Fusion protein and application thereof in treatment of multiple sclerosis

Abstract

The invention belongs to the technical field of fusion proteins, and particularly relates to a fusion protein and application thereof in treatment of multiple sclerosis, the fusion protein is prepared by fusing myelin basic protein, myelin oligodendroglia glycoprotein and proteolipid protein, key epitopes of three main myelin sheath antigens, namely MBP, MOG and PLP, are fused by using an escherichia coli recombinant expression technology to prepare a tolerogen, a multivalent tolerogen is formed, immune tolerance is more comprehensively induced, the risk of immune escape is reduced, an escherichia coli expression system has a mature industrial production basis, fusion protein can be efficiently produced in a large scale at low cost, and the application prospect is wide. In the antigen-specific treatment of multiple sclerosis, the tolerogen of the fusion protein can be directly injected, does not need individual preparation, is safer, more conforms to a clinical conventional administration mode, and is convenient for wide application.

Description

Technical Field

[0001] The present invention belongs to the technical field of fusion proteins, and in particular relates to a fusion protein and its application in treating multiple sclerosis. Background Art

[0002] Multiple sclerosis (MS) is an autoimmune disease characterized by chronic inflammatory demyelinating lesions of the central nervous system. It primarily affects patients between the ages of 20 and 40, with the incidence in women being two to three times higher than in men. The impairment of neurocognitive function ultimately leads to disability, making it a leading cause of disability in young and middle-aged adults, second only to trauma. The disease primarily results from the abnormal activation of autoreactive T cells, which attack the myelin sheath proteins in the axons of the nervous system. This leads to myelin sheath loss, disrupting nerve signaling and causing dysfunction. Approximately 2.5 million people with MS live worldwide.

[0003] Because MS is an autoimmune disease caused by overactivation of autoreactive T cells that attack the myelin sheath, antigen-specific therapies—targeted elimination of autoreactive T cells in the immune system through the induction of immune tolerance—are the optimal treatment strategy. Targeting pathogenic T cells is highly specific, leaving non-antigen-specific lymphocytes unaffected and unactivated, thus preventing the suppression of normal immune system function and potential side effects.

[0004] Numerous clinical studies have shown that T cells infiltrating MS lesions are not specific to a single myelin antigen. Due to the polyclonal complexity of autoreactive T cells in MS, attempts to treat autoimmune diseases with single epitope peptides have failed in clinical trials. In MS patients, a phase III clinical trial showed that a 17-amino acid synthetic peptide derived from MBP had no therapeutic effect. In addition, systemic administration of high-dose soluble peptide tolerance agents can cause allergic reactions in mice and primates, raising safety concerns about soluble peptide strategies. Autologous antigen-presenting cells (APCs) loaded with antigenic peptides have shown therapeutic efficacy in phase I clinical trials, but this approach faces challenges in isolating autologous APCs, selecting, and coupling appropriate peptides to APC cells, and its clinical application is limited. Summary of the Invention

[0005] In order to solve the problems in the prior art, the present invention provides a fusion protein and its application in the treatment of multiple sclerosis, so as to achieve the purpose of multivalent antigen coverage in antigen-specific treatment of multiple sclerosis, overcome the limitations of single target, induce immune tolerance more comprehensively, and reduce the risk of immune escape.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] The present invention aims to provide a fusion protein, which is prepared by fusing myelin basic protein, myelin oligodendrocyte glycoprotein and proteolipid protein.

[0008] Fusion expression involves fusing a target protein or peptide with the DNA sequence of another protein or peptide fragment for intracellular expression. By tandemly expressing the functional regions of an antigen, multiple antigens can be expressed simultaneously, facilitating protein purification. Extensive clinical evidence indicates that T cells infiltrating the lesions of multiple sclerosis patients exhibit polyclonal characteristics, targeting at least three key myelin antigens: MBP, MOG, and PLP.

[0009] Furthermore, the amino acid sequence of myelin oligodendrocyte glycoprotein is such as SEQ ID NO.1, the amino acid sequence of myelin basic protein is such as SEQ ID NO.2, and the amino acid sequence of proteolipid protein is such as SEQ ID NO.3.

[0010] Furthermore, myelin basic protein, myelin oligodendrocyte glycoprotein and proteolipid protein were fused in tandem using Escherichia coli.

[0011] Among various expression systems, the E. coli expression system is currently the most mature. Its advantages, such as rapid cell proliferation, high yields, and relatively simple IPTG-induced expression, make it the most commonly used system for recombinant protein production. Currently, the most commonly used E. coli expression system is the BL21-PET expression system, which has been commercialized and widely used by major laboratories and biotechnology companies. Advantages of this expression system include clear genetic background; rapid propagation, low cost, and strong resistance to contamination; high expression yields, relatively simple isolation and purification of the expression product, and good stability; and a wide range of commercial vectors and strains, with a wide range of applications.

[0012] Furthermore, the fusion tag protein uses SUMO.

[0013] SUMO (small molecule ubiquitin-like modifier) ​​as a fusion tag protein has greater advantages than tags such as MBP (maltose binding protein), GST (glutathione thiol transferase), GFP (green fluorescent protein), and TrX (thioredoxin): 1. It promotes soluble expression of the target protein; 2. It acts as a chaperone protein to promote correct protein folding; 3. It has strong tolerance to heat and proteases; 4. There is a matching specific protease that can remove the tag ULP1, accurately identifying the structure and having better specificity than the enzyme cleavage site based on the protein amino acid sequence; 5. The tag has a small molecular weight and a high proportion relative to the target protein. One embodiment of the fusion protein of the present invention adds a SUMO tag to increase the stability of the protein.

[0014] Furthermore, the amino acid sequence of the fusion protein is such as SEQ ID NO.6 or SEQ ID NO.7.

[0015] Use of a fusion protein in preparing a drug for multiple sclerosis.

[0016] The invention discloses a use of a fusion protein in preparing a composition for preventing and treating autoimmune diseases of the autoantibody type.

[0017] Compared with the prior art, the beneficial technical effects of the present invention are:

[0018] Compared with the MS antigen-specific treatment strategies currently in the clinical stage, the present invention utilizes E. coli recombinant expression technology to fuse key epitopes of three major myelin antigens, MBP, MOG, and PLP, to prepare a tolerogen, which has the following significant advantages:

[0019] (1) Multivalent antigen coverage overcomes the limitations of single targets. The autoimmune response of MS patients is highly heterogeneous. Peptide therapies targeting a single antigen (such as MBP) (such as MBP17 peptide) failed in Phase III clinical trials, proving that a single epitope cannot effectively inhibit polyclonal T cell responses. By using E. coli fusion expression technology, multiple dominant epitopes of MBP, MOG, and PLP are simultaneously integrated to form a multivalent tolerogen, which can more comprehensively induce immune tolerance and reduce the risk of immune escape.

[0020] (2) Efficient and low-cost scalable production. Chemically synthesized peptides are expensive, especially long peptides (>20aa), which have low synthesis efficiency and are difficult to meet clinical needs. The E. coli expression system has a mature industrial production foundation and can produce fusion proteins on a large scale with high efficiency and low cost, reducing treatment costs and improving accessibility.

[0021] (3) Avoiding the complex process of autologous APC therapy. Autologous APC therapy based on loaded antigen peptides requires individualized cell separation, modification, and reinfusion, which is complex and extremely costly, making it difficult to promote. The recombinant protein tolerogen of the present invention can be directly injected without the need for individualized preparation, making it safer, more consistent with conventional clinical administration methods, and easier to apply.

[0022] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the PET28a-S-P170GP recombinant plasmid of the present invention.

[0024] Figure 2 Schematic diagram of the PET28a-PGPe recombinant plasmid in the present invention.

[0025] Figure 3 This is the WB detection of PET28a-PGPe protein expression in the present invention.

[0026] Figure 4 This is the WB detection of PET28a-P170GP protein expression in the present invention.

[0027] Figure 5 This is a diagram of the purification of PET28a-PGPe protein in the present invention.

[0028] Figure 6 This is a diagram of the purification of PET28a-P170GP protein in the present invention. DETAILED DESCRIPTION

[0029] The technical solutions of the present invention are further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0030] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0031] 1. Fusion protein design:

[0032] Protein sequences were obtained using the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). Fusion proteins were generated by tandemly combining functional regions of three proteins: myelin oligodendrocyte glycoprotein (MOG) (SEQ ID NO. 1), myelin basic protein (MBP) (SEQ ID NO. 2), and proteolipid protein (PLP) (SEQ ID NO. 3).

[0033] The functional regions of the three antigens in the CDS region of the plasmid can be arranged in any combination and can be full-length or contain dominant antigenic epitopes. The functional region derived from MBP is named antigen A, the functional region derived from MOG protein is named antigen B, and the functional region derived from PLP protein is named antigen C. The fusion protein can be any of the following six types:

[0034] Sumo-A-Linker-B-Linker-C;

[0035] A-Linker-C-Linker-B;

[0036] B-Linker-A-Linker-C;

[0037] B-Linker-C-Linker-A;

[0038] C-Linker-A-Linker-B;

[0039] C-Linker-B-Linker-A.

[0040] 2. Gene synthesis of recombinant fusion protein:

[0041] According to the codon preference of E. coli for expressing heterologous proteins, the nucleotide sequences of two fusion proteins were designed and synthesized into the PET28a plasmid. The specific codon sequences are shown in SEQ ID NO.4 and SEQ ID NO.5 in the sequence listing. The whole gene synthesis was completed by Sangon Biotech (Shanghai) Co., Ltd., and the recombinant plasmid vector was named PET28a-S-P170GP (reference Figure 1 ), PET28a-PGPe (reference Figure 2 ). PET28a-S-P170GP and PET28a-PGPe both used the Sumo-A-Linker-B-Linker-C combination. PET28a-S-P170GP used SUMO and 8*His tags, while PET28a-PGPe used a His tag for purification.

[0042] 3. DE3 E. coli transformation:

[0043] Remove 200 μl of competent cell suspension from a -80°C freezer and thaw at room temperature. Immediately place on ice. Add the plasmid DNA solution (no more than 10 ng, no more than 10 μl), shake gently, and place on ice for 30 minutes. Heat shock in a 42°C water bath for 45 seconds, then quickly cool on ice for 3-5 minutes. Add 1 ml of LB liquid medium (without Kan resistance) to the tube, mix thoroughly, and incubate at 37°C with shaking for 1 hour to allow the bacteria to resume normal growth and express the antibiotic resistance gene (KanR) encoded by the plasmid. Shake the above bacterial suspension and spread 100 μl onto a Kan resistance selection plate. Once the bacterial suspension has been completely absorbed by the medium, invert the plate and incubate at 37°C for 16-24 hours.

[0044] 4. Monoclonal screening:

[0045] Use a sterile toothpick to inoculate the corresponding colonies grown on the LB agar plate into 2 ml of LB liquid medium. Incubate at 37°C for 6 hours. Take an appropriate amount of the bacterial solution and send it to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. Keep the corresponding glycerol stock. Once the sequencing results are returned, save the corresponding glycerol stock.

[0046] 5. Small-scale expression of recombinant fusion proteins:

[0047] The screened transformants were inoculated into 10 mL of LB medium (LB medium: 1% tryptone, 0.5% yeast powder, 1% NaCl), cultured at 37°C, 220 rpm, and shaken for 24 h. They were then transferred as primary seeds into 300 mL of ZYM5052 medium (1% tryptone, 0.5% yeast powder, 25 mmol / L Na2HPO4, 25 mmol / L KH2PO4, 50 mmol / L NH4Cl, 5 mmol / LNa2SO4, 2 mmol / L MgSO4, 0.5% glycerol, 0.05% glucose, 0.2% lactose) and cultured at 37°C, shaking for 24 h.

[0048] E. coli was sonicated in Lysis buffer (500 mM NaCl, 20 mM Tris / Cl pH 8.0, 10% Glycerin, 0.1% Triton X-114) for 1 hour and centrifuged at 12,000 rpm for 40 minutes. The lysed precipitate was collected to obtain inclusion bodies. The inclusion bodies were washed with 30 ml of Inclusion Body Washing Buffer (500 mM NaCl, 20 mM Tris / Cl pH 8.0, 5 mM EDTANA2, 2 mM β-ME) for 1 hour and centrifuged at 12,000 rpm for 40 minutes. The inclusion bodies were solubilized using Inclusion Body Solubilization Buffer (8 M urea, 0.5 M NaCl, 50 mM Tris / Cl pH 8.0, 5 mM EDTA-NA2, 2 mM β-ME).

[0049] Nickel column purification: Use 5-10 column volumes of Binding Buffer (8M Urea, 500mM NaCl, 50mM Tris / Cl pH8.0, 5% glycerol) to balance the column by gravity or low speed (1-2mL / min) to ensure that Ni 2 The sample was fully bound to the buffer. Filter the sample through a 0.45 μm filter and apply it to the column. The flow rate was controlled at 0.1-0.5 mL / min. The binding time was extended (1-1.5 hours) to improve the binding efficiency. Subsequently, a gradient elution was performed using different concentrations of imidazole (10, 20, 40, 80, 160, and 130 mM).

[0050] Use WB to detect whether the protein expression is the target protein (refer to Figure 3 and Figure 4 ), and protein purification was detected by SDS-PAGE (refer to Figure 5 and Figure 6 ).

[0051] Sequence Listing

[0052]

[0053]

[0054]

[0055] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0056] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein is prepared by fusion of myelin basic protein, myelin oligodendrocyte glycoprotein and proteolipid protein.

2. A fusion protein according to claim 1, characterized in that: The amino acid sequence of myelin oligodendrocyte glycoprotein is shown in SEQ ID NO.1, the amino acid sequence of myelin basic protein is shown in SEQ ID NO.2, and the amino acid sequence of proteolipid protein is shown in SEQ ID NO.

3.

3. A fusion protein according to claim 2, characterized in that: Myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein were fused in tandem using Escherichia coli.

4. A fusion protein according to claim 3, characterized in that: The fusion tag protein uses SUMO.

5. A fusion protein according to claim 3, characterized in that: The amino acid sequence of the fusion protein is such as SEQ ID NO.6 or SEQ ID NO.

7.

6. Use of a fusion protein according to any one of claims 1 to 5 in the preparation of a drug for multiple sclerosis.

7. Use of a fusion protein according to any one of claims 1 to 5 in preparing a composition for preventing and treating autoimmune diseases of the autoantibody type.

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