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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
  • A61P37/02—Immunomodulators
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/74—Inducing cell proliferation
• • 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

Definitions

• Fusion protein with dual functions of inhibiting tumor microenvironment angiogenesis and activating adaptive immune response, and gene and application thereof
• the invention belongs to the field of bioengineering technology.
• the present invention relates to a fusion protein CD137L-Tumstatin having a dual function of inhibiting tumor microenvironment angiogenesis and activating an adaptive immune response, and an expression vector containing the same, and a strain transformed from the vector, and the fusion
• a method for preparing a protein and relates to the use of a protein having dual functions of CD137L and Tumsstatin for the preparation of a medicament for inhibiting tumor microvascular regeneration and related oncological diseases (eg, melanoma, rectal cancer, lung cancer, etc.) and activating an adaptive immune response drug,
• the route of administration e.g., oral administration, spray administration, etc.
• Cancer is a process of multi-gene cooperation and participation by different signaling pathways. By its very nature, cancer is a molecular condition. At present, single-molecule molecular therapy strategies have gradually shown many drawbacks, and multi-target, multi-mechanical combination therapy has shown better therapeutic effects.
• Tumor growth and metastasis depend on microenvironmental angiogenesis.
• the theory suggests that by inhibiting tumor microenvironmental angiogenesis, cutting off the nutrient and oxygen supply of the tumor can inhibit the growth and metastasis of tumor cells. Since its publication, the doctrine has been supported by a large number of laboratory and clinical data worldwide, and has become a new strategy in the field of cancer therapy.
• Human tumor suppressor (Tumstatin) is a tumor angiogenesis inhibitory factor derived from the C-terminal end of the vascular basement membrane type IV collagen ⁇ 3 chain, consisting of 244 amino acids.
• the three-dimensional network skeleton formed by type IV collagen is the main component of scaffolding, which promotes cell adhesion, migration, differentiation and growth. It is encoded by six unique genes to generate six chains ⁇ 1 ⁇ ⁇ 6 and form a trimer with different or identical ⁇ chains to further form a network structure.
• Each ⁇ 3 chain of type IV collagen consists of three functional regions (7S domain, triple helix region, non-collagen peptide NC1 domain), distributed in glomeruli, alveolar capillaries, cochlea, lens capsule, ovary And the basement membrane of the testes.
• Tumstatin specifically inhibits the synthesis of tumor vascular endothelial cell proteins, leading to apoptosis of endothelial cells, inhibiting angiogenesis, thereby inhibiting tumor growth and metastasis.
• Tumstatin also has a property of directly acting on tumor cells and inhibiting their proliferation.
• the cells of the immune system of a tumor patient are capable of recognizing antigens expressed by tumor cells, such as tissue differentiation antigens, carcinoembryonic antigens, and products of mutant genes, and the like.
• tumor antigen recognition and immune response mechanisms studies have shown that providing immune co-stimulatory signals through helper molecules can increase the anti-tumor immune response. Since tumor antigen-specific tau cells require synergistic stimulation to assist the first antigen signal to stimulate the function of effector cells, adjuvant therapy with synergistic stimulation molecules can be used to modulate immune responses against malignant tumors.
• TCR T cell receptor
• APC antigen-presenting cells
• the peptide signal first signal
• the synergistic stimulus signal ie, the second signal
• CD137 and its ligand CD137L are another important pair of T cell synergistic stimulation signal molecules newly discovered in addition to CD28/B7.
• the conjugated molecules can be divided into two categories: Tumor Necrosis Factor Receptor (TNFR) and immunoglobulin superfamily.
• TNFR Tumor Necrosis Factor Receptor
• CD137 is a member of the tumor necrosis factor receptor family. It plays an important role in regulating cell proliferation, differentiation, and apoptosis.
• Its ligand CD137L is also a member of the TNF family, which is a type II transmembrane protein with similar C-terminal amino acids to other T F families.
• the gene encoding human CD137L is located at 19p3.3, and its product is 254 amino acids, of which 28 amino acids in the cytoplasmic region, 21 amino acids in the transmembrane region, and 205 amino acids in the extracellular region.
• CD137L was first isolated from mouse thymoma cells by Goodwin et al. using expression screening techniques and subsequently isolated in human CD4+ T cell clones.
• CD137/CD137L The interaction between CD137 and its ligand CD137L (CD137/CD137L) has been well recognized for its important role in T cell activation. It was observed in both murine and human T cells that CD137 can induce T cell proliferation, synthesize cytokines (such as IFN- ⁇ ), and prolong the survival of activated cells in the presence of CD3 antibodies (first signal). Synergistic stimulation signals enhance effector function by increasing the number of antigen-specific and effector CD8+ T cells; however, in the absence of CD3 antibody signaling, stimulation of CD137 molecules does not alter T cell function, suggesting that CD137 interacts with CD137L. All that is provided is a synergistic stimulus signal.
• CD137 monoclonal antibody-mediated anti-tumor immunity is dependent on the involvement of CD4+T and CD8+ T cells.
• CD137 mediates the activation of F-kB, which in turn up-regulates the expression of bcl-xL and bfl-1 molecules, prolongs the survival of CD8+T and CD4+ T cells and promotes their proliferation.
• Melero et al. used activated murine CD137 monoclonal antibodies for CD137-targeted immunotherapy. The results indicate that CD137 mAb can eliminate P815 solid tumors that have been inoculated in mice.
• CD137L synergistically stimulates cytotoxicity Cytotoxic lymphocyte (CTL) and anti-tumor effects.
• CTL Cytotoxic lymphocyte
• CD137L-/- mice well demonstrate the important role of the CD137/CD137L system in T cell-mediated immune responses to viruses and tumors.
• Studies in CD137 or CD137L deficient mice indicate that the synergistic stimulation of CD137/CD137L is important for graft versus host disease, antiviral cellular responses of T cells.
• the stimulation signal provided by CD137/CD137L can synergize with CD28/B7 molecules to further activate T cells and maintain the proliferation and survival of CD8+ T cells.
• Tumstatin can effectively inhibit the formation of tumor blood vessels and block the survival and metastasis of tumor cells. Therefore, the fusion protein T U m S t a tin-CD137L of the present invention can inhibit tumor growth and metastasis from both inhibition of angiogenesis and enhancement of anti-tumor immune response, thereby avoiding drug tolerance caused by single treatment, for treatment Tumor patients offer new strategies.
• the present invention synthesizes an anti-angiogenic active fragment of tumstatin and a CD137L extracellular domain protein by a short flexible linker peptide, and has a bifunctional, dual target molecule which enhances T cell immunity and antiangiogenesis; and is selected in the present invention.
• Tumstatin 45th to 98th amino acid fragment of Tumstatinl, 60th to 132nd amino acid fragment of Tumstatin2, 60th to 98th amino acid fragment of Tumstatin3, 74th to 98th amino acid of Tumstatin7 Fragment
• CD137L extracellular domain protein amino acid sequence CD137L1 amino acid fragment 46 to 254, CD137L4 50th to 240th amino acid fragment, CD137L5 83th to 254th amino acid fragment, CD137L6 46th to the
• the ligated fragment of amino acid 85 and amino acid 167 to 254, as shown in Figure 2 is prepared by ligation of a linker peptide, using a prokaryotic or eukaryotic expression system, which is simple to prepare and avoids full-length tumstatin and The side effect of full length CD137L, the present invention will be in the preparation of angiogenesis inhibitors, various Tumor-related diseases (such as melanoma, prostate cancer, lung cancer, colon cancer, kidney
• Another object of the present invention is to provide a gene encoding the above-described protein having the functions of Tumstatin and CD137L.
• Another object of the present invention is to provide a process for the preparation of the above-described protein having the functions of Tumstatin and CD137L.
• Another object of the present invention is to provide the above-mentioned protein having Taustatin and CD137L functions and genes thereof for treating oncology-related diseases (e.g., melanoma, rectal cancer, lung cancer, etc.) and administration routes (e.g., oral administration, spraying Application of the route of administration, etc.).
• oncology-related diseases e.g., melanoma, rectal cancer, lung cancer, etc.
• administration routes e.g., oral administration, spraying Application of the route of administration, etc.
• a bifunctional recombinant protein having tumstatin and CD137L having an amino acid sequence of a Tumstatin active fragment and The CD137L extracellular domain protein fragment amino acid sequence, the CD137L extracellular domain protein fragment amino acid sequence and the Tumstatin active fragment amino acid sequence are fused by a flexible linker peptide, and the Tumstatin active fragment amino acid sequence is selected from the group consisting of SEQ ID N0.65 to SEQ ID One of the amino acid sequences shown in N0.68, wherein the amino acid sequence of the CD137L extracellular domain protein fragment is selected from one of the amino acid sequences shown in SEQ ID N0.77 to SEQ ID NO.
• the amino acid sequence of the above-described linker peptide can be designed by a conventional technique in the art, preferably one of SEQ ID N0.69 to SEQ ID N0.76.
• the bifunctional recombinant protein amino acid sequence of the present invention is preferably one of SEQ ID N0.25 to SEQ ID N0.48, and the recombinant protein is schematically shown in Figure 1.
• the present invention also provides a gene encoding the above bifunctional recombinant protein having Tumsstatin and CD 137L, comprising a gene encoding a CD 137L extracellular domain protein fragment, a gene encoding a linker peptide, and a gene encoding a Tumsstatin active fragment, wherein the encoding CD137L extracellular
• the gene for the region protein fragment is selected from one of SEQ ID N0.61 - SEQ ID NO. 64, and the gene encoding the active fragment of Tumsstat is selected from one of SEQ ID N0.49 - SEQ ID N0.52.
• the above gene encoding the linker peptide is preferably one of SEQ ID N0.53-SEQ ID NO.
• the above recombinant protein gene preferably has the nucleotide sequence of one of SEQ ID NO: 0.1 to SEQ ID NO: 4.
• the present invention also provides a gene encoding the above recombinant protein T U m S t a tin-CD137L, which has 70% or more homology with the above nucleotide sequence, and can encode the recombinant protein of the present invention. Or a conservative variant polypeptide thereof or an active fragment thereof or a reactive derivative thereof.
• the present invention also provides the above preparation method of the bifunctional recombinant protein having Tumstatin and CD137L, comprising the following steps:
• the recombinant protein having the activity of Tusumatin and CD137L of the present invention is isolated and purified.
• the above expression system may select a prokaryotic expression system or a eukaryotic expression system, and the prokaryotic expression system is preferably an E. coli expression system or a Bacillus subtilis expression system, and the two expression systems are generally adapted to the expression of the recombinant protein of the present invention, wherein the Escherichia coli system
• the medium expression vector is preferably pET-lla, pET-22b, and the B. subtilis expression system expression vector is preferably pP43;
• the eukaryotic expression system is preferably a yeast expression system, the expression vector is preferably pPIC9K, pPICZaA, and the yeast host cell is preferably GS115 or SMD1168.
• a preferred embodiment of the above preparation method is: the gene encoding the above-mentioned recombinant protein Tumsstatin-CD137L having TumMatin activity and CD137L activity is digested with Ndel and Nhel, and then ligated to the expression vector pET-lla.
• the enzyme cleavage site was transformed into E. coli BL21 (DE3), and the target protein in the form of inclusion bodies was obtained by liquid culture engineering bacteria.
• the inclusion body protein was subjected to dilution and renaturation, that is, the inclusion body protein was washed several times with a solution containing a low concentration of urea, and then the inclusion body was dissolved at 50 ° C with a denatured solution containing 8 M urea, and finally 0.4 M L-Arg was used.
• the refolding body is diluted with the refolding inclusion body (the purity of the target product is more than 80%), and the recombinant protein of the present invention is obtained.
• a preferred embodiment of the above preparation method is: the gene encoding the above-mentioned recombinant protein Tumsstatin-CD137L having TumMatin activity and CD137L activity is digested with Pstl and Hindlll, and then ligated to the corresponding restriction site of the expression vector pP43, and then electrotransferred.
• Bacillus subtilis WB800 a protein of interest secreted into an extracellular soluble form was obtained by liquid culture engineering bacteria. Purification by DEAE anion exchange method, the purity is over 80%, and the recombinant protein of the present invention is obtained.
• a preferred embodiment of the above preparation method is: the gene encoding the above-mentioned recombinant protein Tumsstatin-CD137L having Tumatatin activity and CD137L activity is double-digested with EcoRI and Notl, and then ligated to the corresponding restriction site of the expression vector pPICZaA, and then electrotransferred.
• Pichia pastoris GS115 the target protein secreted into the extracellular soluble form was obtained by liquid culture engineering bacteria. Purification by DEAE anion exchange, the purity is over 90%, and the recombinant protein of the present invention is obtained.
• the present invention also provides the above-mentioned dual-function recombinant protein with tumstatin and CD137L in the preparation of tumor microenvironment-inhibiting angiogenesis and related oncological diseases (eg, melanoma, rectal cancer, lung cancer, etc.), regulation of body immunity, T cell proliferation and body The application of cytokine synthesis and secretion of drugs.
• the present invention also provides the above-mentioned dual-function recombinant protein gene with tumstatin and CD137L for preparing tumor microvascular angiogenesis and related oncological diseases (eg, melanoma, rectal cancer, lung cancer, etc.), regulating body immunity, T cell proliferation, and The application of the synthesis and secretion of cytokines in the body.
• tumor microvascular angiogenesis and related oncological diseases eg, melanoma, rectal cancer, lung cancer, etc.
• body immunity eg, melanoma, rectal cancer, lung cancer, etc.
• T cell proliferation e.g, T cell proliferation
• the recombinant protein of the present invention may be used alone or in the form of a pharmaceutical composition.
• the pharmaceutical composition comprises the recombinant protein of the present invention as an active ingredient and a pharmaceutically acceptable carrier.
• the pharmaceutical composition has from 0.1 to 99.9% by weight of the recombinant protein of the present invention as an active ingredient.
• the "pharmaceutically acceptable carrier” does not destroy the pharmaceutical activity of the recombinant protein of the present invention, and its effective amount, i.e., the amount which can be used as a drug carrier, is not toxic to the human body.
• “Pharmaceutically acceptable carrier” includes, but is not limited to, ion exchange materials, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-vitamin E polyethylene glycol 1000 succinate, Tween Or other surfactants such as a polymeric medium, serum proteins such as human serum albumin, buffer substances such as phosphate, glycine, sorbic acid, potassium sorbate, saturated glycerides of saturated plant fatty acids, water, salt, Electrolytes such as protamine, dibasic hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium silicate, and the like.
• SEDDS self-emulsifying drug delivery systems
• excipients such as fillers (such as anhydrous lactose, starch, lactose beads and glucose), binders (such as microcrystalline cellulose), disintegrants (such as croscarmellose sodium, cross-linked carboxylates) Sodium methylcellulose, low-substituted hydroxypropylcellulose and cross-linked PVP), lubricants (such as magnesium stearate), absorption enhancers, flavoring agents, sweeteners, diluents, excipients, wetting agents Solvents, solubilizers, colorants and the like may also be added to the pharmaceutical compositions of the invention.
• fillers such as anhydrous lactose, starch, lactose beads and glucose
• binders such as microcrystalline cellulose
• disintegrants such as croscarmellose sodium, cross-linked carboxylates
• lubricants such as magnesium stearate
• absorption enhancers such as magnesium stearate
• flavoring agents such as sweeteners,
• any dosage form that can be optionally used.
• oral administration forms such as tablets, capsules, granules, powders or liquid preparations, or parenteral administration forms such as injections, topical products or suppositories, which may be formulated in a conventional manner or unconventional.
• parenteral administration forms such as injections, topical products or suppositories, which may be formulated in a conventional manner or unconventional.
• Methods such as liposomes and the like.
• the recombinant protein of the present invention When used as a therapeutic agent, its use amount is in the range of about 0.01 mg to lg per day for an adult, depending on the age, sex, body weight and degree of symptoms of each patient, and the daily dose can be divided into Several doses.
• the recombinant protein of the present invention further comprises a modified protein which modifies the recombinant protein of the present invention by a conventional method in the prior art.
• modified peptide drugs can significantly reduce immunogenicity, reduce toxic side effects, increase water solubility, prolong the time of action in vivo, and change Its bio-distribution and so on, significantly improve the efficacy of the drug.
• the commonly used modification methods of the recombinant protein of the present invention include modification of intermediate residues, amino acid substitution, glycosylation modification and PEG modification, etc.
• the basic principle is to increase the relative molecular weight and steric hindrance of the polypeptide molecule, and to increase the peptide hydrolase. Stability, reducing glomerular filtration. Replacing a few amino acids in a peptide chain is another way of delaying the degradation of the polypeptide to extend the half-life of the polypeptide drug.
• the replacement object is usually an easily digestible amino acid in the peptide chain.
• the intermediate residues of the recombinant protein may be glycosylated, phosphorylated, methylated, acetylated, nitrated, sulfonated or linked to PEG modified or coupled to a protein, wherein:
• glycosylation-modified peptide is preferably one or more of the oxygen-linked to the sugar at one or more Tyr, Ser or Thr residues in the amino acid sequence of the recombinant protein of the present invention or the amino acid sequence of the recombinant protein of the present invention.
• the amide nitrogen of the asparagine side chain is attached to the sugar.
• the phosphorylated modified peptide is preferably phosphorylated at one or more Tyr, Ser or Thr sites in the amino acid sequence of the recombinant protein of the present invention.
• the methylated modified peptide includes a side chain methylation modified peptide and an N-terminal methylated modified peptide, and the side chain methylation is preferably one or more Lys, Tyr or Arg side chains in the amino acid sequence of the recombinant protein of the present invention.
• Methylation such as Lys (For), Lys (Me), Lys (Me) 2, Lys (Me) 3, Arg (Me) 2 symmetrical, D-Tyr (Me), D-Tyr (Et);
• the acetylated modified peptide is preferably subjected to one or more Lys or Ser side chains in the amino acid sequence of the recombinant protein of the present invention. Acylation, such as Ser (Ac) or Lys (Ac).
• the nitrating or sulfonating modified peptide is preferably nitrolated or sulfonated on one or more Tyr side chains in the amino acid sequence of the recombinant protein of the present invention, Tyr(3-N0 2 ), Tyr(S0 3 H 2 ).
• the PEG modification of the intermediate residue is preferably PEG-modified with one or more amino groups of the Lys side chain in the amino acid sequence of the recombinant protein of the present invention, and the PEG molecular weight is preferably from 2,000 to 10,000.
• one or more amino acids in the amino acid sequence of the recombinant protein of the present invention or the above modified protein thereof are replaced with corresponding amino acid derivatives or special amino acids, such as alanine instead of ⁇ -alanine, high Amphetamine or naphthylalanine, replacing proline with hydroxyproline, leucine with norleucine, valine with normal valine, threon with other threonine,
• the leucine is replaced by a different leucine
• the asparagine is replaced by 2-acetamido-2-deoxy- ⁇ -D-glucopyranosyl asparagine (Asn (GlcNac(Ac)3-e-D))
• Lysine is replaced by Lys (palmitoyl).
• one or more amino acids in the amino acid sequence of the recombinant protein of the present invention or the above-described modified protein thereof are replaced with the corresponding D-type amino acid.
• the gene encoding the above bifunctional recombinant protein having Tusumatin activity and CD137L activity (GenBank: AAF72632.1 and P_003802.1) in the present invention is obtained by a whole gene synthesis, a PCR method, or a combination thereof, using a conventional strategy, wherein CD137L
• the full-length sequence vector template can be prepared by the method disclosed in the literature (Wang shuzhen. J Ind Microbiol Biotechnol. 2012 Mar; 39(3): 471-6. doi: 10.1007/sl0295-011-1045-l.).
• the present invention constructs an expression vector containing the above-described protein gene encoding the dual function of Tumsstatin and CD137L, which is obtained by double-digesting the Numdel and Nhel gene fragments by conventional PCR and restriction enzyme digestion and ligation. , and connected to the corresponding restriction sites of the prokaryotic expression vector pETlla, and verified by sequencing to obtain the correct expression vector.
• the present invention constructs a genetically engineered bacteria containing the above expression vector, that is, by transforming the target gene into BL21, a positive engineering strain expressing the target protein is screened by a small amount of liquid culture medium.
• the invention provides a method for obtaining a protein having dual functions of Tumsstatin and CD137L, which is to carry out culture fermentation of a positive engineering strain and frequently induce it to express a protein having dual functions of Tumsstatin and CD137L, and then collect the bacteria, high pressure.
• the disrupted cells, the pellet obtained by centrifugation were denatured and diluted and renatured to obtain a recombinant protein having a dual function of Tumsstatin and CD137L.
• the activity of the recombinant protein of the present invention having dual functions of tumstatin and CD 137L was carried out by conducting a human umbilical vein endothelial cell activity assay (HUVEC assay) and a mouse T cell activation assay. Among them, the human umbilical vein endothelial cell activity test results showed that the proliferation of endothelial cells was significantly inhibited.
• Mouse T cell activation assays have shown that the recombinant protein maintains the biological activity of CD137L and synergizes with anti-CD3 and anti-CD28 monoclonal antibodies to stimulate T cell proliferation. Colonization.
• the present invention shows that a prokaryotic expression system is used to express a recombinant protein which is active from the extracellular domain of Tumsstatin and CD137L, and a protein which has both the activity of Tumsstatin and CD137L can be produced.
• the Tumstatin-Linker-CD137L recombinant protein (a protein having both Tumstatin and CD137L functions) produced by the present invention has the advantages of high expression efficiency, large expression amount, short expression period, and easy purification.
• the fusion protein provided by the present invention has a significant inhibitory effect on the proliferation of human umbilical vein endothelial cells in a dose-dependent manner, and synergistically anti-CD3 and anti-CD28 monoclonal antibodies can stimulate the proliferation of mouse T cells. Therefore, the present invention provides a new and safe way for large-scale production of recombinant proteins of Tumstatin and CD137L, and lays a solid foundation for further research and development as a new generation of anti-tumor drugs, and has broad application prospects in the pharmaceutical industry.
• Figure 1 is a schematic view showing the structure of a recombinant protein having dual functions of Tumstatin and CD137L according to the present invention.
• the flexible linker amino acid sequence is selected from one of SEQ ID N0.69 - SEQ ID N0.76.
• Figure 2 is a schematic representation of the region of CD137L1 (SEQ ID N0.77), CD137L5 (SEQ ID N0.78), CD137L6 (SEQ ID N0.79) and CD137L4 (SEQ ID NO. 80) in the full length sequence of CD137L amino acid.
• Figure 3 shows the expression of the recombinant protein Tumstatin-linker peptide-CD137L in Escherichia coli with the amino acid sequence as shown in SEQ ID N0.25-SEQ ID N0.48. Lanes 1-24 correspond to SEQ ID N0.25-48, respectively.
• Figure 4 shows the expression of the representative Tumstatin-linker peptide-CD137L protein amino acid sequence (SEQ ID N0.29-SEQ ID N0.38) in Bacillus subtilis. Lanes 1-8 correspond to SEQ ID NO. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, respectively.
• Figure 5 is a representation of the representative amino acid sequence of the Tumstatin-linker peptide-CD137L protein (SEQ ID NO. 29 - SEQ ID NO. 36) in yeast. Lanes 1-16 correspond to the protein amino acid sequences SEQ ID NO. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, respectively.
• Figure 6 is a graph showing the results of SDS-PAGE electrophoresis of a representative Tumtatin-Linker-CD137L protein sample after dilution renaturation.
• the amino acid sequence of the first lane sample is SEQ ID N0.25; the amino acid sequence of the second lane sample is SEQ ID N0.26; the amino acid sequence of the third lane sample is SEQ ID N0.27; the amino acid sequence of the fourth lane sample is SEQ ID N0.37; the amino acid sequence of the fifth lane sample is SEQ ID N0.38; the amino acid sequence of the sixth lane sample is SEQ ID N0.41; the amino acid sequence of the seventh and tenth lane samples is SEQ ID N0.43;
• the amino acid sequence of the 11 lane sample is SEQ ID N0.44; the amino acid sequence of the 9th and 12th lane samples is SEQ ID N0.48; the amino acid sequence of the 13th and 15th lane samples is SEQ ID N0.45; lanes 14 and 16
• Figure 7 is a graph showing the effect of a representative Tumstatin-linker peptide-CD137L protein sample on the proliferation of human umbilical vein endothelial cells.
• Figure 8 is a graph showing the effect of a representative Tumstatin-linker peptide-CD137L protein sample on the proliferation of mouse T lymphocytes.
• the invention has designed 24 bifunctional recombinant proteins having Tumsstatin activity and CD137L activity, and the nucleotide sequence thereof is the sequence shown in SEQ ID NO. 1 to SEQ ID N0.24, and the translation encoding amino acid sequence is SEQ ID N0.25 to The sequence shown in SEQ ID N0.48.
• the histone has amino acid fragments 45 to 98 of Taustantin, amino acid fragments 60 to 132 of Tumstatin 2, amino acid fragments 60 to 98 of Tumstatin 3 or amino acid fragments 74 to 98 of Tumatatin7 and CD137L cells.
• amino acid sequence of the outer region protein (amino acid fragment 46 to 254 of CD137L1, amino acid fragment 83 to 254 of CD137L5, and the framing fragment of amino acids 46 to 85 of CD137L6 and amino acids 167 to 254
• One of the two is fused by a flexible linker peptide, wherein the amino acid sequence of the linker peptide is as shown in SEQ ID N0.69 to SEQ ID N0.76.
• the present invention is linked to the above-described Tumstatin active fragment gene and the linker peptide gene.
• the 5' end and 3' of the nucleotide sequence (shown as SEQ ID N0.81-SEQ ID N0.94) were designed with EcoRI and BamHI restriction sites, and submitted to Shanghai Jierui Bioengineering Co., Ltd. for total synthesis.
• the pBluescriptll SK(+) vector with the corresponding restriction site was inserted, and the recombinant vector was provided by Shanghai Jierui Bioengineering Co., Ltd.
• Various expression vectors were provided by Novagen ToplO and BL21 (DE3) strains were purchased from Invitrogen.
• pMD18-T vector, solution I (Cat. No.
• Example 1 Expression of recombinant protein Tumstatin-CD137L with Tumstatin activity and CD137L activity in Escherichia coli expression system
• CD137L1, CD137L4, CD137L5, and CD137L6 are derived from amino acids 46-254, 50-240 amino acids, 83-254 amino acids, and amino acids 46-85 and 167-254, respectively, of the full-length amino acid sequence of CD137L. Amino acid ( Figure 2).
• the nucleotide sequence of the protein is a sequence of 8£010 ⁇ «).1 to 8£010 ⁇ «).24, and the amino acid sequence of the translation encodes the sequence of SEQ ID N0.25 to SEQ ID N0.48, and the linker peptide One selected from the sequences shown in SEQ ID N0.69 to SEQ ID N0.76.
• CD137L Encoding the extracellular domain of CD137L (amino acid fragment 46 to 254 of CD137L1, amino acid fragment 50 to 240 of CD137L4, amino acid fragment 83 to 254 of CD137L5, amino acid 46 and 85 of CD137L6 and
• the nucleotide sequence of the amino acid sequence from position 167 to position 254 is shown in SEQ ID N0.61-SEQ ID N0.64, and amplified by PCR, wherein the CD137L full-length sequence template reference (Wang shuzhen. J The method disclosed in Ind Microbiol Biotechnol. 2012 Mar; 39(3): 471-6. doi: 10.1007/sl0295-011-1045-l.) was prepared.
• the CD137L template used in this patent was prepared by the method mentioned in the above literature. Using the upstream primer of SEQ ID NO. 61 (SEQ IDN0.95) and the downstream primer of PSEQ ID NO. 61 (SEQ IDN0.96) as a primer, rTaq DNA polymerase catalyzed amplification to obtain a CD137L1 (SEQ ID NO. 61) gene fragment (this time The restriction sites at both ends of the gene were 5' BamHI and 3' Notl), respectively, and the product was ligated to the pMD18-T vector.
• the CD137L1 gene fragment was ligated from the pMD18-T vector by restriction enzyme ligation and other conventional molecular biological methods, and the Tumstatin and the linker peptide (SEQ ID NO. 87 - SEQ ID N0. 94) on the pBluescriptll SK(+) vector.
• the entire fusion protein gene (SEQ ID N0.5 - SEQ ID N0.12) has been integrated into the pBluescriptll SK(+) vector.
• the upstream primer SEQ ID N0.97 and the downstream primer SEQ ID N0.98 were used to amplify the fusion protein gene by PCR.
• the 5' cleavage site was Nhel, and the 3' cleavage site was Ndel.
• Linked to the expression vector pETlla The CD137L template used in this patent was prepared by the method mentioned in the above literature. Using the upstream primer (SEQ ID NO. 100) of SEQ ID NO. 62 and the downstream primer (SEQ ID NO. 100) of SEQ ID NO. 62 as primers, rTaq DNA polymerase catalyzes the CD137L5 (SEQ ID NO. 62) gene. The fragment (in this case, the restriction sites of the two ends of the gene are 5' BamHI and 3' Notl, respectively), and the product was ligated to the pMD18-T vector.
• the CD137L5 gene fragment was ligated from the pMD18-T vector by restriction enzyme ligation and other conventional molecular biological methods, and the Tumstatin and the linker peptide (SEQ ID N0.81-SEQ ID NO. 86) on the pBluescriptll SK(+) vector.
• the entire fusion protein gene (SEQ ID NO. 1, SEQ ID NO, SEQ ID NO. 13 - SEQ ID NO. 16) has been integrated into the pBluescriptll SK (+) vector.
• SEQ ID NO. 15 - SEQ ID NO. 16 upstream primer 104 SEQ ID NO. 1 SEQ ID NO. 3, SEQ ID NO. 13 - SEQ ID NO. 16 downstream primer SEQ ID NO. 105, amplification of the fusion protein gene by PCR
• the product 5' cleavage site is Nhel
• the product 3' cleavage site is Ndel
• ligated to the expression vector pETlla The CD137L template used in this patent was prepared by the method mentioned in the above literature.
• the primer 1 (SEQ ID NO. 107) of the upstream primer 1 (SEQ ID NO. 106) SEQ ID NO. 63 of SEQ ID NO. 63 is used as the primer
• 63 SEQ ID NO. .108
• the downstream primer 2 SEQ ID NO. 109 of SEQ ID N0.63 are primers
• rTaq DNA polymerase catalyzes the amplification of two gene fragments of CD137L6 (SEQ IDN0.63)
• the agarose electrophoresis gel recovers the two Segment gene.
• the recombinant two-stage gene was used as a template, and the upstream primer 1 (SEQ ID NO. 107) of CD137L6 and the downstream primer 2 (SEQ ID NO. 109) of CD137L6 were used as primers.
• the final CD137L6 (SEQ ID N0.63) fragment (both restriction sites 5' BamHI and 3' Notl) was ligated and ligated into the pMD18-T vector.
• the CD137L6 (SEQ IDN0.63) gene fragment was ligated from the pMD18-T vector and ligated to the already synthesized Tusstatin and the linker peptide (SEQ ID N0.8U SEQ ID NO. 82, SEQ ID NO. 84) synthesized by Shanghai Jierui Company. , SEQ ID NO. 86) on pBluescriptll SK (+) vector.
• the entire fusion protein gene (SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO.
• the primers involved in the above experimental procedures are:
• the correctly sequenced fragment was ligated to the prokaryotic expression vector pET1 la.
• the reaction system was T4 ligase 10 X buffer 1 ⁇ , ⁇ ⁇ , target gene 7 ⁇ , ⁇ 4 ligase 1 ⁇ , reaction condition 16 ° C, 16 h. After double enzyme digestion, it was transformed into E. coli expression strain BL21 (DE3).
• the wet weight of the cells was added to 10 mL of PBS and suspended, and the cells were lysed by a high-pressure cell disrupter. The effluent was collected, centrifuged at 4 ° C, 12000 rpm, 30 min, the supernatant was discarded, and the precipitate was left for SDS-PAGE analysis.
• the SDS-PAGE experiment operation flow is as follows:
• the bacterial body precipitate obtained after high-pressure crushing was collected by the above method, and washed repeatedly by washing liquids A and B twice, at 12,000 rpm for 10 minutes, and the supernatant was centrifuged and collected for use.
• the preparation of the washing solution A was 20 mM Tris-HCL, pH 8.5, 2 M urea, 2% Triton X-100 ;
• the washing liquid B formulation was 20 mM Tris-HCL, pH 8.5, 2 M urea, 5 mM EDTA.
• the solution was denatured overnight at 50 ° C with a solution (20 mM Tris, pH 8.5, 8 M urea).
• the denaturant was added to the refolding solution through a constant flow device in a 4 °C chromatography cabinet, and the whole process was continued for 12 h with slow agitation.
• the renaturation solution was formulated as 20 mM Tris-HCL pH 8.5, 2 M urea, 0.4-0.6 M L-Arg, 1 mM EDTA.
• the obtained reconstituted solution containing the sample was concentrated and replaced.
• the apparatus used for concentration was an Amicon Ultra-15 concentrating tube from Millpore, 4500 rpm, 30 min, 4 °C.
• the concentrate was replaced with pH 7.4 in 100 mM PBS buffer to a final volume of 1 mL. Reserve at 4 ° C ( Figure 6). It has been estimated that the purity of the target product after renaturation is over 80%. It can be seen from the figure that the renaturation of the denatured inclusion body protein is good.
• Example 2 Expression of recombinant protein Tumstatin-CD137L with Tumstatin activity and CD137L activity in Bacillus subtilis expression system
• pBluescriptll SK(+) vector containing the complete recombinant protein gene sequence (SEQ ID N0.1-24) constructed in Example 1 as a template, by designing different primers (5 'Pstl, 3 ' Hindlll) and performing PCR, The different fusion protein genes ligated to the pBluescriptll SK(+) vector were amplified and ligated into the expression vector pP43 (Pstl, Hindlll double-cleaved the vector before ligation). The steps of PCR and restriction enzyme ligation are the same as those of the fusion protein gene of Example 1. After the expression vector is constructed, the electroporation operation is carried out (for details, see Bio-Rad electroscopy instructions), and the recombinant strain of B. subtilis expression system WB800 is constructed.
• Seed culture was carried out in a clean bench, and 10 ⁇ L of the above-preserved Bacillus subtilis WB800 strain containing the target gene plasmid was added to 5 mL of test tube medium in LB medium containing 50 ⁇ g/mL kanamycin. , placed on a shaker, incubated at 37 ° C, 250 rpm for 12 h.
• Recombinant WB800 Fermentation Transfer the activated seed broth to a 50 ⁇ g/mL kanamycin sterile 2 X YT medium at a pH of 7.0 at 37 ° C for 96 h after transfer. The supernatant was collected by centrifugation at °C and sampled for SDS-PAGE analysis.
• the protein of interest was purified by GE's DEAE anion exchange method.
• the purification buffer was PBS phosphate buffer, pH 8.5.
• Example 3 Expression of recombinant protein Tumstatin-CD137L with Tumstatin activity and CD137L activity in yeast expression system
• the different fusion protein genes (SEQ ID NOS. 1-24) integrated on the pBluescriptll SK(+) vector were digested with EcoRI and Notl and ligated into pPIC9K to complete the vector construction work. Then carry out the electric operation (for details, see Bio-Rad The electrorotator instructions were used to complete the construction of the recombinant strain of Pichia pastoris GS115.
• a BMGY-enriched cell, BMMY-induced expression strategy was used to ferment to obtain the desired product.
• the expression strain constructed as described above was inoculated to YPD seed medium and cultured overnight at 37 °C.
• the appropriate amount of seed culture solution was transferred to BMGY medium for 48 hours.
• the OD600 was 10
• the medium was changed to BMMY
• the expression was induced for 96 hours, and the supernatant was collected by centrifugation at 12,000 rpm for 30 minutes while sampling for SDS-PAGE analysis.
• Example 4 Effect of recombinant protein with dual functions of Tumstatin and CD137L on proliferation of human umbilical vein endothelial cells.
• MTT colorimetric assay Take logarithmic growth phase cells, inoculate 5000 cells per well in 96-well culture plates, add 100 L of cell suspension to each well, and incubate in a cell culture incubator for 12 h. After adding 100 L of different concentrations of protein samples, the positive control was T P-470, the negative control group was added with the same volume of PBS, and each group was set with 3 duplicate wells for 48 h. Add 4 ⁇ g of 5 mg/mL MTT solution to each well in a 96-well culture plate 4 h before the end of the culture, and continue to incubate for 4 h in a cell culture incubator, then gently aspirate the medium, then add 150 ⁇ L of DMSO to each well and shake for 10 min. The blue-violet precipitate is fully soluble. The absorbance (A490) was measured with a microplate reader.
• T P-470 is a positive control
• "115” is Tumstatin1-linked peptide 1-CD137L5 (SEQ ID N0.25)
• 155 is Tumstatin1-linked peptide 5-CD137L5 (SEQ ID N0.27)
• “116” is the Tumstatin1-linker peptide 1-CD137L6 (SEQ ID N0.26)
• "215" is the Tumstatin2-linker peptide 1-CD137L5 (SEQ ID N0.37)
• “255” is the Tumstatin2-linker Peptide 5-CD137L5 (SEQ ID N0.38)
• “256” is Tumstatin2-linker peptide 5-CD137L6 (SEQ IDN0.41)
• “711” is Tumstatin7-linker peptide 1-CD137L1 (SEQ ID N0.29)
• “721 Tumstatin7-linked peptide 2-CD137L1 (SEQ ID NO.
• Example 5 Effect of recombinant protein with dual function of tumtatin and CD137L on proliferation of mouse spleen T lymphocytes.
• the human CD3 monoclonal antibody was coated in 96-well plates at 7.5 g/mL, overnight at 4 °C, and the isolated mouse spleen T cells were added to 100 ⁇ l/well (10 5 cells) on the second day, and divided into 4 experimental groups: 1) T cell blank group (Medium + T cell) without any antibody stimulation; 2) Anti-CD3 (7.5 g/mL) and anti-CD28 monoclonal antibody (2.5 g/mL) combined with stimulator group (CD3/CD28); 3) anti-CD137 monoclonal antibody combined with anti-CD3 and anti-CD28 monoclonal antibody stimulator group (CD3/CD28/anti-CD137mAb); 4) fusion protein samples combined with anti-CD3 and anti-CD28 monoclonal antibody stimulator group (CD3/CD28) /Tumstatinl-CD137L), 3 replicate wells per group.
• Tumstatin2-linker peptide 1-CD137L5 SEQ ID N0.37
• 255 is the Tumstatin2-linker peptide 5-CD137L5
• 256 is the Tumstatin2-linker peptide 5-CD137L6 (SEQ. IDNO.41)
• 711 is Tumstatin7-linked peptide 1-CD137L1 (SEQ ID N0.29)
• 721 is Tumstatin7-linked peptide 2-CD137L1
• 114 is the Tumstatin1-linker peptide 1-CD137L4 (SEQ ID N0.43)
• 214 is the Tumstatin2-linker peptide 1-CD137L4 (SEQ IDN0.44)
• 314" is the Tumstatin3-linker peptide 1-CD137L4 (SEQ IDN0) .45)
• 154 is Tumstatin1-linking peptide 5-CD137L4 (SEQ ID N0.46)
• 254" is Tumstatin2-linked peptide 5-CD137L4

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