WO2005085291A1 - Proteine fusionnee, gene codant pour elle, sa methode d'expression et ses utilisations - Google Patents
Proteine fusionnee, gene codant pour elle, sa methode d'expression et ses utilisations Download PDFInfo
- Publication number
- WO2005085291A1 WO2005085291A1 PCT/CN2004/000449 CN2004000449W WO2005085291A1 WO 2005085291 A1 WO2005085291 A1 WO 2005085291A1 CN 2004000449 W CN2004000449 W CN 2004000449W WO 2005085291 A1 WO2005085291 A1 WO 2005085291A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fusion protein
- seq
- protein
- sequence
- amino acid
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/16—Otologicals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/862—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving IgE or IgD
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/868—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving autoimmunity, allergy, immediate hypersensitivity, delayed hypersensitivity, immunosuppression, or immunotolerance
Definitions
- Fusion protein coding gene and expression method and application thereof
- the invention relates to a fusion protein, a coding gene and an expression method and application thereof in the field of genetic engineering and immunology, in particular to a fusion protein, a coding gene and an expression method thereof, and anti-allergy using the fusion protein as an active ingredient drug.
- Allergic disease is the sixth leading cause of acute and chronic diseases, and it has a genetic chance of 30-60%.
- Common diseases include allergic rhinitis, asthma, allergic dermatitis, allergic arthritis, ramie, anaphylactic shock and other diseases. About 10-15% of Chinese and about 20% of Americans suffer from allergic diseases.
- the mechanism of an allergic reaction is: When an antigen (allergen, allergen) enters the body, it binds to IgE molecules (cytophilic antibodies) attached to mast cells and basophils, triggering the cell's Immunoreceptor Tyrosine-based Activation Motif (ITAM) activation activates cells to release bioactive substances, causing smooth muscle contraction, increased vascular permeability, and increased serum secretion and other clinical pathological changes.
- IgE molecules cytophilic antibodies
- ITAM Immunoreceptor Tyrosine-based Activation Motif
- Fc E RI affinity IgE receptors
- Fc Y RII inhibitory receptors are expressed on the surface of mast cells and basophils.
- the object of the present invention is to provide a fusion protein and a coding gene thereof, which protein has anti-allergic activity.
- the fusion protein provided by the present invention is a protein having the sequence of the amino acid residues of SEQ ID NO : 2 in the sequence listing, or the sequence of the amino acid residues of SEQ ID: 2 is A protein derived from SEQ ID No : 2 with the substitution, deletion or addition of one or several amino acid residues and having the same activity as the amino acid residue sequence of SEQ ID N 2: 2.
- the amino acid residue sequence of Sequence 2 in the Sequence Listing is a protein consisting of 554 amino acid residues.
- the structure of FP4 is shown in Figure 1. It consists of three parts: the A region (Fc- ⁇ ) (from amino acid residues 1-300 at the amino terminal), and the beta region (Fc- ⁇ ) (from amino terminal 318- 554 amino acid residues) and C region (hinge) from amino acid residues 301-317 at the amino terminus.
- the A region is derived from human IgE immunoglobulin and has a site that binds to the IgE receptor Fc E RI;
- the B region is derived from human IgG immunoglobulin and has a site that binds to the IgG receptor Fc Y RII;
- the C region Human-derived immunoglobulins are stranded structures.
- a fusion protein-encoding gene named / is one of the following nucleotide sequences:
- the DNA sequence in Sequence 1 consists of 1665 bases.
- the open reading frame of the gene is from the 1 'to 1665 bases at the 5' end. ⁇
- Both the expression vector and the cell line containing the gene of the present invention belong to the protection scope of the present invention.
- a primer pair for amplifying any of the 7 / fragments is also within the scope of the present invention.
- a second object of the present invention is to provide a method for expressing the fusion protein FP4.
- the method for expressing the fusion protein FP4 provided by the present invention is to transform an expression vector containing a fusion protein-encoding gene into SP2 / 0 cells to obtain a positive clone, culture the positive clone, and express the fusion protein FP4.
- An expression vector containing a fusion protein-encoding gene can be obtained by ligating ⁇ into an expression vector pSecTag according to a conventional method.
- a third object of the present invention is to provide an anti-allergic drug whose active ingredient is a fusion protein
- the carrier includes a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, etc., which are conventional in the pharmaceutical field, and a fragrance may be added if necessary.
- the medicament of the present invention can be made into an injection for intravenous injection and the like, and used for subcutaneous injection and epidermis Skin preparations for external application, sprays for nasal, larynx, oral cavity, epidermis, mucous membrane, etc., drops for nose, eye, ear, etc., suppositories for anorectal, tablets, powder, granules , Capsules, oral solution, ointment, cream and many other forms.
- the aforementioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field.
- the dosage of the above drugs is generally 0.01-lug / kg / day.
- the course of treatment is generally 10 to 20 days.
- Figure 1 shows the structure of FP4
- Figure 2 is a schematic diagram of the expression process of FP4
- FIG. 3A is an electrophoresis map of Fc s DNA PCR amplification products
- Figure 3B is an electrophoresis map of Fc Y DNA PCR amplification products
- Figure 4 shows the results of FP4 binding to the Fc E RI receptor.
- Figure 5 shows the identification of the binding ability of FP4 to FcyRII receptor
- Figure 6 shows the results of FP4 protein inhibition of histamine release from basophils
- Figure 7 shows the results of FP4 inhibition of allergic reactions in transgenic mice
- the expression process of the fusion protein FP4 is shown in Figure 2. It specifically includes the following steps: Perform the following operations according to conventional methods: Isolate and purify B lymphocytes from human peripheral blood, extract genomic DNA, and use specific primers:
- the Fc Y DNA PCR amplification product was 928bp.
- the PCR products were cloned directly into pCR4-T0P0 (INVITROGEN, CA) to obtain FPEFc- ⁇ and FPGFc- ⁇ for nucleotide sequence analysis. After obtaining a 100% correct nucleotide sequence, what? ⁇ And genes use Sfil-BamHI and
- This plasmid was then transfected into mouse myeloma SP2 / 0 cells using conventional electroporation, and was screened for ZEOCIN resistance and identified by ELISA (a mouse anti-human IgE antibody prepared by a conventional method was used to prepare an ELISA plate, and the sample supernatant was added. Then add goat anti-human IgE enzyme-labeled antibody) to obtain a clone that highly expresses FP4 (its amino acid sequence is sequence 2 in the sequence listing).
- mice myeloma SP2 / 0 cells containing the plasmid pSecTagFP4 were cultured in RPMI1640 (INVITR0GEN, CA) medium at 37 ° C, 5% C0 2 for 15 days. After centrifugation at 2000 rpm, the supernatant was collected and reused.
- Mouse anti-human IgE affinity chromatography cross-linking mouse anti-human IgE to
- IX 10 6 HMC-1 cells were reacted with 5 ⁇ g of the FP4 protein purified in Example 1 at 4 ° C. for 1 hour, and 5 ⁇ l of FITC-labeled anti-human IgG FITC-labeled antibody (CALTAG, CA) was added, and flow cytometry was used. The instrument detects the cells.
- the results are shown in Figure 5, which indicates that FP4 protein can bind to the FcyRII receptor and that HMC-1 cells are FITC positive.
- SSC-H is the number of particles in the cell
- FSC-H is the cell size
- FL2-H is PE
- FL1-H is FITC.
- Hu IgE indicates human Ig £
- NS IgE indicates normal control IgE
- NP-BSA indicates allergen
- + indicates addition, and one indicates not added.
- the components of the fusion protein FP4 of the present invention are all derived from human immunoglobulins. Therefore, the protein enters the human body as a medicine without any immunogenicity of foreign proteins.
- the C region (hinge) of the fusion protein FP4 is flexible and rotatable, which can link Fc E RI and Fc Y RII together, thereby inducing intracellular inhibition signals, preventing cells from releasing various biologically active substances, and inhibiting the occurrence of allergic reactions. .
- Experimental results in vivo and in vitro have proved that the fusion protein FP4 can effectively cross-link Fc E RI and Fc y RII on the surface of mast cells or basophils, thereby inhibiting allergic reactions.
- the fusion protein FP4 of the present invention not only has the function of blocking IgE receptors by IgE monoclonal antibodies, but also more importantly activates the inhibitory system of the allergic reaction in the cell signaling system, thereby strongly inhibiting the allergic response of cells, Will play an important role in the treatment of allergic diseases.
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Pulmonology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Rheumatology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Dermatology (AREA)
- Physical Education & Sports Medicine (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2007502169A JP4676484B2 (ja) | 2004-03-10 | 2004-05-08 | 臨床応用のための融合タンパク質の塩基配列決定及び遺伝子発現 |
US10/592,376 US7544783B2 (en) | 2004-03-10 | 2004-05-08 | Fusion protein for treatment of allergic diseases |
EP04731810A EP1731530A4 (en) | 2004-03-10 | 2004-05-08 | FUSED PROTEIN, GENE ENCODING THE SAME, ITS EXPRESSION METHOD AND USES THEREOF |
AU2004316866A AU2004316866B2 (en) | 2004-03-10 | 2004-05-08 | A kind of fusion protein, gene encoding it, expression method and use thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB2004100064989A CN1317304C (zh) | 2004-03-10 | 2004-03-10 | 一种融合蛋白及其编码基因与表达方法和应用 |
CN200410006498.9 | 2004-03-10 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2005085291A1 true WO2005085291A1 (fr) | 2005-09-15 |
Family
ID=34916975
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2004/000449 WO2005085291A1 (fr) | 2004-03-10 | 2004-05-08 | Proteine fusionnee, gene codant pour elle, sa methode d'expression et ses utilisations |
Country Status (6)
Country | Link |
---|---|
US (1) | US7544783B2 (zh) |
EP (1) | EP1731530A4 (zh) |
JP (1) | JP4676484B2 (zh) |
CN (1) | CN1317304C (zh) |
AU (1) | AU2004316866B2 (zh) |
WO (1) | WO2005085291A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110243944A1 (en) * | 2008-10-08 | 2011-10-06 | Shanghai Kexin Biotech Co., Ltd. | Fusion Protein Inhibiting Osteoclast Formation, Preparation Method and Medicine Compositions Thereof |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5854432B2 (ja) * | 2012-09-28 | 2016-02-09 | 森永乳業株式会社 | アレルギー反応検出法 |
US8961992B1 (en) * | 2014-04-02 | 2015-02-24 | Tunitas Therapeutics, Inc. | Epsigam fusion protein |
CN107635584B (zh) * | 2015-04-09 | 2022-06-17 | 康奈尔大学 | 基因治疗以预防对过敏原的反应 |
CN104922670A (zh) * | 2015-05-06 | 2015-09-23 | 武汉奥斯梅得生物医药有限公司 | 融合免疫蛋白在制备治疗多发性硬化症药物中的应用 |
CN107383200B (zh) * | 2016-05-17 | 2021-04-09 | 深圳先进技术研究院 | 鼠源抗人IgE单克隆抗体的制备方法及其应用 |
CN111454366B (zh) * | 2019-01-21 | 2023-06-16 | 中国科学院深圳先进技术研究院 | 一种融合蛋白及其应用 |
CN111574608B (zh) * | 2020-04-21 | 2021-11-19 | 沈阳农业大学 | 牛细粒棘球蚴病的特异性检测抗原及其应用 |
CN112023062A (zh) * | 2020-09-18 | 2020-12-04 | 北京基因安科技有限公司 | 一个用可溶性IgE受体抑制过敏反应的方法 |
CN116410327A (zh) * | 2021-12-31 | 2023-07-11 | 祝道成 | 一种融合蛋白及其制备方法和应用 |
WO2024120493A1 (en) * | 2022-12-07 | 2024-06-13 | Daocheng Zhu | Formulation and use of a fusion protein |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002088317A2 (en) * | 2001-05-01 | 2002-11-07 | The Regents Of The University Of California | Fusion molecules and methods for treatment of immune diseases |
WO2002102320A2 (en) * | 2001-06-15 | 2002-12-27 | Tanox, Inc. | Fce fusion proteins for treatment of allergy and asthma |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7265208B2 (en) * | 2001-05-01 | 2007-09-04 | The Regents Of The University Of California | Fusion molecules and treatment of IgE-mediated allergic diseases |
-
2004
- 2004-03-10 CN CNB2004100064989A patent/CN1317304C/zh not_active Expired - Fee Related
- 2004-05-08 AU AU2004316866A patent/AU2004316866B2/en not_active Ceased
- 2004-05-08 US US10/592,376 patent/US7544783B2/en not_active Expired - Lifetime
- 2004-05-08 WO PCT/CN2004/000449 patent/WO2005085291A1/zh active Application Filing
- 2004-05-08 EP EP04731810A patent/EP1731530A4/en not_active Ceased
- 2004-05-08 JP JP2007502169A patent/JP4676484B2/ja not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002088317A2 (en) * | 2001-05-01 | 2002-11-07 | The Regents Of The University Of California | Fusion molecules and methods for treatment of immune diseases |
WO2002102320A2 (en) * | 2001-06-15 | 2002-12-27 | Tanox, Inc. | Fce fusion proteins for treatment of allergy and asthma |
Non-Patent Citations (4)
Title |
---|
KEPLEY C.L. ET AL: "FcepsilonRI-FcgammaRII coagregation inhibits IL-16 production from human Langerhans-like dendritic cells.", CLIN.IMMUNOL., vol. 108, no. 2, August 2003 (2003-08-01), pages 89 - 94, XP008091344 * |
See also references of EP1731530A4 * |
YAMADA T. ET AL: "Inhibition of interleukin-4-induced class switch recombination by a human immunoglobulin Fc epsilon chimeric protein.", J.BIOL.CHEM., vol. 278, no. 35, 2003, pages 32818 - 32824, XP002459956 * |
ZHU D. ET AL: "A novel immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation.", NAT.MED., vol. 8, no. 5, 2002, pages 518 - 521, XP002976663 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110243944A1 (en) * | 2008-10-08 | 2011-10-06 | Shanghai Kexin Biotech Co., Ltd. | Fusion Protein Inhibiting Osteoclast Formation, Preparation Method and Medicine Compositions Thereof |
US8460673B2 (en) * | 2008-10-08 | 2013-06-11 | Shanghai Kexin Biotech Co., Ltd. | Fusion protein inhibiting osteoclast formation, preparation method and medicine compositions thereof |
Also Published As
Publication number | Publication date |
---|---|
EP1731530A1 (en) | 2006-12-13 |
JP4676484B2 (ja) | 2011-04-27 |
EP1731530A9 (en) | 2007-05-09 |
US20080015347A1 (en) | 2008-01-17 |
EP1731530A4 (en) | 2008-03-12 |
JP2008500807A (ja) | 2008-01-17 |
AU2004316866B2 (en) | 2009-04-02 |
AU2004316866A1 (en) | 2005-09-15 |
US7544783B2 (en) | 2009-06-09 |
CN1317304C (zh) | 2007-05-23 |
CN1666996A (zh) | 2005-09-14 |
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