and TNF-alpha, and has obvious inhibiting effect. Can also neutralize the expression of IL6 in mast cells and bone marrow cells, neutralize OVA-induced asthma, reduce the total cell number and protein content in bronchoalveolar lavage fluid, and has remarkable effect and the like.

Drawings

FIG. 1 is a schematic sequence diagram of SEQ ID NO. 1;

FIG. 2 is a schematic diagram of vector construction of different fragments in SEQ ID NO.1 sequence;

FIG. 3 is a schematic diagram of the electrophoresis of three fragments induced by IPTG expression;

FIG. 4 is a schematic representation of the binding of three fragments to an antibody;

FIG. 5 is a schematic representation of the binding of fragments 1-9 to an antibody;

FIG. 6 is a diagram showing the binding of four amino acid fragments to an antibody.

FIG. 7 is a schematic representation of IL6 production by macrophages induced by antibody neutralization of IL33 in cooperation with LPS;

FIG. 8 is a graph showing that neutralization of IL33 by antibodies in cooperation with LPS induces TNF- α production by macrophages;

FIG. 9 is a schematic of the purity of induced mast cells;

FIG. 10 is a standard curve of ELISA plotted when the amount of IL6 was measured;

FIG. 11 is a schematic of the testing of IL33 antibody for blocking activity with induced mast cells;

FIG. 12 is another schematic of the testing of IL33 antibody for blocking activity with induced mast cells;

FIG. 13 shows that IL33 antibody neutralizes IL6 expression in bone marrow cells;

figure 14 is a graph reflecting the effectiveness of IL33 antibody in treating an OVA-induced asthma model;

figure 15 is a schematic of antibody neutralization of OVA-induced asthma;

FIG. 16 is a schematic diagram showing antigen-antibody binding;

FIG. 17 is a schematic representation of the binding status of McAb-IL33 at different dilution concentrations;

FIG. 18 is a graph showing the binding of CA-IL33 at various dilution concentrations;

FIG. 19 is a standard curve of a normal distribution fitted;

FIG. 20 is a diagram schematically showing the detection of Tm values of the antibodies in the present application.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. The detailed description of the embodiments of the invention provided below is not intended to limit the scope of the claimed invention, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the scope of protection of the present invention.

Example 1

An epitope peptide, which is: (1) is positioned in SEQ ID NO. 1; or, (2) an amino acid sequence that is at least 80% identical to the sequence of seq id No.1 in (1); that is, SEQ ID No.1 can be an amino acid sequence which is by itself or which has at least 80% identity to itself. The specific sequence of SEQ ID NO.1 is shown in sequence 1 in the sequence table, namely the first sequence.

In this embodiment, seq id No.1 is a sequence in IL33, such as seq id No.1 in humanized IL 33.

In the embodiment, the epitope peptide has immunogenicity, can induce immune response, and has an important role in research of biological medicines, vaccines and the like.

Example 2

On the basis of example 1, preferably, the epitope peptide is located in a continuous amino acid sequence in the amino acid sequence from 65 th to 160 th in SEQ ID NO. 1.

Further preferably, the epitope peptide is a continuous amino acid sequence located in the amino acid sequence from 80 th to 117 th positions in SEQ ID NO. 1.

More preferably, the epitope peptide is a continuous amino acid sequence located in the amino acid sequence from position 100 to position 115 in SEQ ID NO. 1.

In a further preferred embodiment, the epitope peptide is a continuous amino acid sequence located in the amino acid sequence from position 108 to 112 of SEQ ID NO. 1. More preferably, the epitope peptide is a continuous amino acid sequence from 108 th to 112 th in SEQ ID NO.1, i.e. a sequence FVLN.

Meanwhile, the epitope peptide can also be an amino acid sequence which has more than 80 percent of overlapping degree with the continuous amino acids from 108 th to 112 th in SEQ ID NO. 1.

Example 3

In accordance with any of the above embodiments, a fusion protein is formed by fusing an epitope peptide described herein to a carrier protein.

Example 4

A gene molecule capable of encoding the epitope peptide, wherein the gene molecule is a gene sequence or a gene fragment capable of encoding the epitope peptide described in example 1 or 2. It can be used for coding and expressing epitope peptide.

Example 5

An expression vector comprising the gene molecule of example 4. Which can express epitope peptide.

Example 6

A host cell comprising the expression vector of example 5 or having integrated into its genome the genetic or nucleic acid molecule of example 4.

Example 7

A vaccine comprising the epitope peptide of example 1 or 2, the fusion protein of example 3, the nucleic acid molecule of example 4, the expression vector of example 5, or the host cell of example 6, based on any of the above examples.

The vaccine may further comprise an immunologically acceptable carrier and/or adjuvant.

In this example, the epitopes in the present application can be formulated as vaccines.

Example 8

An antibody capable of binding to the epitope peptide of example 1 or 2, based on any of the above examples.

The antibody can inhibit or weaken the epitope peptide described in example 1 or 2, namely, the antibody can treat the corresponding diseases caused by the epitope peptide described in the application, and the antibody can inhibit or weaken IL33 mediated signal transmission because the SEQ ID NO.1 can be derived from IL 33.

Such as: the antibody can be used for preparing medicines for treating or relieving asthma, rheumatoid arthritis, septicemia, heart disease, atherosclerosis or malignant tumor.

Example 9

An isolated antigen antibody complex, wherein the antigen in the complex comprises the epitope peptide of example 1 or the epitope peptide that forms an epitope, or the fusion protein of example 3, wherein the antibody in the complex specifically binds to the epitope, and wherein the antibody can be the antibody of example 8.

Example 10

On the basis of

embodiment

1 or 2, SEQ ID NO.1 is shown as sequence 1 (i.e. the first sequence) in the sequence table, and also see FIG. 1.

The amino acid sequence of SEQ ID NO.1 was divided into fragment A (amino acid fragment 1-80), fragment B (amino acid fragment 81-160) and fragment C (amino acid fragment 65-117), wherein GST (glutathione mercaptotransferase) + IL 33-A: about 36.8kDa, GST + IL 33-B: about 37.0kDa, GST + IL 33-C: about 33.96 kDa. And the three fragments are used for constructing a vector according to a conventional method, as shown in figure 2, and then are induced by IPTG, and as a result, the three fragments are induced to be expressed uniformly by IPTG, as shown in figure 3.

The Western Blot method was used to identify the binding properties of the peptide fragments of the three fragments to the antibodies described in this application, and the antigen binding sites were found to be located in fragment B and fragment C according to the visualization, as shown in fig. 4.

By adopting the method, the 81 th-117 th amino acid fragment is identified and respectively divided into a fragment 1, a fragment 2, a fragment 3, a fragment 4, a fragment 5, a fragment 6, a fragment 7, a fragment 8 and a fragment 9, the sequences of the nine fragments are shown in a sequence 2-10 in a sequence table, namely, the fragment 1 corresponds to the sequence 2, the fragment 2 corresponds to the sequence 3, and by analogy, the fragment 9 corresponds to the sequence 10; the 9 fragments were identified for their binding properties to the antibodies described in this application and, as a result, the epitope regions were found to be located on

fragments

1, 2, 3, 4, 5 and 6, as shown in FIG. 5, based on the visualization.

Similarly, the binding properties of the four sequences FVLHN, FFVLHN, affvlhhn and QAFFVLHN to the antibody described in the present application were identified by the above method, and as a result, the epitope regions were found to be located on the four fragments by development, as shown in fig. 6. Through more detailed research, the epitope is comprehensively determined to be an IL33 sequence containing the FVLN sequence.

Example 11

Based on example 8, the antibody is an antibody or binding fragment capable of inhibiting or attenuating epitope peptide or IL 33-mediated signaling described in any of the above examples. The antibody includes a light chain L chain and a heavy chain H chain.

The antibody comprises hypervariable regions (HVR regions) having immunogenicity, the hypervariable regions of the L chain comprising an L chain first hypervariable region LHVR1, an L chain second hypervariable region LHVR2 and an L chain third hypervariable region LHVR 3; the LHVR1 is selected from any one of sequences SEQ 1-SEQ 12 in LHVR1, the LHVR2 is selected from any one of sequences SEQ 1-SEQ 12 in LHVR2, and the LHVR3 is selected from any one of sequences SEQ 1-SEQ 12 in LHVR 3.

The hypervariable regions of the H chain comprise a first hypervariable region HHVR1 of the H chain, a second hypervariable region HHVR2 of the H chain and a third hypervariable region HHVR3 of the H chain, wherein the HHVR1 is selected from any one of sequences HHVR1 in SEQ 1-SEQ 12, the HHVR2 is selected from any one of sequences HHVR2 in SEQ 1-SEQ 12, and the HHVR3 is selected from any one of sequences HHVR3 in SEQ 1-SEQ 12. See in particular table 1.

TABLE 1 hypervariable region sequences of antibody light chain heavy chains

In a preferred embodiment, the antibody L chain is an L chain as described in any one of the following (1) to (12): (1) an L chain consisting of the LHVR1, LHVR2 and LHVR3 of line SEQ1, (2) an L chain consisting of the LHVR3, LHVR3 and LHVR3 of line SEQ3, (3) an L chain consisting of the LHVR3, LHVR3 and LHVR3 of line SEQ3, (4) an L chain consisting of the LHVR3, LHVR3 and LHVR3 of line SEQ3, (5) an L chain consisting of the LHVR3, LHVR3 and LHVR3 of line SEQ3, (6) an L chain consisting of the LHVR3, LHVR3 and LHVR3 of line SEQ3, (7) an L chain consisting of the LHVR3, LHVR3 and LHVR3 of line SEQ3, (8) an L chain consisting of the LHVR3, LHVR3 and LHVR3 of line SEQ vr3 of line SEQ3 and LHVR3 of line SEQ3, and LHVR (72) an L chain consisting of the LHVR3 and LHVR3 of line SEQ3 and LHVR) and LHVR (72 of line SEQ 3) and LHVR) of line SEQ3 and LHVR) of line 3 of line SEQ3 and LHVR (or LHVR) and LHVR) of line SEQ3 of line SEQ3 of line vr) and LHVR (14 of line SEQ3 of line SEQ3 of line vr) and LHVR) are composed of SEQ3 of line SEQ3 of SEQ3 and LHVR) are composed of SEQ3 of line vr) are composed of SEQ3 of line SEQ3 and LHVR (8) and LHVR) are composed of SEQ3 of line vr (or LHVR) are composed of SEQ3 of line SEQ3 of line vr) and LHVR) are composed of SEQ3 and LHVR).

The antibody H chain is the H chain described in any one of the following (1) to (12): (1) an H chain consisting of HHVR1, HHVR2 and HHVR3 of SEQ1 line, (2) an H chain consisting of HHVR3, HHVR3 and HHVR3 of SEQ3 line, (3) an H chain consisting of HHVR3, HHVR3 and HHVR3 of SEQ3 line, (4) an H chain consisting of HHVR3, HHVR3 and HHVR3 of SEQ3 line, (5) an H chain consisting of HHVR3, HHVR3 and HHVR3 of SEQ3 line, (6) an H chain consisting of HHVR3, HHVR3 and HHVR3 of SEQ3 line, (7) an H chain consisting of HHVR3, HHVR3 and HHVR3 of SEQ3 line, (8) an H chain consisting of HHVR3, HHVR3 and HHVR3 of SEQ3 line, (72 and HHVR3 of HHVR3 and HHVR3 of SEQ3 line, and HHVR3 of HHVR3 and HHVR3 of SEQ3 line, (72 and HHVR3 of HHVR3 and HHVR3 of SEQ3 and HHVR3 of HHVR3 and HHVR3 of SEQ3 line, (8) an HHVR3 and HHVR3 of SEQ3 and HHVR3 of HHVR3 and HHVR3 of SEQ3 and HHVR3 of SEQ3 of HHVR3 and HHVR3 of SEQ3 and HHVR3 of SEQ3 and HHVR3 of HHVR3 and HHVR3 of SEQ3 and HHVR3 of SEQ3 and HHVR3 of HHVR3 and HHVR3 of SEQ3 and HHVR3 of HHVR3 and HHVR3 of SEQ3 and HHVR 368) of SEQ3 and HHVR 3612 of SEQ3 and HHVR 3612 of SEQ3 and HHVR 36.

As a further preferred embodiment, the hypervariable region HVR region (i.e., CDR region) of the antibody is the HVR region described in any one of the following (1) to (12): (1) an HVR region composed of an L chain HVR region and an H chain HVR region of the row of SEQ1, (2) an HVR region composed of an L chain HVR region and an H chain HVR region of the row of SEQ2, (3) an HVR region composed of an L chain HVR region and an H chain HVR region of the row of SEQ3, (4) an HVR region composed of an L chain HVR region and an H chain HVR region of the row of SEQ4, (5) an HVR region composed of an L chain HVR region and an H chain HVR region of the row of SEQ5, (6) an HVR region composed of an L chain HVR region and an H chain HVR region of the row of SEQ6, (7) an HVR region consisting of the L chain HVR region and the H chain HVR region of the row of SEQ7, (8) an HVR region consisting of the L chain HVR region and the H chain HVR region of the row of SEQ8, (9) an HVR region consisting of the L chain HVR region and the H chain HVR region of the row of SEQ9, (10) an HVR region consisting of the L chain HVR region and the H chain HVR region of the row of SEQ10, (11) an HVR region consisting of the L chain HVR region and the H chain HVR region of the row of SEQ11, or (12) an HVR region consisting of the L chain HVR region and the H chain HVR region of the row of SEQ 12.

Example 12

On the basis of example 11, the antibody is an antibody comprising a variable region described in any one of the following (1) to (12): (1) the variable region of the H chain of the antibody is shown as a sequence 11 in a sequence table, and the variable region of the L chain is shown as a sequence 23 in the sequence table; (2) the variable region of the antibody H chain is shown as a sequence 12 in the sequence table, and the variable region of the L chain is shown as a sequence 24 in the sequence table; (3) the variable region of the H chain of the antibody is shown as a sequence 13 in a sequence table, and the variable region of the L chain is shown as a sequence 25 in the sequence table; (4) the variable region of the H chain of the antibody is shown as a sequence 14 in a sequence table, and the variable region of the L chain is shown as a sequence 26 in the sequence table; (5) the variable region of the H chain of the antibody is shown as a sequence 15 in a sequence table, and the variable region of the L chain is shown as a sequence table

Sequence 27 in (b); (6) the H chain variable region of the antibody is shown as a sequence 16 in a sequence table, and the L chain

The variable region of (b) is shown as a sequence 28 in a sequence table; (7) the H chain variable region of the antibody is shown in a sequence table

The variable region of the L chain is shown as a sequence 29 in a sequence table; (8) the H chain of the antibody may

The variable region is shown as a sequence 18 in a sequence table, and the variable region of an L chain is shown as a sequence 30 in the sequence table; (9) the variable region of the H chain of the antibody is shown as a sequence 19 in a sequence table, and the variable region of the L chain is shown as a sequence 31 in the sequence table; (10) the variable region of the H chain of the antibody is shown as a sequence 20 in a sequence table, and the variable region of the L chain is shown as a sequence 32 in the sequence table; (11) the variable region of the H chain of the antibody is shown as a sequence 21 in a sequence table, and the variable region of the L chain is shown as a sequence 33 in the sequence table; (12) the variable region of the H chain of the antibody is shown as a sequence 22 in a sequence table, and the variable region of the L chain is shown as a sequence 34 in the sequence table. The sequences 11 to 22 and the sequences 23 to 34 refer to the sequences in the sequence table.

As a further preferred embodiment, the antibody is a variable domain comprising sequence 15 and sequence 27, a variable domain comprising sequence 16 and sequence 28, a variable domain comprising sequence 17 and sequence 29, or a variable domain comprising sequence 18 and sequence 30.

The antibodies in this example are capable of specifically recognizing binding to the epitope peptide sequences described herein. In addition, since the constant regions of antibodies have the same sequence in different antibodies and do not have the property of specifically binding to antigens, the sequence of the constant region is not important for protection in the present application, as seen in the prior art.

Example 13

On the basis of any of the above examples, the antibody comprises a human murine chimeric antibody or a murine monoclonal antibody whose sequence comprises the sequence of the hypervariable region in example 11 or the sequence of the variable region described in example 12. That is, the sequence of the variable region of either the human murine chimeric antibody or the murine monoclonal antibody may be the protein sequence described in example 11 or 12.

The constant region of the murine monoclonal antibody is derived from the constant region of a murine antibody, and the constant region of the human murine chimeric antibody is the constant region of a human antibody. However, the constant regions of both murine and human antibodies have no antigen-binding properties, and the constant regions of murine monoclonal antibodies correspond in sequence to the constant regions of other murine antibodies and the constant regions of human chimeric antibodies correspond in sequence to the constant regions of other human antibodies, so that the sequences of the constant regions can be found in the prior art.

Example 14

A medicament comprising an antibody as described in any one of the above embodiments, which medicament is capable of treating, preventing or ameliorating a disease associated with IL 33.

The medicament may include medicaments for treating or alleviating asthma, rheumatoid arthritis, sepsis, heart disease, atherosclerosis, malignant tumors.

Example 15: IL33 antibody biological neutralization activity assay

IL33 synergizes with LPS (lipopolysaccharide) to induce macrophages to express more IL6 and TNF-alpha (tumor necrosis factor alpha): after mouse macrophage is pre-stimulated by IL33, the expression level of receptor complex MD2, TLR4 and MyD88 of LPS is increased, after IL33 is removed, the macrophage is stimulated by LPS, and the expression level of downstream cell factor is increased.

The method comprises the following steps: 1) c57 mice for 8 weeks, i.p. 2ml of 4% broth; 2) after 4 days, PBS or 1640 medium added with double antibody (double antibody refers to penicillin and streptomycin) is used for washing the abdominal cavity, and abdominal cavity macrophages are collected; 3) after 2 times of rinsing, counting, inoculating 1x106 cells to a 24-well plate, 0.5 ml/well, wherein the used culture medium is 1640+ 10% FBS + double antibody; 4) culturing for 2h in a 5% CO2 incubator at 37 ℃, discarding the supernatant of the original culture medium, rinsing each hole with 0.5ml of the culture medium, removing the cells which are not attached to the wall, and changing the culture medium to 0.5 ml/hole; 5) adding IL33 with the final concentration of 50ng/ml or IL33 pre-stimulation after being incubated by the antibody, detecting TNF at 6h and detecting IL6 at 8 h. Incubation conditions of IL33 with antibody: 1h at room temperature; the antibody is the monoclonal antibody claimed in the application, namely Anti-IL 33; 6) after pre-stimulation, discarding the supernatant, changing the solution to 0.5 ml/hole, adding LPS with the final concentration of 100ng/ml for stimulation for 24h, and collecting the supernatant; 7) ELISA detects IL6, TNF-alpha.

As a result: as shown in the attached figures 7-8, the Anti-IL33 can neutralize IL33 and stimulate macrophage (PM) to induce the biological activity of IL6 and TNF-alpha by cooperating with LPS, and the inhibition effect is obvious.

Example 16: IL33 antibody biological neutralization activity assay

IL33 induces primary mast cells to express large amounts of IL 6: the mast cell expresses a receptor ST2 molecule of IL33, is a main target cell of IL33, and IL33 can activate the mast cell to produce a proinflammatory factor IL 6.

The method comprises the following steps: 1) taking bone marrow mononuclear by conventional methodA cell; 2)1x10⁶Inoculating 1640 complete culture medium at a concentration of one/ml, and co-stimulating with embryonic cytokine (SCF) at a concentration of 50ng/ml and IL 330 ng/ml for two weeks; 3) IL 330 ng/ml was stimulated alone for 5 weeks; 4) in the cell culture process, suspension cells are collected every week, the solution is changed by centrifugation, the suspension cells are transferred to a new culture dish, and adherent cells are discarded; 5) mast cells were stained 7 weeks later with antibody Fc ε R1-FITC and antibody CD117-APC, and purity was checked by flow; 6) adjust the concentration of the master cell to 1 × 10⁶Spreading the mixture in a 96-well plate at a concentration of 200 ul/ml; 7) the stimulation concentration of IL33 is 100ng/ml, and the stimulation time is 24 h; 8) pre-incubation of the antibody group with IL33 for 20Min at room temperature; the antibody group herein includes monoclonal antibody MCAB of murine origin and chimeric antibody CA of human murine origin, wherein McAb-IL 3310 UL (1mg/ml), chimeric antibody CA-IL 3320 UL (30 ug/ml); 9) after 4h, the supernatant was collected and the expression level of IL6 was measured by ELISA.

As a result: referring to FIGS. 9-12, it can be seen from FIG. 9 that both proteins CD117-APC and Fc ε R1-FITC are in the lower left negative region without induction, and that the amount of both proteins is shifted up and to the right after induction, respectively, i.e., mast cells are increased, indicating that mast cells have been efficiently induced. As can be seen from fig. 10, when the amount of IL6 was measured, a standard curve of ELISA was plotted, and the results of the present experiment, which are linear, were valid and reliable. As can be seen from fig. 11, the applicant tested the blocking activity of IL33 antibody with induced mast cells. Since IL33 can stimulate the mass cell to express IL6, and pre-incubation of IL33 antibody can block IL6 produced by such stimulation, indicating that the antibody is active, fig. 11 shows that the monoclonal antibody MCAB-IL33 and the human-mouse chimeric antibody CA-IL33 in the present application have very good activity and can greatly block the expression of IL6, i.e. the antibody in the present application can neutralize the expression of IL6 in mast cells. FIG. 12 shows the two-week induction of mast cells, indicating that MCAB-IL33 and CA-IL33 are very active and can greatly block the expression of IL 6.

In addition, FIG. 13 is a schematic diagram showing that IL33 antibody can neutralize IL6 expression in bone marrow cells, and thus it is known that MCAB-IL33 and CA-IL33 have very good activity and can greatly block the expression of IL6 in bone marrow cells.

Example 17: IL33 antibody biological neutralization activity assay

IL33 mab alleviates OVA-induced asthma model:

1) the establishment method of the asthma model induced by OVA (ovalbumin) is as follows: on day 0, 100ul of 75ug of OVA solution was mixed with 100ul of aluminum, and then injected subcutaneously with 200ul of the mixture, calculated as 75ug of OVA per mouse. Day 10, the operation is the same as day 0; ③ day 21, 22 and 23, tracheal administration of 50ug of OVA per mouse; fourthly, material is taken on the 25 th day; antibody treatment group and isotype control group, 30Min before each administration of OVA, and 150ug of antibody is injected into abdominal cavity. The antibody is the murine monoclonal antibody McAb-IL33(anti-IL 33).

2) Counting the total cell amount in lung lavage fluid

Counting by serum counting plate

3) Detection of Total protein concentration in Lung lavage fluid

BCA method for detecting protein concentration

4) Flow analysis of eosinophil proportion in Lung lavage fluid

Cells in lung lavage fluid were labeled with flow antibodies CD11C-APC and SiglecF-PE, macrophages were labeled with double positive, positive SiglecF, negative CD11C represented eosinophils.

As a result: as shown in fig. 14-15, fig. 14 reflects the effectiveness of IL33 antibody in treating OVA-induced asthma models. The upper left panel "con" is a flow chart of lung lavage fluid cells from normal mice, and both marker proteins, CD11C and siglecF, represent macrophages if both are positive, so the cells in lung lavage fluid from normal mice are mostly macrophages. The upper right panel shows the OVA group without antibody treatment, and the flow pattern shows a large number of siglecF-positive, CD 11C-negative cells representing eosinophils, which is indicative of asthma, indicating successful modeling. The lower left panel shows the IL33 antibody treatment group (Ova + anti-IL33), i.e., with McAb-IL33 added, which shows similar to normal mice, the degree of asthma is significantly reduced and the effect is significant, as macrophages account for the majority and eosinophils account for the minority of lung lavage fluid. The lower right panel shows the control treated group of mouse IgG (Ova + mIgG) with a large proportion of eosinophils, indicating ineffective treatment. Fig. 15 is a graph showing that antibody neutralization of OVA-induced asthma is shown, and it can be seen from fig. 15 that antibody anti-IL33 is capable of reducing total cell number in bronchoalveolar lavage fluid, and has a significant effect compared to control mIgG.

Example 18: identification of antibody affinity

Surface Plasmon Resonance (SPR) is an optical physical phenomenon that can be centered on a biosensor chip and applied to detecting the interaction between molecules and monitoring the whole process of the interaction of biomolecules in real time. The main principle is that when a beam of parallel light enters the end face of the prism, plasma wave is generated between the prism and the metal film, and when the propagation constant of the light wave is matched with that of the plasma wave, free electrons in the metal film are caused to generate resonance, namely surface plasma resonance.

An antigen (such as human IL33) containing the epitope peptide claimed in the application is coated on a metal film, and the affinity of the antigen and IL33 monoclonal antibody McAb-IL33 and IL33 chimeric antibody CA-IL33 is tested. A schematic representation of antigen-antibody binding is shown in FIG. 16.

The different curves in fig. 17 and 18 are for antibodies that were diluted at equal fold to bind to the antigen containing FVLHN sequences as described herein. Theoretically, fig. 17 and 18 should first rise to a plateau and then fall, but the binding of the antibodies of the present application is too strong, resulting in no fall of the curve. In FIG. 17, for McAb-IL33, its Ka (1/Ms): 1.469E +5, Kd (1/s):2.541E-5, KD ═ Kd/Ka ═ 1.730E-10(M) ═ 173.0 pM; in FIG. 18, for CA-IL33, Ka (1/Ms): 1.447E +5, Kd (1/s):1.691E-6, KD ═ Kd/Ka ═ 1.169E-11(M) ═ 11.69 pM; the KD value can directly and powerfully reflect the strength of the binding force of the antibody and the antigen, the lower the concentration is, the higher the binding force is reflected, the KD value of the McAb-IL33 in the application is 173.0pmol, and the KD value of the CA-IL33 in the application is 11.69 pmol.

Example 19: detection of Tm value of antibody

Using NANO DSC instrument from TA, the monoclonal antibody concentration was about 1mg/ml, and the volume was about 1 ml. The temperature is gradually increased within the range of 20-95 ℃ at 1 ℃/min, and the control group is PBS solution.

Graphs of Tm values, as shown in fig. 19-20, are measured for thermal stability of antibodies by DSC (differential scanning calorimetry) principle, and fig. 19 is a fitted normal distribution standard curve. FIG. 20 is a graph of the Tm values measured in the present application, which substantially coincides with the graph of FIG. 19, and the Tm values are directly shown in FIG. 20. The detection of the Tm value revealed that the Tm value of the present invention is high and the antibody has good thermal stability.

It should be noted that the detection of neutralization activity, affinity, Tm value, and the like (i.e., the detection in examples 13 to 17) in the present application are all detected by taking as an example an antibody a murine monoclonal antibody and/or a human murine chimeric antibody comprising an H chain variable region consisting of sequence 17 (see sequence listing) and an L chain variable region consisting of sequence 29; the antigen is the epitope peptide claimed by the application.

The epitope peptide sequence and related auxiliary subjects, the antibody sequence and related auxiliary subjects and the like provided by the application belong to the invention of the application.

In this embodiment, the antigen can be prepared by artificial synthesis, isolation or other biological methods; the preparation method of the antibody comprises the following steps: artificial synthesis, biological methods or combination of biological and chemical methods, etc. may be performed according to the antibody sequence. The specific method may be any method known in the art, as long as the sequence is ensured to be identical to the antibody sequence in the above examples.

The above description is only a preferred embodiment of the invention and is not intended to limit the invention, and various modifications and changes may be made to the invention by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the invention should be included in the protection scope of the invention.

Sequence listing

<110> Nanjing Sai New Biotechnology Co., Ltd

<120> novel epitope peptide of IL 33-related diseases and application thereof

<130> 1800009-I

<160> 34

<170> SIPOSequenceListing 1.0

<210> 1

<211> 160

<212> PRT

<213> Synthesis (Synthesis)

<400> 1

Ser Ser Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu

1 5 10 15

Ser Thr Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser

20 25 30

Tyr Glu Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu Lys Lys Asp Lys

35 40 45

Val Leu Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly

50 55 60

Asp Gly Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys

65 70 75 80

Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His

85 90 95

Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn

100 105 110

Met His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro Gly Val

115 120 125

Phe Ile Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val Asp Ser

130 135 140

Ser Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys Leu Ser Glu Thr

145 150 155 160

<210> 2

<211> 37

<212> PRT

<213> Synthesis (Synthesis)

<400> 2

Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His

1 5 10 15

Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn

20 25 30

Met His Ser Asn Cys

35

<210> 3

<211> 30

<212> PRT

<213> Synthesis (Synthesis)

<400> 3

Asn Lys Glu His Ser Val Glu Leu His Lys Cys Glu Lys Pro Leu Pro

1 5 10 15

Asp Gln Ala Phe Phe Val Leu His Asn Met His Ser Asn Cys

20 25 30

<210> 4

<211> 26

<212> PRT

<213> Synthesis (Synthesis)

<400> 4

Ser Val Glu Leu His Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe

1 5 10 15

Phe Val Leu His Asn Met His Ser Asn Cys

20 25

<210> 5

<211> 20

<212> PRT

<213> Synthesis (Synthesis)

<400> 5

Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn Met

1 5 10 15

His Ser Asn Cys

20

<210> 6

<211> 14

<212> PRT

<213> Synthesis (Synthesis)

<400> 6

Asp Gln Ala Phe Phe Val Leu His Asn Met His Ser Asn Cys

1 5 10

<210> 7

<211> 32

<212> PRT

<213> Synthesis (Synthesis)

<400> 7

Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His

1 5 10 15

Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn

20 25 30

<210> 8

<211> 27

<212> PRT

<213> Synthesis (Synthesis)

<400> 8

Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His

1 5 10 15

Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe

20 25

<210> 9

<211> 23

<212> PRT

<213> Synthesis (Synthesis)

<400> 9

Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His

1 5 10 15

Lys Cys Glu Lys Pro Leu Pro

20

<210> 10

<211> 17

<212> PRT

<213> Synthesis (Synthesis)

<400> 10

Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His

1 5 10 15

Lys

<210> 11

<211> 105

<212> PRT

<213> Synthesis (Synthesis)

<400> 11

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Asp Trp Gly Gly Asp Tyr

20 25 30

Pro Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser His Trp Trp Gly Ala Ala Tyr Asp Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Gln Arg Cys Tyr Ala Asn Gln Val Ser

100 105

<210> 12

<211> 105

<212> PRT

<213> Synthesis (Synthesis)

<400> 12

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Trp Asn Asn Asp Tyr

20 25 30

Pro Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ala Lys Tyr Thr Leu Pro Tyr Asp Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Thr Arg Arg Asp Ala Ala Val Val Ser

100 105

<210> 13

<211> 105

<212> PRT

<213> Synthesis (Synthesis)

<400> 13

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Thr Ala Ser Ser Phe Leu Phe Ile Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Gln Gln Gly Gly Arg Trp Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Val Cys Leu Pro Ala Pro His Val Ser

100 105

<210> 14

<211> 105

<212> PRT

<213> Synthesis (Synthesis)

<400> 14

Glu Val Lys Leu Val Glu Ser Gly Gly Val Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Met Leu Ser Cys Thr Ala Ser Gly Phe Ala Phe Asn Asp Gly

20 25 30

Gly Met Ser Trp Val Val Gln Thr Pro Glu Lys Ile Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Cys Gly Gly Arg Tyr Ile Tyr Tyr Pro Pro Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Met Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Pro Arg Leu Pro Trp Pro Phe Val Ala

100 105

<210> 15

<211> 105

<212> PRT

<213> Synthesis (Synthesis)

<400> 15

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Ala Thr Phe Asn Trp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Val Gly Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Thr Arg Leu Pro Lys Pro Phe Val Ser

100 105

<210> 16

<211> 112

<212> PRT

<213> Synthesis (Synthesis)

<400> 16

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Leu Lys Leu Ser Cys Ser Ser Lys Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ile Arg Ser Gly Gly Leu Ile Phe Tyr Leu Asp Ser Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Met Ser Glu Asp Thr Ala Phe Tyr Tyr Cys

85 90 95

Val Arg Val Tyr Tyr Pro Tyr Gly Gly Ser Pro Tyr Tyr Phe Asp Tyr

100 105 110

<210> 17

<211> 105

<212> PRT

<213> Synthesis (Synthesis)

<400> 17

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Thr Arg Leu Pro Ala Pro Phe Val Ser

100 105

<210> 18

<211> 105

<212> PRT

<213> Synthesis (Synthesis)

<400> 18

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Asp Trp Asn Gly Asp Tyr

20 25 30

Pro Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser His Thr Trp Gly Ala Ala Tyr Asp Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Gln Arg Cys Pro Ala Asn Gln Val Ser

100 105

<210> 19

<211> 112

<212> PRT

<213> Synthesis (Synthesis)

<400> 19

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ala Gly Lys Asp Phe Arg Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Ser Asp Val Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Trp Gly Leu Ile Phe Tyr Leu Asp Ser Leu

50 55 60

Lys Pro Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Met His Val Asp Thr Ala Phe Tyr Tyr Cys

85 90 95

Ala Cys Val Ile Ser Tyr Tyr Gly Gly Ser Pro Tyr Tyr Phe Val Ser

100 105 110

<210> 20

<211> 112

<212> PRT

<213> Synthesis (Synthesis)

<400> 20

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ala Gly Phe Thr Phe Arg Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Gly Leu Ile Phe Tyr Leu Asp Ser Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Met Ser Glu Asp Thr Ala Phe Tyr Tyr Cys

85 90 95

Val Arg Val Ile Ser Tyr Tyr Gly Gly Ser Pro Tyr Tyr Phe Asp Tyr

100 105 110

<210> 21

<211> 113

<212> PRT

<213> Synthesis (Synthesis)

<400> 21

Glu Val Ser Leu Val Glu Ser Gly Gly Gly Leu Val Gly Pro Gly Ala

1 5 10 15

Ser Leu Lys Leu Ser Val Ala Ala Ala Gly Phe Thr Phe His Arg Asp

20 25 30

Tyr Ala Met Ser Trp Val Arg Thr Thr Ser Asp Lys Arg Leu Glu Trp

35 40 45

Val Ala Ser Ile Ser Phe Gly Gly Gly Leu Ile Phe Tyr Leu Asp Ser

50 55 60

Leu Lys Val Arg Phe Thr Ile Ser Arg Glu Asn Trp Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Gln Ser Leu Met Ser Glu Asp Thr Ser Phe Tyr Tyr

85 90 95

Cys Val Arg Val Ile Gln Tyr Tyr Gly Gly Ser Pro Trp Tyr Phe Asp

100 105 110

Tyr

<210> 22

<211> 112

<212> PRT

<213> Synthesis (Synthesis)

<400> 22

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ala Gly Ser Trp Gly Gly Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile His Asn Met Trp Thr Leu Ile Phe Tyr Leu Asp Ser Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Met Ser Glu Asp Thr Ala Phe Tyr Tyr Cys

85 90 95

Val Arg Val Ile Ser Tyr Tyr Val Ile Ser Asn Thr Tyr Val Ser Tyr

100 105 110

<210> 23

<211> 95

<212> PRT

<213> Synthesis (Synthesis)

<400> 23

Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly Asp Arg

1 5 10 15

Val Thr Val Thr Cys Gln Gln Asn Gln Ser Val Asn Asn Asp Val Ala

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ser Ser Val Ser Phe

35 40 45

Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Gly Gly

50 55 60

Gly Val Thr Asp Phe Thr Phe Thr Ile Thr Thr Val Gln Ala Glu Asp

65 70 75 80

Leu Ala Val Tyr Phe Cys Ser Trp Val Tyr Ser Phe Pro Leu Ala

85 90 95

<210> 24

<211> 95

<212> PRT

<213> Synthesis (Synthesis)

<400> 24

Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly Asp Arg

1 5 10 15

Val Thr Val Thr Cys Lys Ala Ser Gln Ser Val His Val Asp Val Ala

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Ser Phe

35 40 45

Ala Ser Asn Arg Trp Lys Gly Val Pro Asp Arg Phe Thr Gly Gly Gly

50 55 60

Tyr Gly Thr Asp Phe Thr Phe Thr Ile Thr Thr Val Gln Ala Glu Asp

65 70 75 80

Leu Ala Val Tyr Phe Cys Gln Gln Val Val Ser Lys Ala Leu Ala

85 90 95

<210> 25

<211> 95

<212> PRT

<213> Synthesis (Synthesis)

<400> 25

Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly Asp Arg

1 5 10 15

Val Thr Val Thr Cys Lys Ala Ser Gln Ser Val Asn Asn Asp Val Ala

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Ser Phe

35 40 45

Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Gly Gly

50 55 60

Tyr Gly Thr Asp Phe Thr Phe Thr Ile Thr Thr Val Gln Ala Glu Asp

65 70 75 80

Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Phe Pro Leu Ala

85 90 95

<210> 26

<211> 95

<212> PRT

<213> Synthesis (Synthesis)

<400> 26

Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly Asp Arg

1 5 10 15

Val Thr Val Thr Cys Lys Ala Ser Thr Thr Ser Trp Val Asp Val Ala

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Ser Phe

35 40 45

Ala Ser Asn Arg Ala Ser Gly Val Pro Asp Arg Phe Thr Gly Gly Gly

50 55 60

Tyr Gly Thr Asp Phe Thr Phe Thr Ile Thr Thr Val Gln Ala Glu Asp

65 70 75 80

Leu Ala Val Tyr Phe Cys Ser Ser Asp Tyr Ser Phe Leu Cys Thr

85 90 95

<210> 27

<211> 95

<212> PRT

<213> Synthesis (Synthesis)

<400> 27

Val Met Val Val Thr Pro Lys Phe Leu Leu Val Ser Ala Gly Asp Arg

1 5 10 15

Val Thr Val Thr Cys Lys Ala Ser Gln Ser Thr Thr Ser Asp Val Ala

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Ser Phe

35 40 45

Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Gly Gly

50 55 60

Tyr Gly Thr Asp Phe Thr Phe Thr Ile Thr Thr Val Gln Ala Glu Asp

65 70 75 80

Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Phe Pro Leu Ala

85 90 95

<210> 28

<211> 95

<212> PRT

<213> Synthesis (Synthesis)

<400> 28

Val Met Thr Gln Thr Pro Lys Phe Leu Lys Asn Ser Ala Gly Asp Arg

1 5 10 15

Val Thr Val Thr Cys Lys Ala Ser Gln Ser Val Asn Asn Asp Val Ala

20 25 30

Trp Tyr Gln Val Ser Pro Gly Gln Ser Pro Lys Leu Leu Ile Ser Phe

35 40 45

Ala Ser Asn Arg Tyr Thr Gly Val Pro Phe Arg Phe Thr Gly Gly Gly

50 55 60

Tyr Gly Thr Trp Ser Thr Phe Thr Ile Thr Thr Val Gln Ala Glu Asp

65 70 75 80

Leu Ala Val Tyr Phe Cys Gln Ile Asp Tyr Ser Phe Pro Leu Arg

85 90 95

<210> 29

<211> 101

<212> PRT

<213> Synthesis (Synthesis)

<400> 29

Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Arg Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Asp Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr

100

<210> 30

<211> 101

<212> PRT

<213> Synthesis (Synthesis)

<400> 30

Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser His Asn Val Asp Gly Tyr

20 25 30

Ser Asn Ser Ala Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Val Ser Asn Leu Asp Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Cys Asp Pro Leu Ala

100

<210> 31

<211> 101

<212> PRT

<213> Synthesis (Synthesis)

<400> 31

Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser His Asn Ser Asp Gly Tyr

20 25 30

Ser Asn Ser Ala Met His Trp Tyr Gln Gln Lys Lys Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Val Ser Asn Leu Asp Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Cys Asp Pro Trp Ser

100

<210> 32

<211> 101

<212> PRT

<213> Synthesis (Synthesis)

<400> 32

Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu Thr Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Trp Ser Val Asp Arg Tyr

20 25 30

Gly Val Ser Phe Met His Trp Tyr Gln Gln Tyr Pro Gly Gln Pro Pro

35 40 45

Lys Leu Trp Ile Tyr Arg Ala Val Asn Leu Asp Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Ser Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val His Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Ser Ser Asn

85 90 95

Glu Asp Pro Leu Trp

100

<210> 33

<211> 101

<212> PRT

<213> Synthesis (Synthesis)

<400> 33

Asp Ile Val Val Phe Gln Ser Pro Ala Ser Leu Ala Cys Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Val Arg Ala Ser Glu Ser Val Trp Arg Tyr

20 25 30

Gly Asn Gly Phe Met His Trp Tyr Gln Cys Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Thr Ala Ser Asn Leu Asp Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Val Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Trp

65 70 75 80

Pro Val Glu Ala Asp Asn Val Ala Thr Tyr Tyr Cys Phe Gln Ser Asn

85 90 95

Ala Asp Pro Leu Ser

100

<210> 34

<211> 101

<212> PRT

<213> Synthesis (Synthesis)

<400> 34

Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gly Gly Val Asp Arg Tyr

20 25 30

Gly Asn Ser Val Met His Trp Tyr Gln Gln Lys Pro Cys Ser Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg His Ser Asn Leu Asp Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Thr Ala Asp Asp Val Ala Thr Tyr Tyr Ser Thr Gln Ser Asn

85 90 95

Glu Val Asn Leu Thr

100

Claims

1. An epitope peptide, characterized by: the epitope peptide is: the amino acid sequence is a short peptide shown as FVLNH.

2. A fusion protein formed by fusing the epitope peptide of claim 1 to a carrier protein.

3. A genetic molecule capable of encoding said epitope peptide, characterized in that: the gene molecule is a gene sequence or a gene fragment capable of encoding the epitope peptide according to claim 1.

4. An expression vector comprising the gene molecule of claim 3.

5. A host cell comprising the expression vector of claim 4 or having integrated in its genome the genetic molecule of claim 3.

6. An IL33 antibody characterized by: a murine monoclonal antibody or a human murine chimeric antibody having an H chain variable region consisting of SEQ ID NO 17 and an L chain variable region consisting of SEQ ID NO 29.

7. An isolated antigen-antibody complex, wherein an antigen forming the complex comprises an epitope peptide of claim 1 or the fusion protein of claim 2, wherein the epitope peptide forms an epitope, and wherein the antibody specifically binds to the epitope.

8. A vaccine comprising the epitope peptide of claim 1, the fusion protein of claim 2, the nucleic acid molecule of claim 3, the expression vector of claim 4, or the host cell of claim 5.

9. The vaccine of claim 8, wherein: the vaccine also comprises an immunologically acceptable carrier and/or adjuvant.