Fusion protein capable of enriching and proliferating regulatory t cells in the intestinal tract and application

Abstract

The application relates to a fusion protein capable of enriching and proliferating regulatory T cells in the intestinal tract and application. The amino acid sequence of the fusion protein is shown as SEQ ID NO:1, which can selectively promote the proliferation of regulatory T cells in the human body, thereby effectively reducing the inflammatory response in the body; meanwhile, the fusion protein can be enriched in the intestinal tract, thereby selectively reducing the inflammatory response in the intestinal tract of the body, and effectively avoiding the systemic side effects caused by the fusion protein after entering the body. Therefore, the fusion protein can be better applied in the prevention and treatment of inflammatory bowel disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein engineering, and particularly relates to a fusion protein capable of enriching and proliferating regulatory T cells in the intestinal tract and application thereof. BACKGROUND

[0002] Inflammatory Bowel Disease (IBD), consisting of Ulcerative Colitis (UC) and Crohn's Disease (CD), is a chronic and recurrent disease characterized by intestinal inflammation, tissue damage, abdominal pain, frequent / continuous diarrhea, weight loss, rectal bleeding, fatigue, etc. IBD belongs to immune system diseases (not generally considered as autoimmune diseases, not attacking the self, but immune system disorder), and the etiology and pathogenesis of IBD are not clear at present, and it is generally believed that it is related to environmental factors, infection, genetic susceptibility and abnormal activation of immune response. IBD was first discovered in developed western countries, and in recent years, the incidence in China has been rising, and has become an important pathogenic factor of colorectal cancer. Inflammatory bowel disease generally shows structural intestinal epithelial dysfunction and abnormal activation of the immune system caused by various factors. Traditional treatments include aminosalicylic acid, corticosteroid hormones, immunosuppressants, etc., and the main purpose is to induce remission (eliminate symptoms) and maintain remission (prevent recurrence) and control intestinal inflammation. A considerable number of patients cannot achieve clinical remission after treatment, or lose response over time. In recent years, with the deepening of the research on the pathogenesis of IBD, there has been a surge in the development of new drugs for inflammatory bowel disease. The development of new drugs for UC mainly has two directions, one is to interfere with the role of inflammatory cytokines, and the other is to block the large number of lymphocytes from entering the intestinal lamina propria tissue. However, no matter which mechanism acts, the main site of drug action is mainly in the gastrointestinal tract, and systemic action may bring greater side effects, so the targeted drug for intestinal inflammatory tissue is particularly important.

[0003] In the treatment of IBD, reducing the inflammatory response can be achieved by activating immune-suppressive cells in vivo. Regulatory T cells (Tregs) are a class of cells with significant immunosuppressive effects and are the main controllers of self-tolerance. Regulatory T cells can secrete anti-inflammatory cytokines such as IL-4, IL-10, TGF-β, etc., to prevent the occurrence of pathological immune responses that cause tissue destruction. Interleukin-2 (IL-2) is a T cell growth factor, studies have shown that low-dose IL-2 can promote the selective activation and expansion of regulatory T cells in the human body, and preliminary clinical results have also shown the effectiveness of IL-2 in the treatment of inflammatory bowel disease. However, IL-2 has certain systemic side effects. Therefore, it is necessary to provide a fusion protein that can proliferate regulatory T cells while reducing systemic side effects. SUMMARY

[0004] In order to solve the problems of the prior art, the present application provides a new fusion protein which can enrich in the intestinal tract and selectively promote the proliferation of regulatory T cells in the human body, thereby selectively reducing the inflammatory response in the intestinal tract, and thus can be better applied in the prevention and treatment of inflammatory bowel disease.

[0005] To this end, the first aspect of the present application provides a fusion protein which can enrich in the intestinal tract and proliferate regulatory T cells.

[0006] In the present application, the fusion protein can selectively promote the proliferation of regulatory T cells in the human body, thereby effectively reducing the inflammatory response in the body; at the same time, the fusion protein can enrich in the intestinal tract, thereby selectively reducing the inflammatory response in the intestinal tract of the body, and effectively avoiding the systemic side effects caused by the fusion protein entering the body. Therefore, the fusion protein of the present application can be better applied in the prevention and treatment of inflammatory bowel disease.

[0007] In the present application, the term "can enrich in the intestinal tract" means that after the fusion protein of the present application enters the body, the content of the fusion protein in the intestinal tract is much higher than that in other tissues in the body.

[0008] In some preferred embodiments, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 1.

[0009] In the present application, the preparation of the fusion protein with the amino acid sequence as shown in SEQ ID NO: 1 can be achieved by the recognized recombinant DNA technology, including polymerase chain reaction (PCR), preparation of cloning vector, enzymolysis of DNA by restriction endonuclease, preparation of expression vector, transfection of host cell by expression vector, culture of host cell, etc. In addition, the preparation of the fusion protein with the amino acid sequence as shown in SEQ ID NO: 1 can also be achieved directly by means of gene synthesis. By means of gene synthesis, the nucleic acid molecule designed for encoding the fusion protein is first synthesized in vitro, then the synthesized nucleic acid molecule is connected to the expression vector, finally the expression vector is transfected into the host cell, and the host cell is induced for expression, thereby obtaining the fusion protein.

[0010] The second aspect of the present application provides a nucleic acid molecule for encoding the fusion protein as described in the first aspect of the present application.

[0011] In the present application, the nucleic acid molecule includes any nucleic acid molecule known in the art by translating the amino acid sequence of the fusion protein provided in the present application into polynucleotide sequence.

[0012] In some preferred embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 2.

[0013] The third aspect of the present application provides an expression vector comprising the nucleic acid molecule as described in the second aspect of the present application.

[0014] In the present application, the construction of the expression vector can be achieved by steps of restriction endonuclease digestion, DNA molecule connection, E. coli transformation, etc. For those skilled in the art, any known expression vector can be used as the expression vector in the present application, and the expression vector can be selected according to the properties of the target host cell.

[0015] The fourth aspect of the present application provides a host cell comprising the expression vector as described in the third aspect of the present application.

[0016] In the present application, the host cell is a eukaryotic cell, for example, a yeast, an insect cell, an animal cell; preferably an animal cell; more preferably a mammalian cell.

[0017] In the present application, the expression vector can be introduced into the host cell by transfection. The present application does not have explicit limitation on the mode adopted during transfection, which can be routinely selected by those skilled in the art. For example, the expression vector can be introduced into the host cell by means of calcium phosphate transfection, viral infection, DEAE-dextran-mediated transfection, liposome transfection or electroporation, etc.

[0018] The fifth aspect of the present application provides a pharmaceutical composition comprising the fusion protein according to the first aspect of the present application.

[0019] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0020] In the present application, the pharmaceutically acceptable excipient can be, for example, a salt solution, a glucose solution, a preservative, a solubilizer, an analgesic, etc.

[0021] The sixth aspect of the present application provides use of the fusion protein according to the first aspect of the present application, or the pharmaceutical composition according to the fifth aspect of the present application in the preparation of a medicament for preventing or treating an immune system disease.

[0022] In the present application, the immune system disease is not an autoimmune disease, but a disease caused by disorder of the immune system.

[0023] In the present application, "prevention" can include, but is not limited to, any action of blocking a disease symptom, or inhibiting or delaying a symptom, using the medicament described in the present application. "Treatment" can include, but is not limited to, any action of improving or beneficially changing a disease symptom, using the medicament described in the present application.

[0024] In some embodiments, the immune system disease is an inflammatory bowel disease.

[0025] In the present application, the inflammatory bowel disease consists of ulcerative colitis and Crohn's disease.

[0026] Beneficial technical effects of the present application: the present application provides a new fusion protein, which can selectively promote the proliferation of regulatory T cells in the human body, thereby effectively reducing the inflammatory response in the body; at the same time, the fusion protein can be enriched in the intestinal tract, thereby selectively reducing the inflammatory response in the intestinal tract of the body, effectively avoiding the systemic side effects caused by the fusion protein entering the body. Therefore, the fusion protein described in the present application can be better applied in the prevention and treatment of inflammatory bowel disease. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Polyacrylamide gel electrophoresis map of the fusion protein HG28 described in the present application after expression and purification in mammalian cells.

[0028] Figure 2 Content distribution map of the fusion protein HG28 described in the present application in different tissues of transgenic mice. DETAILED DESCRIPTION

[0029] In order to make the present application more easily understood, the present application will be further described in detail below in conjunction with examples, which are only illustrative and do not limit the scope of application of the present application. The raw materials or components used in the present application can be prepared by commercial or conventional methods if not otherwise specified.

[0030] Example 1: Preparation of fusion protein HG28

[0031] First, a nucleic acid molecule for encoding the fusion protein HG28 was prepared by way of total gene synthesis, the nucleotide sequence of which is shown as SEQ ID NO: 2, and then the nucleic acid molecule was ligated to a corresponding expression vector to construct an expression vector containing the nucleic acid molecule.

[0032] The constructed expression vector was transfected into mammalian cells to prepare mammalian cells containing the expression vector. Then the mammalian cells were cultured, the expression of the fusion protein HG28 was induced, and the expressed fusion protein was purified to prepare the fusion protein HG28. The prepared fusion protein HG28 has an amino acid sequence shown as SEQ ID NO: 1.

[0033] The fusion protein HG28 in the present application was prepared by a third-party biological company, and therefore the preparation details are not described in detail.

[0034] The prepared fusion protein HG28 was subjected to polyacrylamide gel electrophoresis, and the electrophoretogram thereof is shown as Figure 1 . Figure 1 In the figure, "M" represents a protein molecular weight marker (protein Marker), "R" represents an electrophoretogram of the reduced fusion protein HG28, and "N-R" represents an electrophoretogram of the fusion protein HG28 without reduction.

[0035] As can be seen from Figure 1 , after reduction, the fusion protein HG28 is degraded into two fragments, and two bands appear in the electrophoretogram; without reduction, the electrophoretogram of the fusion protein HG28 only has one band, and the electrophoretic combination is consistent with the expectation.

[0036] Example 2: Enrichment of fusion protein HG28 in the intestinal tract

[0037] The fusion protein HG28 prepared in Example 1 was injected into a transgenic mouse by intravenous injection, and the content of the fusion protein HG28 in the heart, lung, liver, kidney, intestinal tract and gallbladder of the transgenic mouse was detected, and the results are shown as Figure 2 .

[0038] As can be seen from Figure 2It can be seen that the content of fusion protein HG28 in the mouse intestine is much higher than that in other tissues, indicating that the fusion protein HG28 prepared in this application can be enriched in the intestine.

[0039] Example 3: Effect of fusion protein HG28 on the proliferation of regulatory T cells

[0040] The fusion protein HG28 was added to cell CD4. + CD25 + and cell CD4 + CD25 - In the cell culture medium, the concentration of the fusion protein HG28 was 100 U / mL. After 6 hours of culture, the cells were harvested, and PCR was used to detect CD4+ in the cultured cells. + CD25 + and cell CD4 + CD25 - To detect the expression of the FOXP3 gene, the primers used were: 5′-GGACGTCCCTTTCTGACTG-3′ (SEQ ID NO:3) and 5′-TCACCTACCACATCCACCAG-3′ (SEQ ID NO:4).

[0041] The test results showed that after adding the fusion protein HG28 to the cell culture medium, the cell CD4... + CD25 + The expression level of the FOXP3 gene increased 5.6-fold in the middle; while in the cell CD4... + CD25 - In the study, FOXP3 gene expression was almost undetectable. This indicates that the fusion protein HG28 of this application has the effect of inducing regulatory T cell proliferation, and thus can reduce the inflammatory response in the body's intestine.

[0042] It should be noted that the embodiments described above are only for explaining this application and do not constitute any limitation on this application. This application has been described with reference to typical embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to this application within the scope of the claims, and revisions can be made to the invention without departing from the scope and spirit of this application. Although the application described herein relates to specific methods, materials, and embodiments, it does not mean that this application is limited to the specific examples disclosed herein; on the contrary, this application can be extended to all other methods and applications with the same function. sequence list <110> Hangzhou Gaotian Biopharmaceutical Co., Ltd. <120> Fusion proteins capable of enriching and proliferating regulatory T cells in the gut and their applications <130> 2022 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 401 <212> PRT <213> The amino acid sequence (artificial sequence) of the fusion protein HG28. <400> 1 Gln Ala Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Ser Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Arg Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Ser Arg Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Leu Glu Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu 130 135 140 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Thr Ser Gly 145 150 155 160 Tyr Thr Phe Thr Gly Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly 165 170 175 Gln Gly Leu Glu Trp Met Gly His Ile Ser Pro His Ser Gly Gly Thr 180 185 190 Asp Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr 195 200 205 Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Val Tyr Gly Met Asp Arg Trp 225 230 235 240 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Gly Gly Gly Gly Gly 245 250 255 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Pro Thr Ser 260 265 270 Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp 275 280 285 Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu 290 295 300 Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu 305 310 315 320 Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu 325 330 335 Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp 340 345 350 Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu 355 360 365 Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu 370 375 380 Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu 385 390 395 400 Thr <210> 2 <211> 1295 <212> Ms <213> Fusion protein HG28's nuclear sequence <400> 2 gcggccgcaa actacagac agacttgcaa agaaggcat gcacagctca gcactgctct 60 gttgcctggt cctcctgact ggggtgaggg cccaggctgt gctgacacag cctccttctg 120 cttccggaac ccctggacag agagtcacaa tctcctgctc cggctcctcc tctacatcg 180 gatctaactc cgtcaactgg taccaccagc tccccgac tgctcctaa ctcctgatct 240 acctctaacaa ccagaggccc tccggcgtgc ctgacaggtt ctccggaagc aaatccggca 300 cctccgcatc tctggctatc tctggactcc agtctgaaga tgaagccgac tactactgcg 360 ccgcctggga cgactccctg aacggaaggg tgctgttcgg tggaggaca aagctgaccg 420 tgctgggttc caggggaggt ggtggcggat caggtggggg aggctggt ggaggcggta 480 gtctggaaat ggcccaggtg cagctggtgc agtctggagc tgagtgag aaacccggag 540 cctctgtgaa agtgagctgc agaccctg gatacacctt caccggctac tatatccact 600 gggtgaggca ggcacccgga cagggactgg agtggatggg accatctcc cctcattccg 660 gcggaacaga ttacgctcag aaattccagg gcagagtcac aatgaccagg ccaccaagca 720 tcagcacagc atacatggag ctgagcaggc tgaggagcga cgatacagct gtctactatt 780 gcgccagggg cgtgtacgga atggacaggt ggggacaggg aacactggtg acagtgagct 840 ccgcaggagg tggtggcggt ggatctggag gcggaggtag tggtggggga ggttcaatgc 900 caacctcctc ctctaccaag aaaacccagc tgcagctgga acacctgctg ctggatctgc 960 agatgatcct gaacggcatc aacaactaca aaaaccccaa gctcaccaga atgctgacct 1020 tcaagttcta catgcccaaa aaggccaccg agctcaagca cctgcagtgt ctggaagaag 1080 agctgaaacc cctggaggaa gtgctgaacc tggcccagtc aaaaaacttc cacctgaggc 1140 ccagggacct gatctccaac atcaacgtga tcgtgctgga gctgaagggc tccgagacaa 1200 ccttcatgtg cgagtacgcc gacgagacag ccaccatcgt ggagttcctg aacaggtgga 1260 tcaccttctc ccagtccatc atctccaccc tgacc 1295 <210> 3 <211> 19 <212> DNA <213> Primer (Artificial Sequence) <400> 3 ggacgtccct ttctgactg 19 <210> 4 <211> 20 <212> DNA <213> Primers (artificial sequences) <400> 4 tcacctacca catccaccag 20

Claims

1. A fusion protein, characterized in that, The fusion protein is capable of accumulating and proliferating regulatory T cells in the intestine, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:

1.

2. A nucleic acid molecule for encoding the fusion protein as described in claim 1.

3. An expression vector comprising the nucleic acid molecule as described in claim 2.

4. A host cell comprising the expression vector as described in claim 3.

5. A pharmaceutical composition comprising the fusion protein as described in claim 1.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

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