A fusion protein and a preparation method and application thereof

Abstract

The application discloses a fusion protein, a coding gene and an expression method and application thereof, and aims to provide a fusion protein, a coding gene and an expression method thereof and an anti-allergic drug taking the fusion protein as an active ingredient. The fusion protein provided by the application is a protein with amino acid residues as shown in SEQ ID NO:3. The fusion protein of the application can inhibit the cell allergic reaction through the inhibition system of the intracellular signal transmission system of the allergic reaction, thereby effectively inhibiting the cell allergic reaction and playing an important role in the treatment of allergic diseases.

Description

Technical Field

[0001] This invention relates to a fusion protein, its encoding gene, expression method, and applications in the fields of genetic engineering and immunology, and particularly to a fusion protein, its encoding gene, expression method, and an anti-allergy drug with the fusion protein as an active ingredient. Background Technology

[0002] Allergic diseases are the sixth leading cause of acute and chronic illnesses, and they have a strong genetic predisposition. Common allergic diseases include allergic asthma, allergic rhinitis, hay fever, allergic dermatitis, allergic arthritis, anaphylactic paralysis, and anaphylactic shock. Approximately 10-15% of Chinese people and about 20% of Americans suffer from allergic diseases.

[0003] Currently, most medications used to treat allergic diseases control clinical symptoms, such as corticosteroids, antihistamines, decongestants, sodium tryptophan, and bronchodilators. Recently, the US FDA approved an anti-IgE monoclonal antibody in clinical trials. However, these drugs all have varying degrees of side effects; therefore, many drug research institutions and pharmaceutical companies both domestically and internationally are dedicated to finding new types of anti-allergy drugs.

[0004] The mechanism of allergic reactions is as follows: When antigens (allergens, sensitizers) enter the body, they bind to IgE molecules (cytotropic antibodies) attached to mast cells and basophils, triggering the activation of the immunoreceptor tyrosine-based activation motif (ITAM) on these cells. This leads to the release of bioactive substances from the cells, causing smooth muscle contraction, increased vascular permeability, and increased serous fluid secretion, among other clinical and pathological changes. In this pathological process, the affinity IgE receptor (FcεRI) on mast cells and basophils plays a crucial role in allergic diseases. Simultaneously, when two IgG molecules bind to the FcγRIIB inhibitory receptor expressed on the cell membrane surface of mast cells and basophils, they polymerize, triggering phosphorylation of the immunoreceptor tyrosine-based inhibition motif (ITIM) and inhibiting the activation signal of FcεRI. Studies have shown that cross-linking FcγRII receptors with FcεRI receptors can induce inhibitory signals in mast cells or basophils, blocking intracellular activation pathways and thus inhibiting the release of active mediators, further suppressing allergic reactions (Zhao W, Kepley CL, Morel PA, et al., FcγRIIa, Not FcγRIIb, Is Constitutively and Functionally Expressed on Skin-Derived Human Mast Cells. The Journal of Immunology, 2006, 177(1):694–701). Therefore, using this reaction mechanism to study a fusion protein that activates inhibitory signals in allergic reactions should be a novel approach to treating allergies.

[0005] Recent research indicates that due to polymorphism in human FcγRII gene expression, a minority of human subcutaneous mast cells do not express FcγRIIB, but instead express FcγRIIA. The intracellular terminal of the FcγRIIA receptor contains an immunoreceptor tyrosine activation motif (ITAM). Direct crosslinking of FcγRIIA to FcεRII (resulting in the fusion protein FP4) can induce intracellular phosphorylation in allergic cells, thereby activating intracellular activation pathways, leading to degranulation of allergic cells and the development of allergic diseases. The fusion protein FP4 cannot overcome the allergic reaction induced by FcγRIIA expression. Summary of the Invention

[0006] To overcome the defect in existing technologies where the fusion protein FP4 binds to the FcγRIIA receptor and causes degranulation of allergic reaction cells, this invention provides a fusion protein and its encoding gene. This fusion protein binds only to the FcγRIIB receptor, and its affinity is significantly better than that of the fusion protein FP4 (amino acid sequence as shown in SEQ ID NQ:1; DNA sequence encoding it as shown in SEQ ID NQ:2), while maintaining its affinity for the IgE receptor FcεRI.

[0007] The fusion protein provided by this invention, named MEG, is a protein having amino acid residues as shown in SEQ ID NO:3, or a protein derived from SEQ ID NO:3 by substituting or deleting one or more amino acid residues of the amino acid residue sequence of SEQ ID NO:3 and having the same or better activity as the amino acid residue sequence of SEQ ID NO:3.

[0008] The amino acid sequence of SEQ ID NO:3 is a protein composed of 552 amino acid residues. The structure of MEG is as follows: Figure 1 As shown, it consists of two parts: region A (Fc-ε) (amino acid residues 1-320 from the amino terminus) and region B (Fc-γ) (amino acid residues 321-552 from the amino terminus). Region A is derived from human IgE immunoglobulin and has a binding site for the IgE receptor FcεRI; region B is derived from human IgG immunoglobulin and has a binding site for the IgG receptor FcγRII.

[0009] The technical solution of the present invention is as follows:

[0010] One of the technical solutions provided by the present invention is: a fusion protein, the amino acid sequence of which is shown in SEQ ID NO:3.

[0011] The second technical solution provided by this invention is: a gene encoding a fusion protein, the nucleotide sequence of which is selected from:

[0012] (1)SEQ ID NO:4;

[0013] (2) Encoding a polynucleotide of the fusion protein as described in one of the technical solutions.

[0014] In some preferred embodiments, the nucleotide sequence of the gene is shown in SEQ ID NO:4.

[0015] The third technical solution provided by the present invention is: a recombinant expression vector, which includes the gene as described in the second technical solution.

[0016] In some preferred embodiments, the backbone plasmid of the recombinant expression vector is pSecTag.

[0017] The fourth technical solution provided by the present invention is: a transformant, which includes the recombinant expression vector as described in the third technical solution; preferably, the host of the transformant is a mammalian cell.

[0018] In a preferred embodiment, the mammalian cell is a CHO cell.

[0019] The fifth technical solution provided by the present invention is: a method for preparing the fusion protein as described in the first technical solution, wherein the method involves culturing the transformant as described in the fourth technical solution to express the fusion protein; preferably, the protein is separated and purified by affinity chromatography; more preferably, the affinity chromatography is mouse anti-human IgE affinity chromatography.

[0020] The sixth technical solution provided by the present invention is: a drug whose active ingredient includes the fusion protein as described in one of the technical solutions.

[0021] The seventh technical solution provided by the present invention is: the application of the fusion protein as described in one of the technical solutions in the preparation of anti-allergy drugs.

[0022] The positive and progressive effects of this invention are as follows:

[0023] The fusion protein MEG of this invention is composed of 99.9% human immunoglobulins. Therefore, when this protein enters the human body as a drug, it has no immunogenicity due to the presence of any foreign protein. In vitro and in vivo experiments have demonstrated that the fusion protein MEG can effectively cross-link FcεRI and FcγRII on the surface of mast cells or basophils, thereby inhibiting allergic reactions. The fusion protein MEG of this invention primarily inhibits cellular allergic reactions by activating the inhibitory system of the intracellular signal transduction system in allergic reactions, thus playing an important role in the treatment of allergic diseases. Attached Figure Description

[0024] Figure 1 Schematic diagram showing the structural differences between fusion protein FP4 and fusion protein MEG.

[0025] Figure 2 Comparison of SDS-PAGE of supernatant and purified MEG.

[0026] Figure 3 Figure: Identification results of the binding ability of the fusion protein MEG to the FcεRI receptor.

[0027] Figure 4 Figure: Identification results of the binding ability of the fusion protein MEG to the FcγRII receptor.

[0028] Figure 5Figure 1: Analysis of the binding of MEG fusion protein to FcγRIIA receptor (A) and FcγRIIB receptor (B).

[0029] Figure 6 MEG fusion protein inhibits allergen-induced degranulation in vivo. Detailed Implementation

[0030] The present invention is further illustrated below by way of examples, but these examples do not limit the invention to the scope of the embodiments described. Experimental methods not specifically described in the following examples were performed according to conventional methods and conditions, or as selected according to the product instructions. All reagents and raw materials used in this invention are commercially available.

[0031] Example 1: Expression and purification of MEG molecules

[0032] The expression of the fusion protein FP4, as shown in CN1317304C, specifically includes the following steps:

[0033] The following procedures were performed using standard methods: B lymphocytes were isolated and purified from human peripheral blood, and genomic DNA was extracted. The Fcε and Fcγ genes were amplified by PCR using specific primers. The Fcε DNA PCR amplification product was 1155 bp, and the Fcγ DNA PCR amplification product was 928 bp. The PCR products were cloned into pCR4-TOPO (INVITROGEN, CA) to obtain FPEFc-ε and FPGFc-γ, respectively, and nucleotide sequence analysis was performed. After obtaining 100% correct nucleotide sequences, the Fcε and Fcγ genes were digested with SfiI-BamHI and BamHI-NotI restriction endonucleases (NEW ENGLAND BIOLABS), respectively. The pSecTag (INVITROGEN, CA) expression vector was then digested with the restriction endonuclease SfiI-NotI, and ligated to obtain the plasmid pSecTagMEG. Nucleotide sequence analysis of the pSecTagMEG plasmid showed that the inserted foreign gene sequence was as shown in SEQ IDNQ:2. The plasmid was then transfected into mouse myeloma SP2 / 0 cells using conventional electroporation. After ZEOCIN resistance screening and ELISA identification (ELISA plates were prepared with mouse anti-human IgE antibody using conventional methods, the sample supernatant was added, and then goat anti-human IgE enzyme-labeled antibody was added), a clone expressing FP4 (its amino acid sequence is as shown in SEQ ID NQ:1) was obtained.

[0034] MEG (MEG structure is shown in...) Figure 1 The construction of ) differs from FP4, the difference being:

[0035] (1) MEG genes were synthesized based on the sequences of human immunoglobulin IgE Fcε and IgG Fcγ. The corresponding sequences were cloned into transient expression vectors using SOE PCR and enzyme digestion. After being verified by sequencing, the vectors were transfected into mammalian CHO cells.

[0036] (2) High-purity protein was obtained after two-step purification by GE Mab-select affinity chromatography and cation exchange. SDS-PAGE (results are shown in [see image]). Figure 2 The purity of the protein sample was greater than 90% as determined by HPLC. The protein sample was quantified after ultrafiltration with buffer replacement and filtration.

[0037] (3) MEG has undergone an amino acid mutation at the 8th amino acid residue of Fcγ relative to FP4, changing from P to D.

[0038] The amino acid sequence of FP4:

[0039] FTPPTVKILQSSCDGGGFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRK EEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGKGS EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NQ:1)

[0040] The underlined part represents Fc-γ in region B.

[0041] Nucleotide sequence encoding the FP4 gene:

[0042]

[0043] The amino acid sequence of the MEG fusion protein:

[0044] FTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTR KEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK EPKSCDKTHTCPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:3)

[0045] The underlined part represents Fc-γ in region B.

[0046] The nucleotide sequence of MEG:

[0047]

[0048] Example 3: Experiments on the binding of MEG to FcεRI and FcγRII receptors, respectively

[0049] 1×10 6 CHO 3D10 cells (expressing FcεRI receptor) or HMC-1 cells (expressing FcγRII receptor) were reacted with 5 μg of MEG protein purified in Example 2 at 4°C for 1 hour. After washing, 5 μL of anti-human IgG Fc-FITC labeled antibody or anti-human IgE Fc-FITC labeled antibody (CALTAG, CA) was added, and the cells were detected by flow cytometry. The results showed that MEG protein can bind to FcεRI (such as FcγRII receptor). Figure 3 (as shown) and binds to FcγRII receptors (such as Figure 4 (As shown).

[0050] Example 4: ELISA analysis of the binding properties of MEG to FcγRII receptors

[0051] 100 μL of MEG at an appropriate concentration (2 μg / mL) was added to an ELISA plate and coated overnight. The supernatant was discarded, and the plate was blocked with PBS containing 1% BSA for 2 hours. The plate was washed twice with PBST (PBS containing 0.05% Tween 20). Then, an appropriate concentration of biotin-tagged FcγRII extracellular domain protein (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was added and incubated at room temperature for 1 hour. The supernatant was discarded, and the plate was washed three times with PBST. Streptavidin-HRP (BD Biosciences) was added and incubated at room temperature for 1 hour to detect biotin protein. The supernatant was removed, and the plate was washed four times with PBST. After adding chromogenic buffer and developing for 5-40 minutes, the absorbance (A650) at 650 nm was measured. The results showed that MEG protein does not bind to FcγRII (e.g., ...). Figure 5 As shown in Figure A), it binds only to the FcγRIIB receptor, and MEG has a significantly higher affinity for FcγRIIB than FP4 (as shown in Figure A). Figure 5 (As shown in B).

[0052] Example 5: MEG protein inhibits allergen-induced degranulation response in vivo.

[0053] In transgenic mice (mouse IgE receptor FcεRIα chain knockout, human IgE receptor FcεRIα chain implantation), specific human anti-NP-IgE (Serotech) was subcutaneously injected. Four hours later, 100 μg of the allergen NP and 1% Evans blue dye were intravenously injected. If a degranulation allergic reaction occurred at the sensitization site, the blue dye would seep from the blood vessels into the interstitial space due to increased vascular permeability, resulting in a blue reaction in the subcutaneous mucosa. When an equal amount of MEG protein was added simultaneously with sensitization at the corresponding skin site, the degranulation allergic reaction was completely inhibited, with no local skin reaction. However, an equal amount of human IgG control showed no inhibitory reaction (e.g., ...). Figure 6 (As shown). SEQUENCE LISTING <110> Zhu Daocheng <120> A fusion protein, its preparation method and application <130> P21019840C <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 554 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of FP4 <400> 1 Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly 1 5 10 15 Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr 20 25 30 Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val Met 35 40 45 Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala 50 55 60 Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp 65 70 75 80 Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp 85 90 95 Ser Thr Lys Lys Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr 100 105 110 Leu Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr 115 120 125 Ile Thr Cys Leu Val Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn 130 135 140 Leu Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg 145 150 155 160 Lys Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu 165 170 175 Pro Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg 180 185 190 Val Thr His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys 195 200 205 Thr Ser Gly Pro Arg Ala Ala Pro Glu Val Tyr Ala Phe Ala Thr Pro 210 215 220 Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu Ala Cys Leu Ile Gln 225 230 235 240 Asn Phe Met Pro Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val 245 250 255 Gln Leu Pro Asp Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys 260 265 270 Gly Ser Gly Phe Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu 275 280 285 Trp Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala Ala 290 295 300 Ser Pro Ser Gln Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys 305 310 315 320 Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 325 330 335 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 340 345 350 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 355 360 365 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 370 375 380 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 385 390 395 400 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 405 410 415 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 420 425 430 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 435 440 445 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 450 455 460 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 465 470 475 480 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 485 490 495 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 500 505 510 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 515 520 525 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 530 535 540 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 545 550 <210> 2 <211> 1665 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding the FP4 gene <400> 2 ttcaccccgc ccaccgtgaa gatcttacag tcgtcctgcg acggcggcgg gcacttcccc 60 ccgaccatcc agctcctgtg cctcgtctct gggtacaccc cagggactat caacatcacc 120 tggctggagg acgggcaggt catggacgtg gacttgtcca ccgcctctac cacgcaggag 180 ggtgagctgg cctccacaca aagcgagctc accctcagcc agaagcactg gctgtcagac 240 cgcacctaca cctgccaggt cacctatcaa ggtcacacct ttgaggacag caccaagaag 300 tgtgcagatt ccaacccgag aggggtgagc gcctacctaa gccggcccag cccgttcgac 360 ctgttcatcc gcaagtcgcc cacgatcacc tgtctggtgg tggacctggc acccagcaag 420 gggaccgtga acctgacctg gtcccgggcc agtgggaagc ctgtgaacca ctccaccaga 480 aaggaggaga agcagcgcaa tggcacgtta accgtcacgt ccaccctgcc ggtgggcacc 540 cgagactgga tcgaggggga gacctaccag tgcagggtga cccaccccca cctgcccagg 600 gccctcatgc ggtccacgac caagaccagc ggcccgcgtg ctgccccgga agtctatgcg 660 tttgcgacgc cggagtggcc ggggagccgg gacaagcgca ccctcgcctg cctgatccag 720 aacttcatgc ctgaggacat ctcggtgcag tggctgcaca acgaggtgca gctcccggac 780 gcccggcaca gcacgacgca gccccgcaag accaagggct ccggcttctt cgtcttcagc 840 cgtctagagg tgaccagggc cgaatgggag cagaaagatg agttcatctg ccgtgcagtc 900 catgaggcag ctagcccctc acagaccgtc cagcgagcgg tgtctgtaaa tcccggtaaa 960 ggatccgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 1020 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 1080 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 1140 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 1200 gagcagtaca acagcacgta ccgggtggtc agcgtcctca ccgtcctgca ccaggactgg 1260 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1320 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1380 tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1440 ccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1500 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1560 aagagcaggt ggcagcaggg gaacgtctttc tcatgctccg tgatgcatga ggctctgcac 1620 aaccactaca cgcagaagag cctctccctg tctccgggta aatga 1665 <210> 3 <211> 552 <212> PRT <213> Artificial Sequence <220> <223> MEG is the source of the data <400> 3 Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly 1 5 10 15 Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr 20 25 30 Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val Met 35 40 45 Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala 50 55 60 Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp 65 70 75 80 Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp 85 90 95 Ser Thr Lys Lys Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr 100 105 110 Leu Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr 115 120 125 Ile Thr Cys Leu Val Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn 130 135 140 Leu Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg 145 150 155 160 Lys Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu 165 170 175 Pro Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg 180 185 190 Val Thr His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys 195 200 205 Thr Ser Gly Pro Arg Ala Ala Pro Glu Val Tyr Ala Phe Ala Thr Pro 210 215 220 Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu Ala Cys Leu Ile Gln 225 230 235 240 Asn Phe Met Pro Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val 245 250 255 Gln Leu Pro Asp Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys 260 265 270 Gly Ser Gly Phe Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu 275 280 285 Trp Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala Ala 290 295 300 Ser Pro Ser Gln Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys 305 310 315 320 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 325 330 335 Pro Glu Leu Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro Lys Pro 340 345 350 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 355 360 365 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 370 375 380 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 385 390 395 400 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 405 410 415 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 420 425 430 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 435 440 445 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 450 455 460 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 465 470 475 480 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 485 490 495 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 500 505 510 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 515 520 525 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 530 535 540 Dear Leu Dear Leu Dear Pro Gly Lys 545,550 <210> 4 <211> 1659 <212> DNA <213> Artificial Sequence <220> <223> MEG is the source of the <400> 4 ttcaccccgc ccaccgtgaa gatcttacag tcgtcctgcg acggcggcgg gcacttcccc 60 ccgaccatcc agctcctgtg cctcgtctct gggtacaccc cagggactat caacatcacc 120 tggctggagg acgggcaggt catggacgtg gacttgtcca ccgcctctac cacgcaggag 180 ggtgagctgg cctccacaca aagcgagctc acctcagcc agaagcactg gctgtcagac 240 cgcacctaca cctgccaggt cacctatcaa ggtcacacct ttgaggacag caccaagaag 300 tgtgcagatt ccaacccgag aggggtgagc gcctacctaa gccggcccag cccgttcgac 360 ctgttcatcc gcaagtcgcc cacgatcacc tgtctggtgg tggacctggc acccagcaag 420 gggaccgtga acctgacctg gtcccgggcc agtgggaagc ctgtgaacca ctccaccaga 480 aaggagga agcagcgcaa tggcacgtta accgtcacgt ccaccctgcc ggtgggcacc 540 cgagactgga tcgaggggga gacctaccag tgcagggtga cccaccccca cctgcccagg 600 gccctcatgc ggtccacgac caagaccagc ggcccgcgtg ctgccccgga agtctatgcg 660 tttgcgacgc cggagtggcc ggggagccgg gacaagcgca ccctcgcctg cctgatccag 720 aacttcatgc ctgaggacat ctcggtgcag tggctgcaca acgaggtgca gctcccggac 780 gcccggcaca gcacgacgca gccccgcaag accaagggct ccggcttctt cgtcttcagc 840 cgcctggagg tgaccagggc cgaatgggag cagaaagatg agttcatctg ccgtgcagtc 900 catgaggcag ctagcccctc acagaccgtc cagcgagcgg tgtctgtaaa tcccggtaaa 960 gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 1020 gggggagact cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 1080 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 1140 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 1200 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1260 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1320 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1380 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1440 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1500 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1560 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1620 tacacgcaga agagcctctc cctgtctccg ggtaaatga 1659

Claims

1. A fusion protein having the amino acid sequence shown in SEQ ID NO:

3.

2. A gene encoding a fusion protein, the nucleotide sequence of which is selected from: 1) SEQ ID NO: 4; 2) A polynucleotide encoding the fusion protein as described in claim 1.

3. The gene as described in claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

4.

4. A recombinant expression vector comprising the gene as described in claim 2 or 3.

5. The recombinant expression vector as described in claim 4, characterized in that, The backbone plasmid of the recombinant expression vector is pSecTag.

6. A transformant comprising the recombinant expression vector as described in claim 4 or 5; said transformant being neither an animal nor a plant species.

7. The transformant as described in claim 6, characterized in that, The host of the transformant is a mammalian cell.

8. The transformant as described in claim 7, characterized in that, The mammalian cells mentioned are CHO cells.

9. A method for preparing the fusion protein as described in claim 1, characterized in that, The transformant as described in any one of claims 6-8 is cultured to express the fusion protein, thus obtaining the product.

10. The method as described in claim 9, characterized in that, The fusion protein was isolated and purified using affinity chromatography.

11. The method as described in claim 10, characterized in that, The affinity chromatography method is the mouse anti-human IgE affinity chromatography method.

12. A drug whose active ingredient comprises the fusion protein as described in claim 1.

13. The use of the fusion protein as described in claim 1 in the preparation of anti-allergy drugs.

Images