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 PDF

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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
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Prior art keywords
fusion protein
seq
protein
sequence
amino acid
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PCT/CN2004/000449
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English (en)
French (fr)
Inventor
Xianggui Wu
Jun Bao
Haiming Chen
Junyu Gao
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Xianggui Wu
Jun Bao
Haiming Chen
Junyu Gao
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Application filed by Xianggui Wu, Jun Bao, Haiming Chen, Junyu Gao filed Critical Xianggui Wu
Priority to AU2004316866A priority Critical patent/AU2004316866B2/en
Priority to US10/592,376 priority patent/US7544783B2/en
Priority to EP04731810A priority patent/EP1731530A4/en
Priority to JP2007502169A priority patent/JP4676484B2/ja
Publication of WO2005085291A1 publication Critical patent/WO2005085291A1/zh

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    • 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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Description

一种融合蛋白及其编码基因与表达方法和应用 技术领域
本发明涉及基因工程和免疫学领域中的一种融合蛋白及其编码基因与表 达方法和应用， 特别是涉及一种融合蛋白及其编码基因与表达方法和以该融 合蛋白为活性成分的抗过敏药物。
背景技术
过敏性疾病是引起急，慢性疾病的第六大因素，它有 30- 60%的遗传机率。 常见的有过敏性鼻炎， 哮喘， 过敏性皮炎， 过敏性关节炎， 蓴麻症， 过敏性 休克等疾病。 约有 10- 15%的中国人及约 20%的美国人受过敏性疾病的困扰。
目前用于治疗过敏性疾病的药物大多为控制临床症状， 包括有类固醇激 素， 抗组胺药物， 减充血剂， 色甘酸钠， 支气管扩张剂； 最近美国 FDA批准 了于临床试验的抗 IgE单克隆抗体。 这些药物均有不同程度的副作用， 各研 究所、 大学及医药公司正在寻找一种新型的抗过敏药物。
过敏反应的机理是： 当抗原 （过敏原， 变应原）进入肌体， 与附着在肥 大细胞和嗜碱性粒细胞上的 IgE分子 （嗜细胞抗体） 结合， 触发该细胞的 Immunoreceptor Tyrosine - based Activation Motif (ITAM)激活， 促使细胞 释放生物活性物质， 引起平滑肌收缩,血管通透性增加， 浆液分泌增加等临床 病理变化。在这一病理过程中，人的肥大细胞和嗜碱性粒细胞上的亲和性 IgE 受体（FcERI ) 在过敏性疾病中起关 作用。 同时肥大细胞和嗜碱性粒细胞膜 表面表达 FcYRII抑制性受体， 当二分子 IgG同该受体结合时， 产生聚合而引 发 Immunoreceptor Tyrosine- based Inhibition Motif (ITIM)憐酸化并抑制 FcERI的激活信号。 最新的研究表明， 如果将 FcYRII受体与 FcERI受体交联， 可诱发肥大细胞或嗜碱性粒细胞内的抑制信号， 阻断细胞内的激活通路， 从 而抑制活性介质的释放， 进一步抑制了过敏反应。 因此， 运用该反应机理来 研究一种具有激活过敏反应中的抑制信号的融合蛋白，是崭新的抗过敏途径。 发明公幵
本发明的目的是提供一种融合蛋白及其编码基因， 该蛋白具有抗过敏活 性。
本发明所提供的融合蛋白， 名称为 FP4， 是具有序列表中 SEQ ID ΝΩ : 2 氨基酸残基序列的蛋白质， 或者是将 SEQ ID : 2的氨基酸残基序列经过一 个或几个氨基酸残基的取代、 缺失或添加且具有与 SEQ ID N2 : 2的氨基酸残 基序列相同活性的由 SEQ ID No : 2衍生的蛋白质。
序列表中序列 2的氨基酸残基序列是由 554个氨基酸残基组成的蛋白质。 FP4的结构如图 1所示， 它由三部分组成： A区 （Fc- ε ) (自氨基端第 1-300 位氨基酸残基） ， Β区 （Fc- γ) (自氨基端第 318-554位氨基酸残基） 和 C 区 （hinge) (自氨基端第 301-317位氨基酸残基） 。 其中， A区来源于人的 IgE免疫球蛋白， 具有与 IgE受体 FcERI结合的部位； B区来源于人的 IgG免 疫球蛋白， 具有与 IgG受体 FcYRII结合的部位； C区来源于人的免疫球蛋白， 为绞连结构。
一种融合蛋白编码基因， 名称为 / 是下列核苷酸序列之一：
1 ) 序列表中 SEQ ID No : 1的 DNA序列；
2 ) 编码序列表中 SEQ ID No: 2蛋白质序列的多核苷酸；
3) 与序列表中 SEQ ID Na: 1限定的 DNA序列具有 95%以上同源性， 且 编码相同功能蛋白质的 DNA序列。
序列 1中的 DNA序列由 1665个碱基组成，该基因的开放阅读框架为自 5' 端第 1到第 1665位碱基。 ·
含有本发明基因的表达载体及细胞系均属于本发 '明的保护范围。
扩增 7/ 中任一片段的引物对也在本发明的保护范围之内。
本发明的第二个目的是提供一种表达融合蛋白 FP4的方法。
本发明所提供的表达融合蛋白 FP4的方法， 是将含有融合蛋白编码基因 的表达载体转化到 SP2/0细胞中， 得到阳性克隆， 培养所述阳性克隆， 表 达融合蛋白 FP4。
含有融合蛋白编码基因的表达载体可按常规方法将 ^ ^连接入表达载体 pSecTag得到。
本发明的第三个目的是提供一种抗过敏药物， 它的活性成分是融合蛋白
FP4。
需要的时候，在上述药物中还可以加入一种或多种药学上可接受的载体。 所述载体包括药学领域常规的稀释剂、 赋形剂、 填充剂、 粘合剂、 湿润剂、 崩解剂、 吸收促进剂、 表面活性剂、 吸附载体、 润滑剂等， 必要时还可以加 入香味剂、 甜味剂等。
本发明的药物可以制成用于静脉注射等的注射剂， 用于皮下注射、 表皮 外敷等的皮剂， 用于喷鼻、 喉、 口腔、 表皮、 粘膜等的喷雾剂， 用于滴鼻、 眼、 耳等的滴剂， 用于肛肠等的栓剂， 片剂， 粉剂， 粒剂， 胶囊， 口服液， 膏剂， 霜剂等多种形式。 上述各种剂型的药物均可以按照药学领域的常规方 法制备。
上述药物的用量一般为 0. 01- lug/kg/day 疗程一般为 10至 20天。
附图说明
图 1为 FP4的结构示意图
图 2为 FP4的表达过程示意图
图 3A为 Fcs DNA PCR扩增产物电泳图谱
图 3B为 FcY DNA PCR扩增产物电泳图谱
图 4为 FP4与 FcERI受体结合能力鉴定结果
图 5为 FP4与 FcyRII受体结合能力鉴定结果
图 6为 FP4蛋白抑制嗜碱性粒细胞释放组胺实验结果
图 7为 FP4抑制转基因老鼠的过敏反应结果
实施发明的最佳方式
实施例 1、 融合蛋白 FP4的表达
融合蛋白 FP4的表达过程如图 2所示， 具体包括以下步骤： 按照常规方 法进行以下操作： 从人外周血中分离、纯化 B淋巴细胞， 提取基因组 DNA, 用 特异性引物：
Pl (Fce) : 5' - GTGGCCCAGCCGGCCTTCACCCCGCCCACCGTGAAG- 3， ；
P2 (FcE) : 5' -GTGGATCCTTTACCGGGATTTACAGACAC-3' ；
P3 (Fcy) : 5 ' -GGGGATCCGAGCCCAAATCTTGTGAC-3 ' ；
P4 (Fcy)： 5， -GTGCGGCCGCTCATTTACCCGGAGACAGGGAGAG- 3 ' 。
PCR扩增 1^(^和 Fcy基因， 将 Fc^a Fcy基因的 PCR产物进行 1 %的琼脂糖 凝胶电泳， 电泳结果如图 3A和图 3B所示， FcE DNA PCR扩增产物为 1155bp;
FcY DNA PCR扩增产物为 928bp。 将 PCR产物分别直接克隆至 pCR4-T0P0 ( INVITROGEN, CA) 得到 FPEFc- ε和 FPGFc- γ， 进行核苷酸序列分析； 在得到 了 100%正确的核苷酸序列后， 将？^和 基因分别利用 Sfil-BamHI 和
BamHI-Notl限制性内切酶 （NEW ENGLAND BIOL ABS ) 进行酶切， 并用限制性内 切酶 Sfil- Notl 酶切 pSecTag ( INVITROGEN, CA) 表达载体， 然后进行连接， 得到质粒 pSecTagFP4，对质粒 PSecTagFP4进行核苷酸序列分析，表明插入的 外源基因序列如序列表中的序列 1所示。 然后将此质粒利用常规电穿孔法转 染至小鼠骨髓瘤 SP2/0细胞， 经 ZE0CIN抗药性筛选及 ELISA鉴定（用常规方 法制备的鼠抗人 IgE抗体包备 ELISA平板， 加入标本上清， 然后加入羊抗人 IgE酶标抗体） ， 得到高表达 FP4 (其氨基酸序列如序列表中的序列 2) 的克 隆。
利用 RPMI1640 (INVITR0GEN， CA)培养基， 在 37°C, 5% C02培养上述阳性 克隆（含有质粒 pSecTagFP4的小鼠骨髓瘤 SP2/0细胞） 15天， 2000rpm离心 后， 收集上清，再用鼠抗人 IgE亲和层析法 （将鼠抗人 IgE交联至
CNBr- ACTIVATED SEPHAROSE 4B (PHARMACIA)分离纯化 FP4蛋白。
实施例 2、 FP4与 FceRI受体结合实验 、
将 1 X 106 CH03D10细胞与 5微克实施例 1中纯化的 FP4蛋白在 4°C反应 1小时， 加入 5微升 FITC标记的抗人 IgE抗体（CALTAG, CA)， 用流式细胞仪 检测细胞。 结果如图 4所示， 表明 FP4蛋白可与 FcERI受体结合， CH03D10细 胞呈 FITC阳性。 图 4中， SSC- H为细胞内颗粒数， FSC- H为细胞大小， FL2 - H 为 PE, FL1- H为 FITC。
实施例 3、 FP4与 FcyRII受体结合实验
将 I X 106 HMC-1细胞与 5微克实施例 1中纯化的 FP4蛋白在 4°C 反应 1 小时， 加入 5微升 FITC标记的抗人 IgG FITC标记抗体 (CALTAG, CA) ， 用流 式细胞仪检测细胞。 结果如图 5所示， 表明 FP4蛋白可与 FcyRII受体结合， HMC-1细胞呈 FITC阳性。图 5中， SSC-H为细胞内颗粒数， FSC- H为细胞大小， FL2- H为 PE， FL1- H为 FITC 。
实施例 4、 FP4 蛋白的体外功能实验
按照常规方法从人外周血中分离、 纯化嗜碱性粒细胞， 将分离的 I X 106 嗜碱性粒细胞与 1-10微克人特异性抗 NP-IgE(SER0TECH)37°C反应 2小时后， 加入 2微克过敏原 NP， 然后用 ELISA检测细胞培养上清中的组胺水平。 结果 如图 6所示， 表明该蛋白 （FP4) 处理后， 组胺释放明显减弱， 呈明显的剂量 依赖的抑制作用。 图 6中， Hu IgE表示人 Ig£， NS IgE表示正常对照 IgE， NP - BSA表示过敏原， +表示加入， 一表示不加入。
实施例 5、 FP4 蛋白的体内功能实验
人 FceRI a链转基因鼠 （购自 University of Calif ornia, San Diego) ， 经局部皮内注射特异性人抗 NP- IgE (SER0TECH) ， 4小时后静脉注入 100微 克过敏原 NP和 1% EVANS氏蓝色染料， 如致敏部位发生过敏反应， 局部皮肤 由于血管通透性增加， 蓝色染料从血管内渗出， 进入组织间隙， 呈蓝色反应。 当在对应皮肤部位致敏的同时加入等量的融合蛋白 （FP4) ， 则过敏反应完全 被抑制， 局部皮肤无任何改变， 如图 7所示。 本实验结果进一步证明 FP4蛋 白在动物体内的抗过敏作用。
工业应用
本发明的融合蛋白 FP4组成成份全部来源于人源 （human)性免疫球蛋白， 因此， 该蛋白作为一种药物进入人体， 没有任何异体蛋白的免疫源性。 融合 蛋白 FP4的 C区 （hinge)灵活， 可转动， 可将 FcERI和 FcYRII交联在一起， 从而 诱发细胞内的抑制信号， 阻止细胞释放各种生物活性物质， 抑制过敏反应的 发生。体内外实验结果均证明融合蛋白 FP4可以有效地交联肥大细胞或嗜碱性 粒细胞表面的 FcERI和 FcyRII， 从而抑制过敏反应。本发明的融合蛋白 FP4不但 具有 IgE单克隆抗体的阻断 IgE受体的作用， 而且更重要的是启动了过敏反应 在细胞内信号传导系统的抑制系统， 从而强有力地抑制了细胞过敏反应， 将 在过敏性疾病的治疗中发挥重要作用。

Claims

权利要求书
1、 一种融合蛋白， 是具有序列表中 SEQ ID No : 2氨基酸残基序列的蛋 白质， 或者是将 SEQ ID Na: 2的氨基酸残基序列经过一个或几个氨基酸残基 的取代、 缺失或添加且具有与 SEQ ID No : 2的氨基酸残基序列相同活性的由 SEQ ID No : 2衍生的蛋白质。
2、 根据权利要求 1所述的蛋白质， 其特征在于： 所述融合蛋白是序列表 中的 SEQ ID Na: 2。
3、 一种融合蛋白编码基因， 是下列核苷酸序列之一：
1 ) 序列表中 SEQ ID Ns: 1的 DNA序列；
2) 编码序列表中 SEQ ID No : 2蛋白质序列的多核苷酸；
3) 与序列表中 SEQ ID N2 : 1限定的 DNA序列具有 95%以上同源性， 且 编码相同功能蛋白质的 DNA序列。
4、 根据权利要求 3所述的基因， 其特征在于： 所述融合蛋白编码基因是 序列表中的 SEQ ID No : 1。
5、 含有权利要求 3所述基因的表达载体和细胞系。
6、 扩增权利要求 3所述基因任一片段的引物对。
7、一种表达权利要求 1所述融合蛋白的方法， 是将含有融合蛋白编码基 因的表达载体转化到 SP2/0中， 得到阳性克隆， 培养所述阳性克隆， 表达融 合蛋白 FP4; 所述融合蛋白编码基因是下列核苷酸序列之一：
1 ) 序列表中 SEQ ID No : 1的 DNA序列；
2) 编码序列表中 SEQ ID Na: 2蛋白质序列的多核苷酸；
3) 与序列表中 SEQ ID No : 1限定的 DNA序列具有 95 %以上同源性， 且 编码相同功能蛋白质的 DNA序列。
8、 根据权利要求 7所述的方法， 其特征在于： 所述含有融合蛋白编码基 因的表达载体是将所述融合蛋白编码基因连接入表达载体 PSecTag 中得到 的。
9、 以权利要求 1所述融合蛋白为活性成分的抗过敏药物。
10、 权利要求 1所述融合蛋白在制备治疗抗过敏药物中的应用。
PCT/CN2004/000449 2004-03-10 2004-05-08 Proteine fusionnee, gene codant pour elle, sa methode d'expression et ses utilisations WO2005085291A1 (fr)

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EP04731810A EP1731530A4 (en) 2004-03-10 2004-05-08 FUSED PROTEIN, GENE ENCODING THE SAME, ITS EXPRESSION METHOD AND USES THEREOF
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

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