CN112390884B

CN112390884B - Semaphorin7A monoclonal antibody and application thereof in preparation of drugs for treating inflammatory diseases

Info

Publication number: CN112390884B
Application number: CN202011274704.XA
Authority: CN
Inventors: 董春升; 熊思东; 胡静平; 孙天乐
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2019-04-09
Filing date: 2019-04-09
Publication date: 2022-07-08
Anticipated expiration: 2039-04-09
Also published as: CN110156890A; CN110156890B; CN112390884A

Abstract

The invention discloses a Semaphorin7A monoclonal antibody and application thereof in preparing medicaments for treating inflammatory diseases, belongs to the field of immunology and cardiovascular disease medicament preparation, and discloses a medicament taking a Semaphorin7A monoclonal antibody as a main active ingredient, which is used for treating a mouse with a Semaphorin7A monoclonal antibody in a viral myocardial inflammation, staphylococcus aureus-induced sepsis inflammation and enterovirus EV71 acute infection model, and can effectively relieve the inflammation and improve the survival rate of the mouse; the application invention has important application prospect and popularization value in the research, development and production of the medicine for treating the viral myocarditis, staphylococcus aureus induced sepsis inflammation and enterovirus EV71 acute infection.

Description

Semaphorin7A monoclonal antibody and application thereof in preparation of drugs for treating inflammatory diseases

Technical Field

The invention belongs to the field of preparation of immunology and cardiovascular disease medicines, and particularly relates to a Semaphorin7A monoclonal antibody and application thereof in preparation of medicines for treating inflammatory diseases.

Background

The avidin Semaphorins are a family of axon-homing molecule proteins, a family of secreted or transmembrane proteins. The classification of the Semaphorin family of proteins is based on the protein domain, which contains an N-terminal extracellular matrix domain (Sema domain) of approximately 500 amino acids that is rich in cysteine-containing residues. The intermediate domain is the plexin-semaphorin (PSI) integrin domain, and the C-terminus is a variable protein domain. Variation of the C-terminal motif is a key factor in the classification of the Semaphorin family. The Semaphorins protein family has a total of 8 subclasses.

Classes

1 and 2 are in the presence of invertebrates, while classes 3 to 7 are in vertebrates, and class 8 molecules are encoded by viruses. Of these, 1, 4-7 are mainly transmembrane proteins, while 2, 3, 8 are mainly secreted proteins. Semaphorins were originally discovered as axon-guiding molecules that mediate cell-cell interactions or chemotaxis, but now act far more than during neurite outgrowth, and were found to be involved in a number of pathophysiological processes, such as organ maturation, regulation of immune cells and angiogenesis.

The gene Semaphorin7A (Sema7A, amino acid sequence shown in SEQ ID NO. 1) was located on human chromosome 15, was first found in erythrocytes, and was first named CDw 108. Yamada et al cloned the human CDw108 cDNA sequence for the first time, which encoded a total of 666 amino acids. The corresponding protein of murine origin consists of 664 amino acids and has 90% homology with human Sema 7A. Until 1998, CDw108 was not confirmed to belong to the family Semaphorin and was named SemaK-1. In 1999, the protein was renamed Sema7A because of its GPI-anchored specificity, which localizes it in a new Semaphorin family subfamily, the only glycosylphosphatidylinositol-anchored protein. Both Sema7A of murine and murine origin contain a 7-paddle β -helix Semaphorin N-terminal domain, a PSI domain, an immunoglobuloid domain and a GPI-anchored C-terminus. Sema7A is typically a dimer formed by the intermediate linkage of a Sema domain to an immunosphere-like structure. Sema7A binds to the virus-encoded Semaphorin protein receptor (VESPR), plexin c 1. May also bind to integrin α 1 β 1.

Current research on Sema7A has focused on inducing neurite outgrowth and modulating inflammatory immune responses. Sema7A has been reported to be involved in the maintenance of the intestinal mucosal immune system, and Kang et al found that Sema7A produced by intestinal epithelial cells induces macrophages to produce IL-10 in the intestinal mucosal immune homeostasis, thereby alleviating the inflammatory response of the intestine, and the effect can be blocked by IL-10 neutralizing antibodies. In addition, Sema7A also plays a major role in skin inflammation, viral infections, and a variety of autoimmune diseases. In the study on skin inflammation, when the keratinocyte epithelial cells and the monocytes interact with each other, Kamata et al find that Sema7A interacts with alpha 1 beta 1 integrin to activate the monocytes, induce the monocytes to generate inflammatory factors such as IL-8 and the like, and aggravate the skin inflammation. Sultana et al, when studying West Nile Virus (WNV), found that WNV infection resulted in increased expression of Sema7A in blood and tissues, that the inflammatory response following WNV infection in mice deficient in Sema7A was significantly reduced, and that the lethality of WNV infection was also significantly reduced in mice deficient in Sema7A or injected intraperitoneally with Sema7A antibody. The Sema7A can promote the progress of autoimmune diseases such as multiple sclerosis, activate the combination of Sema7A on T cells and alpha 1 beta 1 integrin receptor on macrophages, and play a role in proinflammatory. It has also been shown that Sema7A on intestinal epithelial cells binds to its receptor α v β 1 integrin on macrophages, secreting IL-10 and regulating the inflammatory response of the gut.

At present, the development of neutralizing antibodies in genetic engineering for both the Sema7A monoclonal antibody and the Sema7A antibody is in the laboratory research stage, and none of the Sema7A monoclonal antibody neutralizing antibodies is marketed as clinical drugs.

Disclosure of Invention

In order to treat macrophage-related inflammatory diseases, the embodiment of the invention provides a Semaphorin7A monoclonal antibody and application thereof in preparing a medicament for treating inflammatory diseases, and the application and popularization of the Semaphorin7A monoclonal antibody in preparing the medicament for treating inflammatory diseases have important significance.

In one aspect, the embodiments of the present invention disclose a Semaphorin7A monoclonal antibody, wherein the Semaphorin7A monoclonal antibody comprises a heavy chain amino acid variable region and a light chain amino acid variable region, wherein: the 6 CDR amino acid sequences of the Semaphorin7A monoclonal antibody are as follows:

the amino acid sequence of VH CDR1 is SEQ ID NO.2,

the amino acid sequence of VH CDR2 is SEQ ID NO.3,

the amino acid sequence of VH CDR3 is SEQ ID NO.4,

the amino acid sequence of VL CDR1 is SEQ ID NO.6,

the amino acid sequence of VL CDR2 is SEQ ID NO.7,

the amino acid sequence of VLCDR3 is SEQ ID No. 8; or

The amino acid sequence of VH CDR1 is SEQ ID NO.10,

the amino acid sequence of VH CDR2 is SEQ ID NO.11,

the amino acid sequence of VH CDR3 is SEQ ID NO.12,

the amino acid sequence of VL CDR1 is SEQ ID NO.14,

the amino acid sequence of VL CDR2 is SEQ ID NO.15,

the amino acid sequence of VL CDR3 is SEQ ID No. 16;

in some embodiments, the Semaphorin7A monoclonal antibody has at least 90% homology of 6 CDR amino acids.

In some embodiments, the amino acid sequence of the Semaphorin7A monoclonal antibody comprises:

a heavy chain consisting of an amino acid sequence shown in SEQ ID NO.5 and a light chain consisting of an amino acid sequence shown in SEQ ID NO. 9; or;

a heavy chain consisting of the amino acid sequence shown in SEQ ID NO.13 and a light chain consisting of the amino acid sequence shown in SEQ ID NO. 17.

On the other hand, the embodiment of the invention also discloses an application of the Semaphorin7A monoclonal antibody in preparing a medicament for treating inflammatory diseases, wherein the inflammatory diseases at least comprise viral myocardial inflammation, staphylococcus aureus (S.aureus) -induced sepsis inflammation and enterovirus EV71 acute infection.

In some embodiments, the tissue selected when verifying the use of the drug in treating the viral myocardial inflammation comprises at least serum and heart tissue.

In some embodiments, the selected tissue includes at least serum, lung tissue, kidney tissue, and combinations thereof, when the drug is validated for use in treating inflammation of sepsis induced by s.

In some embodiments, when the drug is validated for use in treating acute infection with enterovirus EV71, the selected tissues include at least: skeletal muscle tissue, small intestine tissue, brain stem tissue.

In some embodiments, the medicament for treating inflammatory diseases further comprises a pharmaceutical excipient and a pharmaceutical base.

In some embodiments, the medicament for treating an inflammatory disease is a liquid formulation or a lyophilized formulation.

The technical scheme provided by the embodiment of the invention has the following beneficial effects:

1. according to the invention, by utilizing technologies such as truncated expression and peptide fragment synthesis, the epitope recognized by the Semaphorin7A monoclonal antibodies 2567-1 and 2567-3 is subjected to panning to obtain the consistent epitope sequence, and the sequence comparison result shows that the polypeptide sequence recognized by the Semaphorin7A monoclonal antibody falls into the Semaphorin7A beta helical region. Furthermore, applicants analyzed the light and heavy chain sequences of 2567-1 and 2567-3 monoclonal antibodies that specifically bind to Sema7A using antibody gene sequencing techniques.

2. In the embodiment, viral myocardial inflammation, staphylococcus aureus (s.aureus) -induced sepsis inflammation and enterovirus EV71 acute infection are respectively treated by the Semaphorin7A monoclonal antibody, so that the inflammatory infection can be effectively alleviated and the survival rate of mice can be improved, and the therapeutic effect in the treatment of the inflammatory infection is obvious;

3. the embodiment discloses application of a Semaphorin7A monoclonal antibody in preparation of a medicament for treating viral myocardial inflammation, staphylococcus aureus (S.aureus) -induced sepsis inflammation, enterovirus EV71 acute infection and other inflammatory diseases; in the research, development and production of medicaments for treating the inflammatory diseases, the invention of the application of the Semaphorin7A monoclonal antibody has important significance;

4. the embodiment of the invention establishes a healthy Balb/c male mouse model, which is taken as an inflammatory disease animal model widely accepted and applied, is suitable for clinical application research, and has important application and popularization values for research and development of medicaments for inflammatory diseases such as viral myocarditis, staphylococcus aureus (S.aureus) -induced sepsis inflammation, enterovirus EV71 acute infection and the like.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is the E.coli expression profile of the Sema7A _ truncated protein;

FIG. 2 is a WB validation graph of the Sema7A monoclonal antibody;

FIG. 3 is a Sema7A antibody ELISA validation and GST-1, GST-2, GST-3 affinity map;

FIG. 4 is a Sema7A antibody ELISA validation and peptide fragments P1, P2, P3, P4, P5 affinity panel;

FIG. 5 is a Sema7A antibody ELISA validation and

peptide fragment

2, 3, 4, 5 affinity diagram;

FIG. 6 is a graph of serum calcium changes following treatment with Sema7A antibody in CVB3 infected mice;

FIG. 7 is a photograph of immunohistochemical staining of cardiac tissue sections following treatment of CVB3 infected mice with Sema7A antibody;

FIG. 8 is a graph of mouse cardiac tissue inflammatory factor detection following Sema7A antibody treatment;

FIG. 9 is a graph of the survival rate of CVB3 infected viral myocarditis mice after treatment with Sema7A antibody;

FIG. 10 is a graph of serum inflammatory factor detection of Sema7A antibody in mice treated with Staphylococcus aureus infection;

FIG. 11 is a graph of lung lesions of mice treated with the Sema7A antibody against Staphylococcus aureus infection;

FIG. 12 is a graph of immunohistochemical staining of kidney sections from mice treated with Sema7A antibody for Staphylococcus aureus infection;

FIG. 13 is a graph of survival of mice treated with the Sema7A antibody for Staphylococcus aureus infection;

FIG. 14 is a photograph of immunohistochemical staining of skeletal muscle sections of mice treated with the Sema7A antibody for EV71 infection;

FIG. 15 is a photograph of immunohistochemical staining of small intestine sections of mice infected with EV71 treated with the Sema7A antibody;

FIG. 16 is a photograph of immunohistochemical staining of brainstem sections of mice treated with the Sema7A antibody for EV71 infection;

FIG. 17 is a graph of the detection of inflammatory factors in mice infected with EV71 treated with the Sema7A antibody;

FIG. 18 is a graph of survival of EV71 infected mice treated with the Sema7A antibody;

fig. 19 is a graph of survival rates of EV71 infected mice treated with Sema7A antibody in combination with the antiviral drug acyclovir.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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 protection scope of the present invention.

Example 1

This example provides two Semaphorin7A monoclonal antibodies: 2567-1 and 2567-3, both Semaphorin7A monoclonal antibodies comprise a heavy chain variable region and a light chain variable region.

Wherein, the heavy chain variable region of the Semaphorin7A monoclonal antibody 2567-1 comprises the following 3 CDR amino acid sequences and a nucleotide sequence: the amino acid sequence is VH CDR1 of SEQ ID NO.2, the amino acid sequence is VH CDR2 of SEQ ID NO.3, the amino acid sequence is VH CDR3 of SEQ ID NO.4, and the nucleotide sequence is the nucleotide of SEQ ID NO. 5. The light chain variable region of the Semaphorin7A monoclonal antibody 2567-1 comprises the following 3 CDR amino acid sequences and a nucleotide sequence: the amino acid sequence is VL CDR1 of SEQ ID NO.6, the amino acid sequence is VL CDR2 of SEQ ID NO.7, the amino acid sequence is VL CDR3 of SEQ ID NO.8, and the nucleotide sequence is the nucleotide of SEQ ID NO. 9.

Similarly, the heavy chain variable region of the Semaphorin7A monoclonal antibody 2567-3 comprises the following 3 CDR amino acid sequences and a nucleotide sequence: the amino acid sequence is VH CDR1 of SEQ ID NO.10, the amino acid sequence is VH CDR2 of SEQ ID NO.11, the amino acid sequence is VH CDR3 of SEQ ID NO.12, and the nucleotide sequence is the nucleotide of SEQ ID NO. 13. The light chain variable region of the Semaphorin7A monoclonal antibody 2567-3 comprises the following 3 CDR amino acid sequences and a nucleotide sequence: the amino acid sequence is VL CDR1 of SEQ ID NO.14, the amino acid sequence is VL CDR2 of SEQ ID NO.15, the amino acid sequence is VL CDR3 of SEQ ID NO.16, and the nucleotide sequence is the nucleotide of SEQ ID NO. 17.

The embodiment also provides a preparation method of the Semaphorin7A monoclonal antibody, which specifically comprises the following steps:

(1) firstly, the antigenicity of the mouse Sema7A gene sequence is analyzed by a protean program in a Lasergene software package, and a protein sequence Sema7A _ truncated (SEQ ID NO.18) with better antigenicity is obtained.

(2) As shown in FIG. 1, the Sema7A _ truncated sequence was cloned into pET28a prokaryotic expression vector, and expression was induced by IPTG in E.coli, expressing Sema7A _ truncated antigen protein.

(3) The antigen protein of Sema7A _ truncated is used for subcutaneously immunizing a mouse, and spleen cells of the immunized mouse are taken and fused with sp20 cells to obtain a 3-strain Sema7A specific mouse monoclonal antibody. As shown in the Elisa titer Table of Table 1, in order to verify the neutralizing titer of the antibodies prepared, the plate was coated with 1. mu.g of antigen, and the antibodies prepared were diluted at different concentrations, and it was found that the antibodies of these 3 strains were also well able to bind to the antigen Sema7A _ truncated at a dilution ratio of 1: 80000.

TABLE 1 Elisa Titers of the Semaphorin7A monoclonal antibodies 2567-1, 2567-2, 2567-3

Judgment standard: S/N > 2.1 (S: OD value for antibody detection, N: OD value for blank control detection)

And (4) conclusion: the titer of the obtained antibody meets the standard, and the Elisa detection result is qualified.

(4) As shown in FIG. 2, we also verified the specificity of the obtained 3 strains of antibodies by means of western blot, and the mouse lung tissues were run on SDS-PAGE gel, the expression of Sema7A protein could be detected by using our antibodies 1: 1000 or 1: 5000, the positive control is commercial antibody, and through the above experiments, we showed that the 3 strains of mouse Sema7A specific monoclonal antibodies were successfully prepared.

Further, the present embodiment also provides identification of the epitope of the Semaphorin7A monoclonal antibody and analysis of the antibody sequence, which specifically includes the following steps:

(1) for epitope analysis of the Semaphorin7A monoclonal antibody, the Sema7A _ truncated protein was further divided into 3 fragments: GST-1, GST-2, GST-3 (amino acid sequences are shown in SEQ ID NO.19-SEQ ID NO.21, respectively). By fusion expression with prokaryotic expression vector GST, GST-1, GST-2 and GST-3 proteins expressed and purified are coated on a plate, and then an antibody is used for enzyme-linked immunosorbent assay, and the result shows that the protein (Sema7A-His) using Sema7A _ truncated can be well combined with antibodies No. 2567-1 and No. 2567-3. As shown in FIGS. 3A and 3B, further analysis revealed that the GST-3 protein sequence of Sema7A _ truncated protein was able to bind to 2567-1 and 2567-3, respectively.

(2) Based on the GST-3 amino acid sequence, it was further divided into 5 peptide fragments: p1, P2, P3, P4 and P5, wherein amino acid sequences from P1 to P5 are respectively shown as SEQ ID NO. 22-26, 5 synthesized peptide segments are coated on a plate, an antibody is also used for enzyme-linked immunosorbent assay, and the affinity of the antibodies 2567-1 and 2567-3 with the antibody is detected. As a result shown in fig. 4, it was found that: the P2 peptide sequence has high affinity with the 2567-1 and 2567-3 antibodies, as well as the GST-3 positive control.

(3) As shown in FIG. 5, in order to more accurately analyze the sequence of the P2 peptide fragment combined with the monoclonal antibody, P2 is further decreased from the N end or the C end and divided into 4 peptide fragments 2-5, and the amino acid sequences are respectively shown in SEQ ID NO.27-SEQ ID NO. 30. After biochemical synthesis, the synthesized peptide segment is coated on a plate, the antibodies are also used for enzyme-linked immunosorbent assay, and the affinities of the 2567-1 and 2567-3 antibodies are detected, so that the N-terminal amino acid sequence of the P2 peptide segment plays an important role in the combination of the antibodies. As shown in FIG. 5A, if the C-terminal was deleted by 4 amino acids, the affinity of peptide No.2 and P2 for the 2567-1 and 2567-3 antibodies did not change much, while as shown in FIG. 5B, if the P2 peptide was deleted by 4 amino acids from the N-terminal, the affinity for the 2567-1 and 2567-3 antibodies was greatly reduced. Thus, the above experiment demonstrates that peptide fragment No. 2: FSPDENSLVLFEGDEV are epitopes determined by the 2567-1 and 2567-3 Sema7A monoclonal antibodies we prepared.

(4) In order to analyze the sequence of antigen binding of the 2567-1 and 2567-3 monoclonal antibodies, gene cloning and sequencing were performed on the variable regions of the 2567-1 and 2567-3 monoclonal antibodies provided in this example.

Further: the amino acid sequence of the Semaphorin7A monoclonal antibody comprises:

a heavy chain consisting of the amino acid sequence shown in SEQ ID NO.31 and a light chain consisting of the amino acid sequence shown in SEQ ID NO.32 (2567-1); or;

a heavy chain consisting of the amino acid sequence shown in SEQ ID NO.33 and a light chain consisting of the amino acid sequence shown in SEQ ID NO.34 (2567-3).

In this example, the epitope recognized by the Semaphorin7A monoclonal antibodies 2567-1 and 2567-3 was panned by techniques such as truncated expression and peptide fragment synthesis to obtain a uniform epitope sequence. The alignment of the sequences indicated that the polypeptide sequence recognized by Semaphorin7A fell within the Semaphorin7A beta helix region. Furthermore, the applicant analyzed the light chain and heavy chain sequences of the monoclonal antibodies 2567-1 and 2567-3 and the Sema7A, which are specifically combined, by using an antibody gene sequencing technology.

Example 2

The present example provides the use of the monoclonal antibody (2567-1, 2567-3) to Semaphorin7A in the treatment of viral myocardial inflammation induced by CVB 3.

Firstly, constructing a viral myocarditis model induced by CVB 3; the construction steps of the model specifically comprise:

(1) taking 6-8 week old male Balb/c mice, dividing into 4 groups of 6 mice, and injecting into abdominal cavity of each mouse 10³TCID50 dose of CVB3 virus induced a mouse model of viral myocardial inflammation.

(2) Mice were given 50ug of Sema7a2567-1 or 2567-3 antibody by i.p. injection on the first, third and fifth days of virus infection, respectively, CVB3 group was a virus infection non-treated control group, and control was a healthy mouse group.

As shown in FIG. 6, on day 7 of infection, the myocarditis index in the mice was observed, and compared with the control group, the serum muscle calcium index cTnI content was 2.77ng/ml after the treatment with the antibody 2567-1 in the mouse Sema7A, 4.24ng/ml after the treatment with the antibody 2567-3 in the mouse Sema7A, while the cTnI content in the CVB3 control group was 10.83ng/ml and 0.54ng/ml in the control group in the serum of the healthy mice.

Heart tissue from infected mice was harvested on day 7 post-infection and infiltrated inflammatory cells were stained with Hematoxylin-Eosin (Hematoxylin). As shown in FIG. 7, pathological sections of mouse hearts also showed that the heart of the mice treated with Sema7A2567-1, 2567-3 antibody showed a slight inflammation at day 7, while the heart of the CVB3 mice in the control group was more inflamed, had a large infiltration of lymphocytes, and showed diffuse necrosis of the heart, as indicated by the arrows in the figure, and the staining of the pathological sections of myocardium was consistent with the results of the cTnI serum.

Further, heart tissues of infected mice on the seventh day were ground and cytokines associated with inflammation secreted in the heart tissues were detected, and the detection results are shown in fig. 8, which indicates that: inflammatory cytokines IL-6, IL-1. beta. and TNF-. alpha. were significantly decreased in the 2567-1, 2567-3 antibody-treated group compared to CVB3, while MCP-1 inflammatory factor was not significantly changed in the 2567-1, 2567-3 antibody-treated group compared to CVB 3.

Further referring to fig. 9, the mice in each group were observed to have daily survival rates, and the Sema7a2567-1 antibody group was effective in increasing the survival rate of the mice by 83.3% (5, n ═ 6), the Sema7a2567-3 antibody group was effective in increasing the survival rate of the mice by 67.7% (4, n ═ 6), and the survival rate of the mice in the control group was only 50% (3, n ═ 6).

The technical scheme provided by the embodiment has the following beneficial effects:

1. in the embodiment, the treatment of viral myocarditis induced by CVB3 by using the Semaphorin7A monoclonal antibody can effectively relieve the myocarditis and improve the survival rate of mice, and has obvious curative effect on myocarditis diseases;

2. the embodiment discloses the application of a Semaphorin7A monoclonal antibody in preparing a medicament for treating viral myocardial inflammation induced by CVB 3; the invention has important significance in the research, development and production of the medicine for treating myocardial inflammation, and the application of the Semaphorin7A monoclonal antibody is significant;

3. the embodiment of the invention establishes a healthy Balb/c male mouse model which is taken as a myocarditis animal model widely accepted and applied, is suitable for clinical application research, and has important application and popularization values for research and development of medicines for myocarditis.

Example 3

The present example provides the use of the Semaphorin7A monoclonal antibody (2567-1, 2567-3) for the treatment of s.aureus (s.aureus) induced sepsis inflammation.

A staphylococcus aureus (s. aureus) induced sepsis inflammation model was also constructed; the construction steps of the model specifically comprise:

(1) taking 6-8 week old male C57b1/6 mice, dividing into 4 groups of 6 mice, and intraperitoneally injecting 10 of each mouse⁷Staphylococcus aureus at CFU0 dose induced a sepsis mouse model of inflammation.

(2) Mice were given 50ug of Sema7A2567-1 or 2567-3 antibody by i.p. injection on the first, third and fifth days of bacterial infection, respectively, s.

Inflammatory cytokines were detected in the serum of the infected mice on day 6, and the results are shown in fig. 10. The results show that: the inflammatory cytokines IL-6, MCP-1 and TNF-alpha are significantly reduced in mice treated with 2567-1, 2567-3 antibody compared with S.aureus, while the IL-10 inflammatory factor is not significantly changed in mice treated with 2567-1, 2567-3 antibody compared with CVB 3.

As shown in FIG. 11, we examined the pulmonary organs of mice. The wet/dry weight ratio of lung tissue is an index of lung tissue damage, and we tested the wet/dry weight ratio of lung tissue of each group of mice on the 6 th day of infection, and the test results are shown in FIG. 11A, and the wet/dry weight ratio of lung tissue is significantly reduced in 2567-1, 2567-3 antibody-treated mice compared with S.aureus group, and similarly, the bacterial load in blood is also significantly reduced after 2567-1, 2567-3 antibody treatment, as shown in FIG. 11B. Similarly, as shown in FIG. 11C, simultaneous detection of protein levels in pulmonary lavage fluid also showed a significant decrease following treatment with antibodies 2567-1 and 2567-3. The above results indicate that Sema7A antibody 2567-1, 2567-3 treatment ameliorated S.aureus infected lung injury.

Meanwhile, the kidney organs of the mice are also detected. The results of the detection by HE kidney section are shown in fig. 12: the glomerular structures in the kidney sections of the aureus group were destroyed and appeared irregular or flat, and the interstitial space of the glomerular structures became smaller or disappeared (as indicated by arrows in the figure).

Referring to fig. 13, the mice in each group were observed to have daily survival, and the Sema7a2567-1 antibody group was effective in increasing the survival rate of the mice by 83.3% (5, n ═ 6), the Sema7a2567-3 antibody group was effective in increasing the survival rate of the mice by 67.7% (4, n ═ 6), and the survival rate of the mice in the control group was only 33.3% (2, n ═ 6).

1. the treatment of staphylococcus aureus (s.aureus) -induced sepsis inflammation with the Semaphorin7A monoclonal antibody provided in the embodiment can effectively relieve sepsis inflammation and improve the survival rate of mice, and has an obvious curative effect in the treatment of staphylococcus aureus (s.aureus) -induced sepsis inflammation;

2. the present example discloses the use of the Semaphorin7A monoclonal antibody for the preparation of a medicament for the treatment of s.aureus (s.aureus) induced sepsis inflammation; the invention of the application of the Semaphorin7A monoclonal antibody has important significance in the research, development and production of staphylococcus aureus (S.aureus) induced sepsis inflammation drugs;

3. the embodiment of the invention establishes a healthy Balb/c male mouse model, is used as a widely accepted and applied staphylococcus aureus (S.aureus) induced sepsis inflammation animal model, is suitable for clinical application research, and has important application and popularization values for research and development of staphylococcus aureus (S.aureus) induced sepsis inflammation drugs.

Example 4

The embodiment provides application of a Semaphorin7A monoclonal antibody (2567-1, 2567-3) in treatment of acute enterovirus EV71 infection. The EV7 virus has extremely high infectivity on the central nervous system, and clinically-occurring symptoms comprise encephalitis (encephalitis), aseptic meningitis (aseptic meningitis), acute flaccid paralysis (acute flaccid paralysis), hand-foot-mouth disease (hand-foot-mouth disease), vesicular angina (herpangina), acute hemorrhagic conjunctivitis (acute hemorrhagic conjunctivitis) and the like, and particularly has great harm to infants.

In this example, an acute infection model of enterovirus EV71 was also constructed; the construction steps of the model specifically comprise:

(1) firstly, an animal model of EV71 infected suckling mouse is established, male Balb/c mice of 3-5 days are taken and divided into 4 groups of 6 mice each, and 10 mice are directly injected into the abdominal cavity⁷PFU EV71 virus.

(2) On the first, third and fifth days of viral infection, 50ug of Sema7A2567-1 or 2567-3 antibody was administered intraperitoneally to mice, CVB3 group was a virus-infected non-treated control group, and control was a healthy mouse group, respectively.

On the first, third and fifth days of viral infection, 50ug of Sema7A2567-1 or 2567-3 antibody was administered intraperitoneally to mice, CVB3 group was a virus-infected non-treated control group, and control was a healthy mouse group, respectively.

Infected mouse skeletal muscle tissue was taken on day 5 post-infection and infiltrated inflammatory cells were stained with Hematoxylin-Eosin (Eosin). As shown in fig. 14, skeletal muscle pathology sections of mice showed significantly less infiltration of inflammatory cells in skeletal muscle of mice in Sema7A2567-1, 2567-3 antibody-treated group than in EV 71-infected non-treated group. Meanwhile, we stained the small intestine tissue of the infected mice with HE sections, as shown in FIG. 15, and the disruption of the microvilli structure of the small intestine was improved in the Sema7A2567-1, 2567-3 antibody-treated mice compared with the non-treated mice. In particular, in nerve tissues, as shown in FIG. 16, we also observed the same phenomenon that in the brainstem region of infected mice, the infiltration of inflammatory cells was significantly less in the Sema7A2567-1, 2567-3 antibody-treated group than in the non-treated group of EV 71.

As shown in fig. 17, inflammatory cytokines in skeletal muscle, small intestine and brain stem on day 5 of infected mice were detected by means of quantitative PCR; the detection result shows that: the inflammatory cytokines IL-6, MCP-1 and TNF-alpha in 2567-1, 2567-3 antibody-treated mice showed a significant decrease in skeletal muscle, small intestine and brainstem compared with EV71 non-treated groups.

Further referring to fig. 18, observing the daily survival of each group of mice, while antibody treatment did not ultimately improve the survival of the mice, Sema7A antibody 2567-1 and 2567-3 treatment significantly prolonged the survival time of the mice compared to the non-treated group, which all died on day 7 of infection, by approximately 1-fold compared to the control group.

In addition, we established a mouse model of EV7 virus infection and treatment with a combination of Sema7A antibody and the antiviral drug acyclovir. Male Balb/c mice from 3-5 days were divided into 4 groups of 6 mice each and mice were injected intraperitoneally with 107PFU EV71 virus. On the first, third and fifth days of viral infection, mice were given either Sema7A 2567-.

The daily survival of the mice in each group was observed, and the results, see the survival chart of the mice infected with EV71, treated with Semaphorin7A antibody and antiviral drug acyclovir as shown in fig. 19, indicate that: if only acyclovir drug treatment is given, the survival rate of the mice is 33.4% (n is 6), but if combined treatment is carried out by using Sema7A antibody drug, the survival rate of the mice can be greatly improved by 1-fold to 66.7%, so that the Semaphorin7A monoclonal antibody prepared by the method can be further explained to have great application value in the clinical treatment of EV 71.

1. the acute infection of the enterovirus EV71 is treated by the Semaphorin7A monoclonal antibody, so that the acute infection of the enterovirus EV71 can be effectively relieved, the survival rate of mice is improved, and the curative effect in the treatment of the acute infection of the enterovirus EV71 is obvious;

2. the embodiment discloses application of a Semaphorin7A monoclonal antibody in preparation of a medicament for treating acute enterovirus EV71 infection; in the research and development and production of the medicine for acute enterovirus EV71 infection, the invention of the application of the Semaphorin7A monoclonal antibody has important significance;

3. the embodiment of the invention establishes a healthy Balb/c male mouse model as a widely accepted and applied animal model of acute infection of enterovirus EV71, the model is suitable for clinical application research, and has important application and popularization values for research and development of medicines for acute infection of enterovirus EV 71.

Example 5

The present example further provides a medicament for treating inflammatory diseases, wherein the dosage form of the medicament in the present example is a liquid preparation, and the active ingredient of the liquid preparation comprises the aforementioned Semaphorin7A monoclonal antibody. Wherein the content of the Semaphorin7A monoclonal antibody is 75-120 mg. The medicine for treating inflammatory diseases also comprises medicinal auxiliary materials and medicinal substrates.

Specifically, the pharmaceutical excipients comprise immunologic adjuvants, stabilizers and preservatives. Wherein the stabilizer can be 1% human albumin; in practice, a saccharide-based biological agent stabilizer such as maltose, glucose, sorbitol, etc. is also often used. The immunologic adjuvant is vegetable oil adjuvant, specifically glycerol; the preservative is 0.5% chloroform.

In addition, the pharmaceutical preparations may contain other customary auxiliary substances or additives, such as antioxidants, for example glutathione or ascorbic acid.

Specifically, the matrix including the Semaphorin7A monoclonal antibody as an active ingredient for the preparation of a medicament in the present embodiment is preferably a buffered antibody solution containing sodium chloride, such as an injection or the like. The antibody solution is mixed with an aqueous solution containing an additive sugar, an amino acid and a surfactant while adjusting the pH to 5-8 with an acid or an alkali. Appropriate amounts of phosphoric acid or phosphate and sodium chloride are added to bring the solution to the previously determined concentration.

1. the treatment of the Semaphorin7A monoclonal antibody (2567-1 and 2567-3) for the diseased mouse can effectively relieve viral myocarditis, the septicemia inflammation induced by staphylococcus aureus (S.aureus) and the inflammation caused by acute infection of enterovirus EV71, improve the survival rate of the mouse, and have obvious curative effect on treating partial inflammatory diseases;

2. the embodiment of the invention discloses application of a Semaphorin7A monoclonal antibody (2567-1, 2567-3) in preparation of a medicament for treating inflammatory diseases; the invention of the new application of the Semaphorin7A monoclonal antibody (2567-1 and 2567-3) has important significance in the research, development and production of medicaments for treating inflammation.

Example 6

First, a solution as an injection preparation was prepared by the preparation process of example 5, and then, the solution was filter-sterilized, freeze-dried, to prepare a lyophilized preparation.

In the embodiment of the invention, an unstable aqueous solution containing the freeze-sensitive antibody is added into the medicinal matrix, and is converted into a stable preparation by a freeze drying mode, and the stable preparation is not deteriorated and keeps stable at high temperature. In practice, sugar-type biological agent stabilizers such as maltose, glucose, and sorbitol are also commonly used.

According to the invention, another advantage of the lyophilisate is that: in addition to avoiding antibody damage during freezing, the antibody content of the lyophilizate is not reduced and no aggregates are formed or flocculation occurs even upon long-term storage at 50 ℃. Therefore, the content and purity of the antibody are stable. After regeneration of the lyophilisate with water for injection, low turbidity values indicate that the formation of particles is avoided.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Sequence listing

<110> Suzhou university

<120> Semaphorin7A monoclonal antibody and application thereof in preparing medicines for treating inflammatory diseases

<160> 34

<170> SIPOSequenceListing 1.0

<210> 1

<211> 664

<212> PRT

<213> Homo sapiens

<400> 1

Met Thr Pro Pro Pro Pro Gly Arg Ala Ala Pro Ser Ala Pro Arg Ala

1 5 10 15

Arg Val Leu Ser Leu Pro Ala Arg Phe Gly Leu Pro Leu Arg Leu Arg

20 25 30

Leu Leu Leu Val Phe Trp Val Ala Ala Ala Ser Ala Gln Gly His Ser

35 40 45

Arg Ser Gly Pro Arg Ile Ser Ala Val Trp Lys Gly Gln Asp His Val

50 55 60

Asp Phe Ser Gln Pro Glu Pro His Thr Val Leu Phe His Glu Pro Gly

65 70 75 80

Ser Phe Ser Val Trp Val Gly Gly Arg Gly Lys Val Tyr His Phe Asn

85 90 95

Phe Pro Glu Gly Lys Asn Ala Ser Val Arg Thr Val Asn Ile Gly Ser

100 105 110

Thr Lys Gly Ser Cys Gln Asp Lys Gln Asp Cys Gly Asn Tyr Ile Thr

115 120 125

Leu Leu Glu Arg Arg Gly Asn Gly Leu Leu Val Cys Gly Thr Asn Ala

130 135 140

Arg Lys Pro Ser Cys Trp Asn Leu Val Asn Asp Ser Val Val Met Ser

145 150 155 160

Leu Gly Glu Met Lys Gly Tyr Ala Pro Phe Ser Pro Asp Glu Asn Ser

165 170 175

Leu Val Leu Phe Glu Gly Asp Glu Val Tyr Ser Thr Ile Arg Lys Gln

180 185 190

Glu Tyr Asn Gly Lys Ile Pro Arg Phe Arg Arg Ile Arg Gly Glu Ser

195 200 205

Glu Leu Tyr Thr Ser Asp Thr Val Met Gln Asn Pro Gln Phe Ile Lys

210 215 220

Ala Thr Ile Val His Gln Asp Gln Ala Tyr Asp Asp Lys Ile Tyr Tyr

225 230 235 240

Phe Phe Arg Glu Asp Asn Pro Asp Lys Asn Pro Glu Ala Pro Leu Asn

245 250 255

Val Ser Arg Val Ala Gln Leu Cys Arg Gly Asp Gln Gly Gly Glu Ser

260 265 270

Ser Leu Ser Val Ser Lys Trp Asn Thr Phe Leu Lys Ala Met Leu Val

275 280 285

Cys Ser Asp Ala Ala Thr Asn Arg Asn Phe Asn Arg Leu Gln Asp Val

290 295 300

Phe Leu Leu Pro Asp Pro Ser Gly Gln Trp Arg Asp Thr Arg Val Tyr

305 310 315 320

Gly Val Phe Ser Asn Pro Trp Asn Tyr Ser Ala Val Cys Val Tyr Ser

325 330 335

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

340 345 350

His Met Gly Leu Pro Asn Pro Arg Pro Gly Met Cys Leu Pro Lys Lys

355 360 365

Gln Pro Ile Pro Thr Glu Thr Phe Gln Val Ala Asp Ser His Pro Glu

370 375 380

Val Ala Gln Arg Val Glu Pro Met Gly Pro Leu Lys Thr Pro Leu Phe

385 390 395 400

His Ser Lys Tyr His Tyr Gln Lys Val Val Val His Arg Met Gln Ala

405 410 415

Ser Asn Gly Glu Thr Phe His Val Leu Tyr Leu Thr Thr Asp Arg Gly

420 425 430

Thr Ile His Lys Val Val Glu Ser Gly Asp Gln Asp His Ser Phe Val

435 440 445

Phe Asn Ile Met Glu Ile Gln Pro Phe His Arg Ala Ala Ala Ile Gln

450 455 460

Ala Ile Ser Leu Asp Ala Asp Arg Arg Lys Leu Tyr Val Thr Ser Gln

465 470 475 480

Trp Glu Val Ser Gln Val Pro Leu Asp Met Cys Glu Val Tyr Ser Gly

485 490 495

Gly Cys His Gly Cys Leu Met Ser Arg Asp Pro Tyr Cys Gly Trp Asp

500 505 510

Gln Asp Arg Cys Val Ser Ile Tyr Ser Ser Gln Arg Ser Val Leu Gln

515 520 525

Ser Ile Asn Pro Ala Glu Pro His Arg Glu Cys Pro Asn Pro Lys Pro

530 535 540

Asp Glu Ala Pro Leu Gln Lys Val Ser Leu Ala Arg Asn Ser Arg Tyr

545 550 555 560

Tyr Leu Thr Cys Pro Met Glu Ser Arg His Ala Thr Tyr Leu Trp Arg

565 570 575

His Glu Glu Asn Val Glu Gln Ser Cys Glu Pro Gly His Gln Ser Pro

580 585 590

Ser Cys Ile Leu Phe Ile Glu Asn Leu Thr Ala Arg Gln Tyr Gly His

595 600 605

Tyr Arg Cys Glu Ala Gln Glu Gly Ser Tyr Leu Arg Glu Ala Gln His

610 615 620

Trp Glu Leu Leu Pro Glu Asp Arg Ala Leu Ala Glu Gln Leu Met Gly

625 630 635 640

His Ala Arg Ala Leu Ala Ala Ser Phe Trp Leu Gly Val Leu Pro Thr

645 650 655

Leu Ile Leu Gly Leu Leu Val His

660

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gly Tyr Thr Phe Thr Ser Tyr Tyr

1 5

<210> 3

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ile Asn Pro Ser Asn Gly Asp Thr

1 5

<210> 4

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Thr Thr Pro Ala Arg Ala Thr Phe Ala Tyr

1 5 10

<210> 5

<211> 352

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

caggtccaac tgcagcagtc tggggctgaa ctggtgaagc ctggggcttc agtgaagttg 60

tcctgcaagg cttctggcta caccttcacc agctactata tgtactgggt gaagcagagg 120

cctggacaag gccttgagtg gattggagag attaatccta gcaatggtga tactaacttc 180

aatgagaagt tcaagagcaa ggccacactg actgtagaca aatcctccag cacagcttac 240

atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtac aacaccagct 300

cgggctacgt ttgcttactg gggccaaggg actctggtca ctgtctctgc ag 352

<210> 6

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr

1 5 10

<210> 7

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Lys Val Ser

1

<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Ser Gln Ser Thr His Val Pro Tyr Thr

1 5

<210> 9

<211> 337

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120

tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300

tacacgttcg gaggggggac caagctggaa ataaaac 337

<210> 10

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Tyr Thr Phe Thr Ser Tyr Trp

1 5

<210> 11

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Ile Asp Pro Ser Asp Ser Tyr Thr

1 5

<210> 12

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Ala Arg Asp Gly Gly Tyr

1 5

<210> 13

<211> 340

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

caggtccaac tgcagcagcc tggggctgag cttgtgaagt ctggggcttc agtgaagctg 60

tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg 120

cctggacaag gccttgagtg gatcggagag attgatcctt ctgatagtta tactaactac 180

aatcaaaagt ttaagggcaa ggccacattg actgttgaca aatcctccag cacagcctac 240

atgcagctca gcagcctgac atctgaggag tctgcggtct attactgtgc aagagatggc 300

ggctactggg gccaaggcac cactctcaca gtctcctcag 340

<210> 14

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Gln Asp Val Ser Thr Ala

1 5

<210> 15

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Ser Ala Ser

1

<210> 16

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gln Gln His Tyr Ser Thr Pro Trp Thr

1 5

<210> 17

<211> 322

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcatc 60

atcacctgca aggccagtca ggatgtgagt actgctgtag cctggtatca acagaaacca 120

ggacaatctc ctaaattact aatttactcg gcatcctacc gatacactgg agtccctgat 180

cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagcag tgtgcaggct 240

gaagacctgg cagtttatta ctgtcagcaa cattatagta ctccgtggac gttcggtgga 300

ggcaccaagc tggaaatcag ac 322

<210> 18

<211> 161

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Lys Gly Gln Asp His Val Asp Phe Ser Gln Pro Glu Pro His Thr Val

1 5 10 15

Leu Phe His Glu Pro Gly Ser Phe Ser Val Trp Val Gly Gly Arg Gly

20 25 30

Lys Val Tyr His Phe Asn Phe Pro Glu Gly Lys Asn Ala Ser Val Arg

35 40 45

Thr Val Asn Ile Gly Ser Thr Lys Gly Ser Cys Gln Asp Lys Gln Asp

50 55 60

Cys Gly Asn Tyr Ile Thr Leu Leu Glu Arg Arg Gly Asn Gly Leu Leu

65 70 75 80

Val Cys Gly Thr Asn Ala Arg Lys Pro Ser Cys Trp Asn Leu Val Asn

85 90 95

Asp Ser Val Val Met Ser Leu Gly Glu Met Lys Gly Tyr Ala Pro Phe

100 105 110

Ser Pro Asp Glu Asn Ser Leu Val Leu Phe Glu Gly Asp Glu Val Tyr

115 120 125

Ser Thr Ile Arg Lys Gln Glu Tyr Asn Gly Lys Ile Pro Arg Phe Arg

130 135 140

Arg Ile Arg Gly Glu Ser Glu Leu Tyr Thr Ser Asp Thr Val Met Gln

145 150 155 160

Asn

<210> 19

<211> 50

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Lys Gly Gln Asp His Val Asp Phe Ser Gln Pro Glu Pro His Thr Val

1 5 10 15

Leu Phe His Glu Pro Gly Ser Phe Ser Val Trp Val Gly Gly Arg Gly

20 25 30

Lys Val Tyr His Phe Asn Phe Pro Glu Gly Lys Asn Ala Ser Val Arg

35 40 45

Thr Val

50

<210> 20

<211> 51

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Asn Ile Gly Ser Thr Lys Gly Ser Cys Gln Asp Lys Gln Asp Cys Gly

1 5 10 15

Asn Tyr Ile Thr Leu Leu Glu Arg Arg Gly Asn Gly Leu Leu Val Cys

20 25 30

Gly Thr Asn Ala Arg Lys Pro Ser Cys Trp Asn Leu Val Asn Asp Ser

35 40 45

Val Val Met

50

<210> 21

<211> 60

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Ser Leu Gly Glu Met Lys Gly Tyr Ala Pro Phe Ser Pro Asp Glu Asn

1 5 10 15

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

20 25 30

Gln Glu Tyr Asn Gly Lys Ile Pro Arg Phe Arg Arg Ile Arg Gly Glu

35 40 45

Ser Glu Leu Tyr Thr Ser Asp Thr Val Met Gln Asn

50 55 60

<210> 22

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Ser Leu Gly Glu Met Lys Gly Tyr Ala Pro Phe Ser Pro Asp Glu Asn

1 5 10 15

Ser Leu Val Leu

20

<210> 23

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Phe Ser Pro Asp Glu Asn Ser Leu Val Leu Phe Glu Gly Asp Glu Val

1 5 10 15

Tyr Ser Thr Ile

20

<210> 24

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Phe Glu Gly Asp Glu Val Tyr Ser Thr Ile Arg Lys Gln Glu Tyr Asn

1 5 10 15

Gly Lys Ile Pro

20

<210> 25

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Arg Lys Gln Glu Tyr Asn Gly Lys Ile Pro Arg Phe Arg Arg Ile Arg

1 5 10 15

Gly Glu Ser Glu

20

<210> 26

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Arg Phe Arg Arg Ile Arg Gly Glu Ser Glu Leu Tyr Thr Ser Asp Thr

1 5 10 15

Val Met Gln Asn

20

<210> 27

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Phe Ser Pro Asp Glu Asn Ser Leu Val Leu Phe Glu Gly Asp Glu Val

1 5 10 15

<210> 28

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Phe Ser Pro Asp Glu Asn Ser Leu Val Leu Phe Glu

1 5 10

<210> 29

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Glu Asn Ser Leu Val Leu Phe Glu Gly Asp Glu Val Tyr Ser Thr Ile

1 5 10 15

<210> 30

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Val Leu Phe Glu Gly Asp Glu Val Tyr Ser Thr Ile

1 5 10

<210> 31

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

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

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Thr Thr Pro Ala Arg Ala Thr Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala

115

<210> 32

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 33

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

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

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Glu Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

100 105 110

Ser

<210> 34

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg

100 105

Claims

1. A monoclonal antibody to Semaphorin7A, characterized by: the Semaphorin7A monoclonal antibody comprises a heavy chain amino acid variable region and a light chain amino acid variable region, wherein: the 6 CDR amino acid sequences of the Semaphorin7A monoclonal antibody are as follows:

the amino acid sequence of VH CDR1 is SEQ ID NO.10,

the amino acid sequence of VH CDR2 is SEQ ID NO.11,

the amino acid sequence of VH CDR3 is SEQ ID NO.12,

the amino acid sequence of VL CDR1 is SEQ ID NO.14,

the amino acid sequence of VL CDR2 is SEQ ID NO.15,

the amino acid sequence of VL CDR3 is SEQ ID NO. 16.

2. The monoclonal antibody to Semaphorin7A, according to claim 1, wherein: the nucleotide sequence of the Semaphorin7A monoclonal antibody comprises: a heavy chain variable region consisting of the nucleotide sequence shown in SEQ ID NO.13 and a light chain variable region consisting of the nucleotide sequence shown in SEQ ID NO. 17.

3. Use of the Semaphorin7A monoclonal antibody of claim 1 or 2 for the preparation of a medicament for the treatment of an inflammatory disease, wherein: the inflammatory diseases are viral myocardial inflammation, staphylococcus aureus-induced sepsis inflammation and enterovirus EV71 acute infection.

4. The use of the Semaphorin7A monoclonal antibody according to claim 3, for the preparation of a medicament for the treatment of an inflammatory disease, wherein: the medicine for treating the inflammatory diseases also comprises a medicinal auxiliary material and a medicinal substrate.

5. The use of the Semaphorin7A monoclonal antibody according to claim 3 or 4, for the preparation of a medicament for the treatment of an inflammatory disease, wherein the medicament for the treatment of an inflammatory disease is in a liquid formulation or a lyophilized formulation.