Fusion proteins and their production and use

Abstract

The present invention discloses a fusion protein and its production method and use, and further discloses a gene encoding the fusion protein, a production method for the fusion protein, an antiallergic drug containing the fusion protein as an active ingredient, and use of the fusion protein. The fusion protein provided by the present invention is a protein having the amino acid residues shown in SEQ ID NO: 3. The fusion protein of the present invention activates the inhibitory system of the intracellular signal transduction pathway of allergic reactions, effectively inhibits the allergic reactions of cells, and plays an important role in the treatment of allergic diseases.

Description

[Technical field]

[0001] This application claims priority to Chinese patent application No. 2021116739174, filed on December 31, 2021. This application cites the above Chinese patent application in full.

[0002] The present invention belongs to the fields of genetic engineering and immunology. Specifically, the present invention relates to a fusion protein, a gene encoding the fusion protein, a method for producing the fusion protein, an antiallergic drug containing the fusion protein as an active ingredient, and use of the fusion protein. [Background technology]

[0003] Allergic diseases are the sixth most common cause of acute and chronic diseases, and have a clear genetic predisposition. Common allergic diseases include allergic asthma, allergic rhinitis, hay fever, atopic dermatitis, allergic arthritis, urticaria, anaphylactic shock, etc. About 10-15% of Chinese people and about 20% of Americans suffer from allergic diseases.

[0004] At present, most of the medicines used to treat allergic diseases are aimed at controlling clinical symptoms, such as steroid hormones, antihistamines, decongestants, sodium tryptophan, bronchodilators, etc. Recently, the US FDA has approved clinical trials of anti-IgE monoclonal antibodies. However, these drugs have different degrees of side effects, so many pharmaceutical research and development institutions and pharmaceutical companies at home and abroad are working on the development of new types of anti-allergic drugs.

[0005] The mechanism of allergic reactions is that when an antigen (allergen, allergy-causing substance) enters the body, it binds to IgE molecules (cytoaffinity antibodies) attached to mast cells and basophils, causing the activation of the cell's immunoreceptor tyrosine-based activation motif (ITAM), which promotes the release of physiologically active substances from the cell, resulting in clinical pathological changes such as smooth muscle contraction, increased vascular permeability, and increased serous secretion. In this pathological process, the affinity IgE receptor (FcεRI) on the body's mast cells and basophils plays an important role in allergic diseases. At the same time, when dimeric IgG binds to the FcγRIIB inhibitory receptor expressed on the cell membrane surface of mast cells and basophils, polymerization occurs, inducing phosphorylation of the immunoreceptor tyrosine-based inhibition motif (ITIM), which inhibits the activation signal of Fc_25RI. Research has shown that when FcγRII receptor crosslinks with FcεRI receptor, it induces inhibitory signals in mast cells or basophils, blocking the intracellular activation pathway, inhibiting the release of active mediators, and further inhibiting 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 fusion proteins that activate inhibitory signals in allergic reactions should be a new way to treat allergies.

[0006] Recent studies have shown that due to polymorphisms in human FcγRII gene expression, a small number of human subcutaneous mast cells express FcγRIIA but not FcγRIIB. The intracellular end of the FcγRIIA receptor contains an immunoreceptor tyrosine-based activation motif (ITAM). Direct cross-linking of FcγRIIA to FcεRI (obtaining the fusion protein FP4) may cause phosphorylation in allergic cells, thereby activating the intracellular activation pathway and causing degranulation of allergic cells, resulting in allergic diseases. However, the fusion protein FP4 cannot overcome the allergic response caused by the expression of FcγRIIA. Summary of the Invention

[0007] In order to overcome the deficiencies in the prior art in which the fusion protein FP4 binds to the FcγRIIA receptor and causes degranulation of allergic reaction cells, the present invention provides a fusion protein and its production method and use, which binds only to the FcγRIIB receptor with an affinity significantly superior to that of the fusion protein FP4 (the amino acid sequence of which is shown in SEQ ID NO:1 and the DNA sequence encoding it is shown in SEQ ID NO:2), and the affinity to the IgE receptor FcεRI is unchanged.

[0008] The fusion protein provided by the present invention is named MEG and is a protein having the amino acid residues shown in SEQ ID NO:3, or a protein derived from SEQ ID NO:3 in which the amino acid residue sequence of SEQ ID NO:3 has been substituted or deleted by one or more amino acid residues and has the same or superior activity as the amino acid residue sequence of SEQ ID NO:3.

[0009] The amino acid residue sequence of SEQ ID NO: 3 is a protein composed of 552 amino acid residues. The structure of MEG is shown in FIG. 1 and is composed of two parts, region A (Fc-ε) (amino acid residues 1 to 320 from the amino terminus) and region B (Fc-γ) (amino acid residues 321 to 552 from the amino terminus). Here, region A is derived from human IgE immunoglobulin and has a site that binds to the IgE receptor FcεRI, and region B is derived from human IgG immunoglobulin and has a site that binds to the IgG receptor FcγRII.

[0010] The technical solution of the present invention is as follows:

[0011] The first technical solution provided by the present invention is a fusion protein, the amino acid sequence of which is shown in SEQ ID NO:3.

[0012] The second technical solution provided by the present invention is that the nucleotide sequence is (1) SEQ ID NO: 4, (2) A polynucleotide encoding the fusion protein according to one of the technical solutions. The gene encoding the fusion protein is selected from the group consisting of:

[0013] In some preferred embodiments, the nucleotide sequence of the gene is set forth in SEQ ID NO:4.

[0014] The third technical solution provided by the present invention is a recombinant expression vector, comprising the gene according to the second technical solution.

[0015] In some preferred embodiments, the backbone plasmid of the recombinant expression vector is pSecTag.

[0016] The fourth technical solution provided by the present invention is a transformant comprising the recombinant expression vector according to the third technical solution, preferably the host of the transformant is a mammalian cell.

[0017] In a preferred embodiment, the mammalian cell is a CHO cell.

[0018] The fifth technical solution provided by the present invention is a method for producing a fusion protein according to the first technical solution, the method comprising culturing the transformant according to 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.

[0019] The sixth technical solution provided by the present invention is a medicine, the active ingredient of which comprises the fusion protein according to the first technical solution.

[0020] The seventh technical solution provided by the present invention is the use of the fusion protein according to the first technical solution in the manufacture of an anti-allergic agent.

[0021] The positive advances of the present invention are: The fusion protein MEG of the present invention is 99.9% derived from human immunoglobulin, so that the protein can be introduced into the body as a medicine without the immunogenicity of foreign proteins. In vivo and in vitro experimental results have demonstrated that the fusion protein MEG can effectively crosslink FcεRI and FcγRII on the surface of mast cells or basophils to inhibit allergic reactions. The fusion protein MEG of the present invention mainly activates the inhibitory system of the intracellular signal transduction system of allergic reactions, effectively inhibiting cellular allergic reactions and playing an important role in the treatment of allergic diseases. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram of the structure of the fusion protein MEG. [Diagram 2] FIG. 1 is a comparison of SDS-PAGE of the supernatant and purified MEG. [Diagram 3]FIG. 1 shows the results of identifying the binding ability of the fusion protein MEG to the FcεRI receptor. [Figure 4] FIG. 1 shows the results of identifying the binding ability of the fusion protein MEG to the FcγRIIB receptor. [Diagram 5] FIG. 1 shows the binding analysis of fusion protein MEG to FcγRIIA (ie, FcgRIIA) receptor (A) and FcγRIIB (ie, FcgRIIB) receptor (B). [Figure 6] A comprehensive analysis of the affinity of fusion protein MEG with FcγR (i.e., FcgR), where the affinity of FcγRI (i.e., FcgRI), H subtype of FcγRIIA (i.e., FcgRIIAH) and R subtype (i.e., FcgRIIAR), FcγRIIB (i.e., FcgRIIB), F subtype of FcγRIIIA (i.e., FcgRIIIAF) and V subtype (i.e., FcgRIIIAV) are shown in Figure 6A and 6B, respectively. [Figure 7] These results show that the fusion protein MEG inhibits allergen-induced degranulation responses in vivo. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention will be further described below in the form of examples, but the present invention is not limited to the scope of the examples. In the following examples, the experimental methods for which no specific conditions are described are selected according to the usual methods and conditions or product instructions. The reagents and raw materials used in the present invention are commercially available.

[0024] Example 1 Expression and purification of MEG molecules The expression of the fusion protein FP4 is as shown in CN1317304C, and specifically includes the following steps:

[0025] According to the conventional method, B lymphocytes were isolated and purified from human peripheral blood, genomic DNA was extracted, and Fcε and Fcγ genes were amplified by PCR using specific primers, the PCR amplification product of Fcε DNA was 1155bp, and the PCR amplification product of Fcγ DNA was 928bp. The PCR products were cloned into pCR4-TOPO (INVITROGEN, CA) to obtain FPEFc-ε and FPGFc-γ, respectively, and nucleotide sequence analysis was performed to obtain 100% correct nucleotide sequence. After that, the Fcε and Fcγ genes were enzymatically cleaved using SfiI-BamHI and BamHI-NotI restriction endonucleases (NEW ENGLAND BIOLABS), respectively, and the pSecTag (INVITROGEN, CA) expression vector was enzymatically cleaved using restriction endonucleases SfiI-NotI, and then ligated to obtain plasmid pSecTagMEG, and nucleotide sequence analysis was performed on the plasmid pSecTagMEG, and the inserted foreign gene sequence was as shown in SEQ ID NO:2. The plasmid was then transfected into mouse myeloma SP2 / 0 cells using conventional electroporation, and drug resistance screening was performed using ZEOCIN, followed by identification by ELISA (ELISA plates were coated with mouse anti-human IgE antibodies prepared by conventional methods, sample supernatants were added, and then goat anti-human IgE enzyme-labeled antibodies were added), resulting in clones with high expression of FP4 (the amino acid sequence of which is shown in SEQ ID NO: 1).

[0026] The structure of MEG (MEG structure is shown in Figure 1) is different from FP4, and the differences are as shown below.

[0027] (1) The MEG gene was synthesized based on the sequences of human immunoglobulin IgE Fcε and IgG Fcγ, and the MEG gene was cloned into the pBG eukaryotic expression vector using the methods of SOE PCR and enzyme cleavage. After being confirmed to be correct by sequencing, it was transfected into CHO cells and expressed in 50% CD CHO+50% Dynamis medium.

[0028] (2) Highly pure protein was obtained by a two-step purification process using GE Mab-select affinity chromatography and cation exchange, and the purity was more than 90% by SDS-PAGE (results shown in Figure 2) and HPLC detection. The protein sample was subjected to buffer exchange by ultrafiltration and quantified after filtration.

[0029] (3) Compared with FP4, MEG had an amino acid mutation in an amino acid residue of Fc-γ, where P was mutated to D.

[0030] [ka]

[0031] Here, the underline represents the B region Fc-γ.

[0032] Nucleotide sequence encoding the FP4 gene:

[0033] [ka]

[0034] Here, the underline represents the B region Fc-γ.

[0035] The nucleotide sequence of MEG:

[0036] Example 2 Binding study of MEG to FcεRI and FcγRII receptors, respectively Binding test with FcεRI receptor: 1×10 6 CHO 3D10 cells (expressing FcεRI receptor) were reacted with 5μg hIgE, 5μg FP4 and 5μg purified MEG protein of Example 2 at 4℃ for 1 hour, washed, and then 5μL of 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 receptor compared to hIgE (Figure 3A) and FP4 (Figure 3B).

[0037] Binding test with FcγRII receptor: 1×10 6 HMC-1 cells (expressing FcγRIIB receptor) were reacted with 5 μg of hIgG, 5 μg of FP4, and 5 μg of the purified MEG protein of Example 2 at 4° C. for 1 hour, washed, and then 5 μL of anti-human IgG Fc-FITC labeled antibody (CALTAG, CA) was added, and the cells were detected by flow cytometry. The results showed that MEG protein could bind to FcγRIIB receptor, compared with hIgG (Figure 4A) and FP4 (Figure 4B).

[0038] Example 3 Analysis of the binding properties of MEG to different types of FcγR receptors by ELISA 100μL of MEG at an appropriate concentration (2μg / mL) was added to the enzyme-binding plate and coated overnight, the supernatant was discarded, the plate was blocked with PBS containing 1% BSA for 2 hours, washed twice with PBST (PBS containing 0.05% Twain 20), and then extracellular domain proteins such as biotin-tagged FcγRI, FcγRIIAH, FcgRIIAR, FcgRIIIAF, FcgRIIIAV or FcγRIIB (Sino Biological Inc.) at appropriate concentrations were added, 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, incubated at room temperature for 1 hour to detect biotin protein, the supernatant was discarded, washed four times with PBST, a color-developing solution was added to develop color for 5 to 40 minutes, and the absorbance A650 at 650 nm was measured.

[0039] The first results showed that the MEG protein did not bind to FcγRIIAH (i.e., shown as FcgRIIA in Figure 5A) but only to the FcγRIIB (FcgRIIB) receptor, and that the affinity of MEG for FcγRIIB was significantly greater than that of FP4 (shown in Figure 5B).

[0040] In the second round, further analysis was performed for each subtype of FcγRI, FcγRII and FcγRIII, and the results showed that MEG protein did not bind to FcγRI (i.e., FcgRI) (Figure 6A), H subtype (i.e., FcgRIIAH) (Figure 6B) and R subtype (i.e., FcgRIIAR) (Figure 6C), F subtype (i.e., FcgRIIIAF) (Figure 6E) and V subtype (i.e., FcgRIIIAV) (Figure 6F) of FcγRIIIA, but only bound to FcγRIIB (i.e., FcgRIIB) receptor, and the affinity of MEG to FcγRIIB was significantly better than that of FP4 (Figure 6D).

[0041] Example 4 Inhibition of allergen-induced degranulation in vivo by MEG protein Transgenic mice (mouse IgE receptor FcεRIα chain knockout, human IgE receptor FcεRIα chain grafted) were subcutaneously injected with specific human anti-NP-IgE (Serotech), and 4 hours later, 100 μg of allergen NP and 1% Evans Blue (EVANS) dye were injected iv., and a degranulation allergic reaction occurred at the sensitized site, and the blue dye leaked out of the blood vessels and entered the tissue space due to an increase in vascular permeability of the local skin, and the subcutaneous mucosa showed a blue reaction. When the same amount of MEG protein was added while sensitizing the corresponding skin site, the degranulation allergic reaction was completely inhibited, and there was no reaction in the local skin, but there was no inhibitory reaction with the same amount of human IgG control (see Figure 7).

Claims

1. A fusion protein characterized in that the amino acid sequence is shown in SEQ ID NO:

3.

2. A gene encoding a fusion protein, the nucleotide sequence of said gene being 1) SEQ ID NO:4, 2) A polynucleotide encoding the fusion protein according to claim 1. A gene encoding a fusion protein, characterized in that it is selected from:

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

4.

4. A recombinant expression vector comprising the gene according to claim 2 or 3.

5. The recombinant expression vector according to claim 4, wherein the backbone plasmid of the recombinant expression vector is pSecTag.

6. A transformant comprising the recombinant expression vector according to claim 4 or 5, preferably wherein the host is a mammalian cell.

7. The transformant according to claim 6, wherein the mammalian cell is a CHO cell.

8. A method for producing the fusion protein according to claim 1, comprising culturing the transformant according to claim 6 or 7 to express the fusion protein, and preferably isolating and purifying the fusion protein by affinity chromatography, more preferably, the affinity chromatography being mouse anti-human IgE affinity chromatography.

9. A pharmaceutical composition comprising the fusion protein of claim 1 as an active ingredient.

10. Use of the fusion protein according to claim 1 in the manufacture of an antiallergic agent.

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