WO2017020686A1 - 融合蛋白ifn-elp及其应用 - Google Patents

融合蛋白ifn-elp及其应用 Download PDF

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WO2017020686A1
WO2017020686A1 PCT/CN2016/089739 CN2016089739W WO2017020686A1 WO 2017020686 A1 WO2017020686 A1 WO 2017020686A1 CN 2016089739 W CN2016089739 W CN 2016089739W WO 2017020686 A1 WO2017020686 A1 WO 2017020686A1
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sequence
polypeptide
elastin
single copy
protein
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French (fr)
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高卫平
胡瑾
王贵林
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清华大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to the fusion protein IFN-ELP and its application.
  • Interferon- ⁇ 2 has antiviral replication, anti-tumor proliferation and immunomodulatory effects, and has been successfully used to treat viral diseases (such as hepatitis B, hepatitis C, genital warts, etc.) and related cancers (such as Leukemia, kidney cancer, malignant melanoma, multiple sclerosis, etc.).
  • viral diseases such as hepatitis B, hepatitis C, genital warts, etc.
  • related cancers such as Leukemia, kidney cancer, malignant melanoma, multiple sclerosis, etc.
  • IFN is easily degraded by in vivo proteases and renal excretion after systemic injection, and the circulating half-life is very short, requiring frequent administration to maintain a high blood concentration, resulting in serious toxic side effects and giving patients a heavy economy. burden.
  • Modification of IFN- ⁇ 2 with polyethylene glycol (PEG) can effectively improve its pharmacokinetics, improve drug distribution, and improve its efficacy.
  • PEG polyethylene glycol
  • the current pegylated interferon has disadvantages such as low reaction yield, difficulty in controlling the binding site and coupling stoichiometry, and severely reduced biological activity.
  • the fusion of human serum albumin can effectively prolong the circulating half-life of interferon and effectively control the modification site, but the activity remains only 1%, and the clinical trial effect is not obvious. Therefore, the development of a site-specific modification method with mild reaction conditions, simple steps, rapid and high efficiency is particularly important for interferon and other medicinal proteins.
  • the technical problem to be solved by the present invention is how to prolong the half-life of the protein drug in the living body and/or increase the activity retention rate.
  • the present invention first provides a fusion protein which is a protein of the following b1) or b2):
  • B1) a fusion protein comprising a functional protein and an elastin-like polypeptide; b2) a tagged protein obtained by ligating the N-terminus or C-terminus of b1).
  • the elastin-like polypeptide is an elastin-like polypeptide A or an elastin-like polypeptide B.
  • the elastin-like polypeptide A can be any of the following a1)-a8): a1) a polypeptide having more than one single copy of B, each single copy of B having more than one single copy A; said single copy A
  • the amino acid sequence is XGVPG, X represents any amino acid residue other than a proline residue; a2) in the a1), each single copy B has 10 of the single copy A; a3) the a2)
  • the single copy B is as shown in positions 202 to 251 of Sequence Listing 2; a4) in the a1), the elastin-like polypeptide A has 9 said single copy B; a5) In a4), each single copy B has 10 of the single copy A; a6) of the a5), the single copy B is as shown in the 202th to the 251th of Sequence Listing 2; a7 In the a6), the elastin-like polypeptide A is shown in positions 202 to 651 of Sequence Listing 2; a8) in the
  • the elastin-like polypeptide B may be any of the following a9)-a15): a9) a polypeptide having more than one single copy of butyl, each single copy of which has more than one single copy of C; the single copy The amino acid sequence of C is VPGVG; a10) in the a9), there are 10 such singles in each single copy Copying a; a11) in a10), the single copy is shown as position 202 to position 251 of sequence 5 of the sequence listing; a12) a9), the elastin-like polypeptide B has 9 a single copy of the single copy; a13) in the a12), each single copy of the single copy of 10 of the single copy of the single; a14) of the a13), the single copy of the single sequence of the sequence of the sequence of Positioned to position 251; a15) in a14), the elastin-like polypeptide B is shown in positions 202 to 651 of Sequence Listing 5.
  • any of the above functional proteins may be any one of the following c1)-c20): c1) interferon- ⁇ 2; c2) interferon- ⁇ 2 shown in position 1 to position 166 of sequence 2 of the sequence listing; C3) interferon- ⁇ ; c4) interferon; c5) glucagon-like peptide-1 and its derivatives; c6) hirudin; c7) insulin; c8) monoclonal antibody; c9) blood factor; c10) growth Hormone; c11) interleukin; c12) growth factor; c13) therapeutic vaccine; c14) calcitonin; c15) tumor necrosis factor; c16) enzyme; c17) selected from proteins related to medicine, agriculture, scientific research and other industrial fields, Small peptide or antibody; c18) colony stimulating factor; c19) leptin; c20) c1)-c19) any of said functional proteins are substituted and/or deleted and/or added by one or several amino acid residues
  • any of the above functional proteins include, but are not limited to, asparaginase, glutamate, arginase, arginine deaminase, adenosine deaminase ribonuclease, cytosine deamination Enzyme, trypsin, chymotrypsin, papain, epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF) Bone morphogenetic protein (BMP), fibroblast growth factor, somatostatin, growth hormone, somatostatin, parathyroid hormone, coagulation factor, tumor necrosis factor, gastrointestinal peptide, vasoactive intestinal peptide (VIP) Incretin peptide (CCK), gastrin, secretin, erythropoietin, hormone, vasopressin, octreotide, pancreatic enzyme, superoxide dismutas
  • the positional relationship between the elastin-like polypeptide and the functional protein may be as follows d1), d2) or d3): d1) the elastin-like polypeptide is located upstream of the functional protein Or downstream; d2) the elastin-like polypeptide is fused to the C-terminus or the N-terminus of the functional protein; d3) the elastin-like polypeptide is inserted into the functional protein; the insertion site of the elastin-like polypeptide may It is away from the position of the active site of the functional protein or does not interfere with the position of the active site of the functional protein.
  • amino acid sequence of the fusion protein is shown in positions 1 to 651 of Sequence 2 of Sequence Listing or from position 1 to position 653 of Sequence 2 of Sequence Listing.
  • any of the above-described fusion proteins in the preparation of a product is also within the scope of the invention; the function of the product is to inhibit tumor cell proliferation and/or treat tumors.
  • the inhibition of tumor cell proliferation may specifically inhibit proliferation of human Burkitt's B lymphoma cells and/or human ovarian cancer cells (OVCAR-3).
  • the therapeutic tumor can be a tumor that is caused by human Burkitt's B lymphoma cells and/or human ovarian cancer cells (OVCAR-3).
  • the invention also provides a product, the active ingredient of which is any of the fusion proteins described above.
  • the product can be a drug.
  • nucleic acid molecules encoding any of the fusion proteins described above are also within the scope of the invention.
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • nucleotide sequence encoding the interferon- ⁇ 2 can be as shown in positions 5209 to 5706 of Sequence 1 of the Sequence Listing; the nucleotide sequence encoding the elastin-like polypeptide can be as in the sequence listing. From positions 5812 to 7161 of Sequence 1, or from positions 5812 to 7167 of Sequence 1 of Sequence Listing or from positions 5812 to 5961 of Sequence 1 of Sequence Listing.
  • the nucleic acid molecule may be a DNA molecule as shown in any one of e1) to e10): the nucleotide sequence of e1) comprises a DNA molecule shown in positions 5812 to 7161 of Sequence 1 of the Sequence Listing; The e2) nucleotide sequence comprises the DNA molecule shown in positions 5812 to 7167 of Sequence 1 of the Sequence Listing; the nucleotide sequence of e3) comprises the DNA molecule shown in positions 5812 to 5961 of Sequence 1 of the Sequence Listing; E4) The nucleotide sequence contains the nucleotides 5209 to 5706 of Sequence 1 of the Sequence Listing and the 5812th to 7161th positions of Sequence 1 of the Sequence Listing; e5) The nucleotide sequence contains Sequence 1 of the Sequence Listing The DNA molecules shown in positions 5209 to 5706 and positions 5812 to 7167 of Sequence Listing 1; the nucleotide sequence of e6) contains
  • the DNA molecule shown in positions 5812 to 5961; the nucleotide sequence of e7) may be the DNA molecule shown in positions 5209 to 7161 of Sequence 1 of the Sequence Listing; e8) the nucleotide sequence may be as in the sequence listing
  • the DNA molecule shown in positions 5209 to 7167 of sequence 1; e9) has 75% or more identity with the nucleotide sequence defined in any of e1)-e8)
  • a DNA molecule encoding the fusion protein; e10) hybridizes under stringent conditions to a nucleotide sequence defined by any of e1) to e8), and encodes a DNA molecule of the fusion protein.
  • An expression cassette, expression vector, recombinant microorganism or transgenic cell line containing any of the above nucleic acid molecules is also within the scope of the present invention.
  • the present invention also provides a method of preparing a protein drug having an increased half-life and/or an increased bioactivity retention.
  • the invention provides a method for preparing a protein drug with increased half-life and/or increased bioactivity retention, comprising the steps of: introducing an elastin-like polypeptide into a functional protein to obtain a protein drug having an increased half-life and/or an increase in biological activity retention.
  • the elastin-like polypeptide may be fused to the C-terminus or the N-terminus of the functional protein, or the elastin-like polypeptide is located upstream or downstream of the functional protein, or the elastin-like polypeptide is inserted into the functional protein
  • the insertion site of the elastin-like polypeptide may be located away from the active site of the functional protein or may not interfere with the functional protein The location of the active site.
  • the elastin-like polypeptide is an elastin-like polypeptide A or an elastin-like polypeptide B.
  • the elastin-like polypeptide A may be any one of the following a1)-a8): a1) a polypeptide having more than one single copy of B, each single copy B having more than one single copy A; the single copy
  • the amino acid sequence of A is XGVPG, X represents any amino acid residue other than a proline; a2) in the a1), there are 10 such single copy A in each single copy B; a3) in the a2) , the single copy B is shown in positions 202 to 251 of Sequence Listing 2; a4) in the a1), the elastin-like polypeptide A has 9 of the single copy B; a5) In a4), each single copy B has 10 of the single copy A; a6) in the a5), the single copy B is as shown in the 202th to the 251th of Sequence Listing 2; a7) In the a6), the elastin-like polypeptide A is shown in position 1 to position 651 of Sequence Listing 2; a8)
  • the elastin-like polypeptide B may be any of the following a9)-a15): a9) a polypeptide having more than one single copy of butyl, each single copy of which has more than one single copy of C; the single copy
  • the amino acid sequence of C is VPGVG; a10) in the a9), each of the single copy butyl has 10 of the single copy of C; a11) of the a10), the single copy of the sequence of the sequence 5 202 to 251; a12) in the a9), the elastin-like polypeptide B has 9 of the single-copy butyl; a13) the a12), 10 of each single-copy butyl In the single copy C; a14) in the a13), the single copy butyl is as shown in positions 202 to 251 of Sequence Listing 5; a15) in the a14), the elastin-like polypeptide B As shown in sequence block sequence 5 from 202 to 651.
  • the functional protein is any one of the following c1)-c20): c1) interferon- ⁇ 2; c2) interferon- ⁇ 2 shown in position 1 to position 166 of sequence 2 of the sequence listing; C3) interferon- ⁇ ; c4) interferon; c5) glucagon-like peptide-1 and its derivatives; c6) hirudin; c7) insulin; c8) monoclonal antibody; c9) blood factor; c10) growth Hormone; c11) interleukin; c12) growth factor; c13) therapeutic vaccine; c14) calcitonin; c15) tumor necrosis factor; c16) enzyme; c17) selected from proteins related to medicine, agriculture, scientific research and other industrial fields, Small peptide or antibody; c18) colony stimulating factor; c19) leptin; c20) c1)-c19) any of said functional proteins are substituted and/or deleted and/or added by one or several amino acid residues
  • interferons may be interferon- ⁇ , interferon- ⁇ , interferon- ⁇ or interferon- ⁇ .
  • any of the above functional proteins include, but are not limited to, asparaginase, glutamate, arginase, arginine deaminase, adenosine deaminase ribonuclease, cytosine deamination Enzyme, trypsin, chymotrypsin, papain, epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF) Bone morphogenetic protein (BMP), fibroblast growth factor, somatostatin, growth hormone, somatostatin, parathyroid hormone, coagulation factor, tumor necrosis factor, gastrointestinal peptide, vasoactive intestinal peptide (VIP) Incretin peptide (CCK), gastrin, secretin, erythropoietin, hormone, vasopressin, octreotide, pancreatic enzyme, superoxide dismutas
  • phase transition temperature of any of the above elastin-like polypeptides may be higher than the temperature in the human body (eg, the phase transition temperature of the elastin-like polypeptide A is 47.8 ° C), or may be lower than the temperature in the human body (eg, elastin-like polypeptide B) The phase transition temperature is less than 35 ° C).
  • the fusion protein of any of the above may be higher than the temperature in the human body (as in the embodiment of the present invention, the phase transition temperature of IFN-ELP is 45.3 ° C), which remains in a dissolved state in the human body, effectively increases the molecular weight of the drug, and prolongs its half-life in vivo.
  • systemic treatment intravenous, subcutaneous, intramuscular, intraperitoneal, etc.
  • the fusion protein of any of the above may also be lower than the temperature in the human body (for example, less than 35 ° C), and aggregates in the human body to form a gel-like structure, and the "storage" is slowly released in the human body, and can be used for local treatment (such as the lesion site). Medicine) and systemic treatment (such as subcutaneous, intramuscular, intraperitoneal administration, etc.).
  • the body temperature is 35 to 38 °C.
  • the present invention first provides a fusion molecule which is a fusion molecule containing a functional molecule and the elastin-like polypeptide; the functional molecule is a polynucleotide or a nucleic acid aptamer or other organism molecule.
  • the polynucleotide includes a natural or artificially synthesized, single-stranded or double-stranded polynucleotide or oligonucleotide, and specific examples include, but are not limited to, antisense oligonucleotides, siRNA, anti-miR (target) Genes such as Bcl-2, V2R, EphA2, caveolin 1, TNF- ⁇ , MIF, GFPRAF-1, C-RAF, luciferase, vascular endothelial growth factor, SCV, FAS, INS2, Caspase-8 or HBsAg) .
  • antisense oligonucleotides such as Bcl-2, V2R, EphA2, caveolin 1, TNF- ⁇ , MIF, GFPRAF-1, C-RAF, luciferase, vascular endothelial growth factor, SCV, FAS, INS2, Caspase-8 or HBsAg
  • the nucleic acid aptamers include, but are not limited to, vascular endothelial growth factor (VEGF) aptamer, ricin toxin, cichler protein aptamer, thrombin aptamer, activated plasma protein C adapter , HIV-1 reverse transcriptase aptamer, HIV-1 integrase aptamer, protein kinase C aptamer, human neutrophil elastase aptamer, L-selectin aptamer, P-selectin Aptamer, Yersinia protein tyrosine phosphatase aptamer, phospholipase A2 angiogenin aptamer, rhinovirus capsid protein aptamer, and the like.
  • VEGF vascular endothelial growth factor
  • the other biomolecules include, but are not limited to, oxytocin, vasopressin, adrenocorticotropic hormone, prolactin, luteinizing hormone releasing hormone, luteinizing hormone releasing hormone, growth hormone releasing factor, somatostatin, Glucagon, gastrin, pentagastrin, tetrapeptide gastrin, enkephalin, endorphin, angiotensin, renin, bradykinin, bacitracin, polymyxin, slime , bacillus tyrosin, gramicidin and synthetic analogs, variants and pharmacologically active fragments, monoclonal antibodies and soluble vaccines, and the like.
  • the fusion protein IFN-ELP disclosed in the present invention can increase the biological activity retention rate, prolong the half-life in vivo, prolong the average residence time in vivo, and effectively inhibit tumors.
  • this hair A method for preparing a protein drug with increased half-life and/or increased bioactivity retention is a novel method for modifying protein drugs to improve drug stability, improve pharmacokinetics, and enhance therapeutic efficacy.
  • FIG. 1 is a schematic diagram of an inverse transition cycling (ITC) process.
  • FIG. 2 shows the IFN-ELP obtained by ITC purification.
  • Figure 3 shows the ELP synthesis and purification process.
  • Figure 4 shows the IFN synthesis and purification process.
  • Figure 5 is a MALDI-TOF analysis of the molecular weight of IFN-ELP and IFN.
  • Figure 6 is a graph showing the hydration radius of IFN-ELP and IFN by DLS.
  • Figure 7 shows the phase transition temperature measurement of IFN-ELP and ELP by a microplate reader.
  • Figure 8 is a CD analysis of the secondary structure of IFN-ELP and IFN.
  • Figure 9 shows the in vitro biological activity of IFN-ELP and IFN by MTT assay.
  • Figure 10 is a graph showing the pharmacokinetic parameters of IFN-ELP and IFN using the atrioventricular elimination model in DAS software.
  • Figure 11 shows the distribution of IFN-ELP and IFN in tissues.
  • Figure 12 shows inhibition of tumor growth by IFN-ELP, ELP and IFN.
  • Figure 13 is a survival curve of nude mice injected with IFN-ELP, ELP, IFN or physiological saline.
  • Figure 14 is a graph showing tumor growth after injection of IFN-ELP, ELP, IFN or physiological saline in nude mice.
  • Figure 15 shows changes in body weight after injection of IFN-ELP, ELP, IFN or saline in nude mice.
  • Figure 16 shows the changes of physiological indexes such as lactate dehydrogenase, creatine kinase isoenzyme, alanine aminotransferase, aspartate aminotransferase, creatinine and urea nitrogen after injection of IFN-ELP, ELP, IFN or normal saline in nude mice.
  • physiological indexes such as lactate dehydrogenase, creatine kinase isoenzyme, alanine aminotransferase, aspartate aminotransferase, creatinine and urea nitrogen after injection of IFN-ELP, ELP, IFN or normal saline in nude mice.
  • Figure 17 shows the changes in physiological indexes of red blood cells, white blood cells, hemoglobin and platelets after injection of IFN-ELP, ELP, IFN or normal saline in nude mice.
  • the following examples are provided to facilitate a better understanding of the invention but are not intended to limit the invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. For the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged.
  • the present invention is further described in detail with reference to the preferred embodiments thereof.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • Plasmid pET-25b(+) is a product of Bio-Bioengineering (Shanghai) Co., Ltd. Human Burkitt's B lymphoma cells and human ovarian cancer cells (OVCAR-3) were purchased from the Chinese Academy of Sciences tumor cell bank. RMPI-1640 medium is a product of Gibco. Male SD rats are products of Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.; male SD rats are hereinafter referred to as rats. Female athymic (Nude) nude mice Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. products. Female athymic (Nude) nude mice are referred to hereinafter as nude mice.
  • the TB medium in the following examples was configured as follows: 12 g of peptone, 24 g of yeast extract, and 4 mL of glycerin were added to 900 mL of water, fully dissolved and autoclaved at 121 ° C for 15 min, and the mixture after sterilization was cooled to 60 ° C. Then 100 mL of a sterilized aqueous solution containing 170 mmol/L of KH 2 PO 4 and 0.72 mol/L of K 2 HPO 4 was added.
  • purified IFN-ELP was obtained by inversion transition cycling (ITC) purification. Characterization of molecular weight, phase transition temperature and hydration radius of IFN-ELP and IFN by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), microplate reader, dynamic light scattering (DLS) and circular dichroism (CD) And physical and chemical properties such as secondary structure.
  • ITC inversion transition cycling
  • Human Burkitt's B lymphoma cells were used to test the in vitro biological activity of IFN-ELP and IFN, ie their ability to resist tumor cell proliferation in vitro; using a rat model to test the pharmacokinetics of IFN-ELP and IFN in vivo, using DAS 3.0 Pharmacokinetic analysis software calculates pharmacokinetic parameters; established a nude mouse tumor model to test the anti-tumor effect and drug distribution of IFN-ELP and IFN.
  • the recombinant plasmid pET-25b-IFN-ELP (double-stranded circular plasmid) was artificially synthesized.
  • the nucleotide sequence of the recombinant plasmid pET-25b-IFN-ELP is shown in Sequence Listing 1 of the Sequence Listing.
  • the recombinant plasmid pET-25b-IFN-ELP has an expression cassette A.
  • the nucleotide sequence of expression cassette A is shown in the 5th end of the sequence of sequence 1 from positions 5121 to 7314, wherein the 5th to the 5th end are the T7 promoter, and the 5209 to 5706 are interferon-
  • the coding gene of ⁇ 2 is the recognition region of transpeptidase A
  • the 5812 to 7161 is the coding gene of elastin-like polypeptide
  • the 7265 to 7314 is the T7 terminator.
  • Interferon- ⁇ 2 is hereinafter referred to as IFN.
  • the elastin-like polypeptide is hereinafter referred to as ELP.
  • the protein expressed by expression cassette A is IFN-ELP, hereinafter referred to as IFN-ELP.
  • the amino acid sequence of IFN is shown in positions 1 to 166 of Sequence 2 of the Sequence Listing.
  • the amino acid sequence of ELP is shown in positions 202 to 651 of Sequence 2 of the Sequence Listing.
  • the amino acid sequence of IFN-ELP is shown as position 1 to position 651 of Sequence 2 of the Sequence Listing.
  • the DNA between the NdeI recognition sequence of pET-25b(+) and the EcoRI recognition sequence was replaced with the double-stranded DNA molecule shown in SEQ ID NO: 3 of the sequence listing, and the other sequences of pET-25b(+) were kept unchanged, and the recombinant plasmid pET was obtained.
  • -25b-Sortase The recombinant plasmid pET-25b-Sortase expressed the transpeptidase Sortase shown in Sequence 4 of the Sequence Listing.
  • the transpeptidase Sortase is hereinafter referred to as SrtA.
  • the recombinant plasmid pET-25b-IFN-ELP was introduced into Escherichia coli competent Rosetta-gami (DE3) pLysS (product of Novagen) to obtain recombinant Escherichia coli containing pET-25b-IFN-ELP, and the recombinant Escherichia coli was named Rosetta. /pET-25b-IFN-ELP.
  • ELP-ELP The expression and purification of ELP-ELP was carried out as follows:
  • the Rosetta/pET-25b-IFN-ELP monoclonal was inoculated into 50 mL of TB medium containing 100 ⁇ g/mL ampicillin, cultured at 37 ° C, shaking at 250 rpm for 8 hours to obtain culture broth 1; : 20 (volume ratio) was inoculated into 1 L of TB medium containing 100 ⁇ g / mL ampicillin, and cultured at 200 rpm for 5 h to obtain culture liquid 2; IPTG was added to the culture liquid 2 to make the concentration of IPTG in the system 0.4.
  • the culture liquid 3 was obtained by shaking at 18 ° C and shaking at 200 rpm for 16 hours.
  • the culture broth 3 obtained in the above step (1) was collected in a centrifuge bottle, centrifuged at 3000 g for 5 min, and the cells were collected to obtain the bacterium 1.
  • the cells obtained in the step (2) were resuspended in ice-cold pH 7.4, 10 mM PBS buffer, and sonicated at 4 ° C (ultrasonic power 50 W, ultrasonic time 30 min, 5 S broken 10 S gap) to obtain a bacterial cell disrupted solution.
  • ITC Inverse transition cycling
  • ITC Inverse transition cycling
  • Supernatant 4 contained IFN-ELP, hence the name IFN-ELP solution.
  • the supernatant 4 was subjected to SDS-PAGE to determine the purity of IFN-ELP.
  • concentration of IFN-ELP in supernatant 4 was determined using a NanoDrop 2000 spectrophotometer.
  • the principle of purification of Inverse transition cycling (ITC) is shown in Figure 1.
  • Fig. 2 The expression and purification of IFN-ELP are shown in Fig. 2 (Fig. 2, 1 is the bacterial cell disruption of step (3), 2 is the supernatant A of step (6), and 3 is the precipitate of step (6).
  • the supernatant B, 5 of the step (7) is the precipitate 2, 6 of the step (7), which is a protein standard.
  • step (4) of step 1 the supernatant is obtained, 6.
  • the solution was removed from the column by a desalting column (HiPrep 26/10 Desalting), and then replaced with a pH 7.4, 50 mM Tris ⁇ HCl buffer to obtain a SrtA solution (ie, pH 7.4, 50 mM containing SrtA). Tris ⁇ HCl buffer).
  • the SrtA solution was subjected to SDS-PAGE to determine the purity of SrtA.
  • the concentration of SrtA in the above purified sample was measured using a NanoDrop 2000 spectrophotometer.
  • step 2 the mixed solution 2 was purified by anion exchange chromatography (HiTrap Capto Q 5 mL) on an AKTA protein purification system (AKTA Purifier 10, GE product) to obtain an ELP solution.
  • anion exchange chromatography HiTrap Capto Q 5 mL
  • AKTA Purifier 10 GE product
  • Anion exchange chromatography column is HiTrap Capto Q 5mL; first elute the column with equilibration buffer (pH 4.5, 20 mM acetic acid solution), then load 10mL mixture 2, wash with eluent De-extraction process: A solution is pH 4.5, 20 mM acetic acid aqueous solution, B solution is 1 M NaCl aqueous solution, and the eluent is composed of liquid A and liquid B, and linear washing is carried out using mobile phase B gradually increasing and mobile phase A gradually decreasing.
  • the conditions for removal are as follows: within 0-10 min, the volume percentage of liquid A in the mobile phase is reduced from 100% to 0% at a constant rate.
  • the detection wavelength is 280 nm.
  • the elution volume of 60 mL-65 mL of the post-column solution is collected, ie It was an ELP solution, and was replaced with a pH 7.4, 50 mM Tris ⁇ HCl buffer containing 150 mM NaCl through a HiPrep 26/10 Desalting Desalting Column (product of GE Corporation).
  • step 2 the mixed solution 2 was purified by anion exchange chromatography (HiTrap Capto Q 5 mL) on an AKTA protein purification system (AKTA Purifier 10, GE product) to obtain an IFN solution.
  • Anion exchange chromatography column is HiTrap Capto Q 5 mL; first elute the column with equilibration buffer (pH 7.4, 20 mM Tris ⁇ HCl), then load 10 mL of the mixture 2, and eluate Elution (elution process: liquid A is pH 7.4, 20 mM Tris ⁇ HCl aqueous solution, liquid B is 1M NaCl aqueous solution, eluent consists of liquid A and liquid B, using mobile phase B gradually increasing, mobile phase A gradually decreasing
  • the linear elution conditions are as follows: within 0-10 min, the volume percentage of liquid A in the mobile phase is reduced from 100% to 0%.
  • the detection wavelength is 280 nm.
  • the column is collected.
  • the solution which is an IFN solution, was replaced with a pH 7.4, 50 mM Tris ⁇ HCl buffer containing 150 mM NaCl through a HiPrep 26/10 Desalting Desalting Column (product of GE Corporation).
  • Figure 3 (A in Figure 3 is a chromatogram of the ELP purified from the mixture 2 using an anion exchange column, wherein UV monitoring linearly elutes to obtain the target product ELP; in Figure 3, B is the result of SDS-PAGE analysis in the process of purifying the ELP, 1 is a protein standard, 2 is a mixed solution 1, 3 is a mixed solution 2, 4 is a flow-through solution, 5 is an eluted volume of 43 mL-50 mL to obtain an eluted protein IFN, and 6 is a collection elution volume of 52 mL-58 mL.
  • the obtained eluted proteins IFN-ELP and IFN 7 is an eluted protein ELP obtained by collecting an elution volume of 60 mL-65 mL, and 8 is an eluted protein SrtA obtained by collecting an elution volume of 70 mL-80 mL, and 9 is Step 1 of Example 1.
  • the prepared supernatant 4) is an ELP synthesis and purification process.
  • Figure 4 ( Figure 4 is a chromatogram of the purification of IFN from the mixture 2 using an anion exchange column, wherein UV monitoring linear elution yields the target product IFN;
  • Figure 4 B shows the results of SDS-PAGE analysis in the process of purifying IFN,
  • M is a protein standard
  • 1 is a SrtA solution prepared in the second step of Example 1
  • 2 is a supernatant prepared in the first step of Example 1
  • 3 is a mixed solution
  • 4 is obtained by collecting an elution volume of 0 mL to 40 mL.
  • the flow-through proteins ELP and SrtA, 5 are collected elution volumes of 90 mL-100 mL of the eluted protein IFN) for the synthesis and purification of IFN.
  • MALDI-TOF time of flight mass spectrometry
  • the protein to be tested (IFN-ELP or IFN) was dissolved in pH 7.4, 10 mM PBS buffer, and then filtered through a 0.22 ⁇ m pore size filter to obtain a sample to be tested.
  • Weiping Gao, et al. In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves the sample to be tested on a Malvern Zetasizer Nano ZS90 nanoparticle size potential analyzer (Weiping Gao, et al. Pharmacokinetics and tumor accumulation. Proc Natl Acad Sci USA. 2010 Sep 21; 107(38): 16432-7.)
  • the hydration radius of the protein to be tested (IFN-ELP or IFN) was measured. For the specific measurement steps, refer to the instrument's own manual.
  • the DLS test uses Malvern Zetasizer Nano-zs90. Data processing uses the software Zetasizer software 6.32.
  • the turbidity method is used to determine the phase transition temperature. The specific steps are as follows:
  • the protein to be tested (IFN-ELP or ELP) was dissolved in a pH 7.4, 10 mM PBS buffer to a concentration of 1 mg/mL of the protein to be tested in the system, and then determined by a microplate reader (product of Molecular Devices) at 35- OD 350 nm value in the range of 55 ° C (linearly increasing at 0.5 ° C / min).
  • the temperature at which the OD 350 nm value of the protein to be tested reaches half of the maximum value of the OD 350 value in the above temperature range is the phase transition temperature.
  • the secondary structure of the protein to be tested (IFN-ELP or IFN) is determined by circular dichroism chromatography. The specific steps are as follows:
  • the protein to be tested (IFN-ELP or IFN) was dissolved in water to a concentration of 0.18 mg/mL in the system, and then subjected to ultraviolet scanning analysis using a circular dichroism spectrometer (Applied Photophysics Co., Ltd.) in the wavelength range of 200-260 nm. .
  • the in vitro biological activity of the protein to be tested was determined by the MTT method. Specific steps are as follows:
  • Daudi B Human Burkitt's B lymphoma cells (abbreviated as Daudi B) were cultured in RMPI-1640 medium containing 10% (by volume) FBS, 50 U/mL penicillin and 50 ⁇ g/mL streptomycin to obtain a cell suspension.
  • the protein to be tested is diluted with RMPI-1640 medium containing 10% (by volume) FBS, 50 U/mL penicillin and 50 ⁇ g/mL streptomycin to obtain protein solutions of different concentrations.
  • step 3 Take a 96-well cell culture plate, add 50 ⁇ L of the cell suspension obtained in step 1 (about 10 4 cells per well), and then add the protein solution to be tested in step 2 (50 ⁇ L per well) so that the protein to be tested is
  • the concentration in the well is 1, 2 , 5, 10, 20, 50, 100, 1000 or 10000 pg/mL), 5% CO 2 , 37 ° C for 72 h, then MTT solution (Promega company), 3 h,
  • MTT solution Promega company
  • Positive control wells were placed in 96-well cell culture plates, and 50 ⁇ L of the cell suspension obtained in step 1 and 50 ⁇ L of RMPI containing 10% (by volume) FBS, 50 U/mL penicillin and 50 ⁇ g/mL streptomycin were added to each positive control well.
  • -1640 medium Negative control wells were placed in 96-well cell culture plates, and 100 ⁇ L of RMPI-1640 medium containing 10% (by volume) FBS, 50 U/mL penicillin, and 50 ⁇ g/mL streptomycin was added to each negative control well. The absorbance at 490 nm of the negative control well and the positive control well was measured using a microplate reader.
  • the concentration of the protein to be tested in the well is the abscissa, and the relative activity is the ordinate, and the protein to be tested is compared.
  • the relative activity is calculated according to the following formula:
  • the experimental results are shown in Table 1 and Figure 9.
  • the results showed that the semi-inhibitory concentration (IC50) of IFN-ELP was 32.82 pg/mL, and the relative activity was 41%; the semi-inhibitory concentration (IC50) of IFN was 13.51 pg/mL, and the relative activity was 100%.
  • the results showed that IFN was not significantly reduced in IFN in vitro by ELP modification.
  • IFN group IFN was injected into the tail vein at a dose of 125 ⁇ g/kg body weight
  • IFN-ELP group IFN-ELP was injected into the tail vein at a dose of 125 ⁇ g/kg body weight.
  • step 2 After the injection in step 1 is completed, the timing is started, and the treatment time is different (the time points of the IFN-ELP group are 1 min, 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 24 h, 48 h, 72 h, 96 h, and the time point of the IFN group is Male SD rats after 1 min, 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 24 h) were anesthetized with isoflurane, and then 0.3 mL of blood was collected through the intraocular iliac vein collection tube (the collection tube was heparin sodium in advance) Jiangsu Wanbang Biochemical Pharmaceutical Co., Ltd. product) infiltrated and dried), allowed to stand at room temperature for 1 h, collected at 4 ° C, 3000 ⁇ g, and stored in the low temperature refrigerator at -80 ° C.
  • the time points of the IFN-ELP group are 1 min, 5 min, 15 min,
  • step 2 the IFN concentration in the plasma of each male SD rat in step 2 was measured using a human IFN- ⁇ 2 ELISA kit (product of PBL interferon source). The measurement methods and procedures are carried out according to the instructions.
  • the pharmacokinetic parameters of IFN-ELP and IFN were analyzed by the atrioventricular elimination model in DAS software. The results showed (Table 2 and Figure 10): 1. The half-life of IFN was 0.3 h, and the blood interference occurred after a few minutes of administration. The concentration of the hormone rapidly decreased to less than 50% of the initial dose. After 24 hours of administration, the residual concentration of interferon was less than 0.1% of the initial concentration of interferon; the half-life of IFN-ELP was 8.57 h, and the concentration of interferon in the blood was gradually After 72 hours of administration, the interferon residual concentration was still more than 10% of the initial concentration of interferon. 2.
  • the average in vivo residence time of IFN-ELP is 11.3 times the average residence time of IFN (MRT0- ⁇ ). 3.
  • the drug-time curve area (AUC) of IFN-ELP is 32.9 times the drug-time curve area (AUC) of IFN. It showed that compared with IFN, the half-life and average residence time of IFN-ELP were significantly prolonged, the curve area of the drug was significantly increased, and the bioavailability was significantly improved.
  • Human ovarian cancer cells (OVCAR-3) cultured in RMPI-1640 medium containing 10% FBS, 50 U/mL penicillin and 50 g/mL streptomycin were trypsinized and stripped by pH 7.4, 10 mM. After washing with PBS buffer, the mixture 1 obtained in the step 1 was resuspended to obtain a resuspension.
  • the resuspension obtained in step 2 was inoculated subcutaneously with the back of the femur at the left hind limb of 18 nude mice.
  • the inoculum was 0.2 mL (about 5 ⁇ 10 6 cells) per resuscitation, and 6 nude mice after 30 days. Each formed a solid tumor mass of about 200 mm 3 in size.
  • Six nude mice were randomly divided into an IFN group and an IFN-ELP group, 9 in each group.
  • the IFN-ELP was injected into the tail vein of the nude mice in the IFN-ELP group in step 4, and the IFN group was injected intravenously into the tail vein of nude mice.
  • the injected dose was 10 ⁇ g / 20 g body weight.
  • nude mice treated with IFN-ELP or IFN for different time (2h, 6h, or 24h) were sacrificed by cervical dislocation, collecting heart, kidney, liver, spleen, lung, pancreas, stomach, muscle. Tissues such as small intestine, plasma and tumors.
  • tissue organs herein, kidney, liver, spleen, lung, pancreas, stomach, muscle, small intestine, plasma or tumor
  • the solute of the extraction buffer and its concentration were: 1 mM EDTA, 0.5% Triton X-100, 0.5% sodium deoxycholate, 1 mM PMSF, 1% by volume protease inhibitor mixture (product of Sigma-Aldrich) and 1% Phosphatase inhibitor cocktail (product of Sigma-Aldrich); solvent was pH 7.4, 10 mM PBS buffer.
  • step 6 Quantitatively determine the concentration of IFN in the supernatant extract in step 6 using a human IFN- ⁇ 2 ELISA kit (product of PBL interferon source), and measure the method and procedure according to the instructions, and further obtain the organ of step 5 (heart, kidney). The concentration of IFN in liver, spleen, lung, pancreas, stomach, muscle, small intestine, plasma or tumor.
  • IFN-ELP group was injected with IFN-ELP for different time (2h, 6h, or 24h), and IFN was in heart, kidney, liver, spleen, lung, pancreas, stomach, muscle, small intestine. It can be effectively accumulated in plasma and tumors.
  • the concentration of interferon in the tumor of IFN-ELP group was 29 times higher than that of IFN group tumor, 2 times of injection 2h, 66 times of injection 6h, and 21 times of injection 24h. It shows that IFN-ELP can effectively enhance the permeability and retention effect, make interferon accumulate in tumor tissue, prolong the half-life in blood circulation, thereby improving the bioavailability and anti-tumor efficacy of interferon in vivo.
  • the resuspension prepared in steps 6 and 2 in Example 6 was inoculated into the back of the femur of the left hind limb of 26 nude mice, and the inoculum was 0.2 mL (about 5 ⁇ 10 6 cells)/only. After 30 days, 26 nude mice formed solid tumor masses of approximately 40 mm 3 in size.
  • mice Twenty-six nude mice were randomly divided into four groups: IFN-ELP group (7), IFN group (7), ELP group (6) and saline group (6).
  • the IFN-ELP was injected into the tail vein of the nude mice in the IFN-ELP group in step 2.
  • the nude mice in the IFN group were injected with IFN in the tail vein, and the ELP group was injected with ELP in the tail vein at a dose of 60 ⁇ g/20 g body weight.
  • Normal saline was injected into the tail vein of the saline group in the step 2, and an equal volume of physiological saline was injected.
  • nude mice Each group was injected every three days until all of the nude mice in the saline group, the IFN group, and the ELP group died.
  • the death of nude mice includes natural death and euthanasia.
  • the death of euthanasia refers to the tumor growth of nude mice over 1000mm 3 or weight loss of more than 15%, and was killed by injection of barbiturate.
  • step 3 the body weight and tumor volume of each group of nude mice were measured every three days; blood was taken through the eyeball every 15 days, and lactate dehydrogenase, creatine kinase isoenzyme, alanine aminotransferase, aspartate aminotransferase, Basic physiological indicators such as creatinine, urea nitrogen, red blood cells, white blood cells, platelets, and hemoglobin.
  • the experimental results showed (Fig. 12, Fig. 13 and Fig. 14) that during the experiment, the tumor volume of the nude mice in the saline group and the ELP group gradually increased, and the tumor volume of the nude mice in the saline group and the ELP group on the 39th day after injection exceeded 1000mm 3 , the median survival was only 34.5 days and 36 days; during the experiment, the tumor volume of the nude mice in the IFN group also gradually increased.
  • the tumor volume of the nude mice in the IFN group also exceeded 1000 mm 3 on the 45th day of injection, and the median survival was At 42 days, no significant anti-tumor activity was observed; during the experiment, the tumor volume of the nude mice in the IFN-ELP group did not change substantially, and the median survival was 87 days. It is shown that IFN-ELP can effectively inhibit tumor growth and has very good in vivo antitumor activity.
  • the fusion protein IFN-ELP provided by the invention can be used as a protein drug to increase the biological activity retention rate, prolong the half-life in vivo, prolong the average residence time in vivo, and effectively inhibit tumors. Meanwhile, the present invention provides a method for preparing a protein drug having an increased half-life and/or an increased bioactivity retention rate as a novel method for modifying a protein drug to improve drug stability, improve pharmacokinetics, and enhance therapeutic efficacy.

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Abstract

本发明公开了融合蛋白IFN-ELP，是如下b1)或b2)的蛋白质：b1)包括功能蛋白和弹性蛋白样多肽融合得到的融合蛋白；b2)将b1)的N端或C端连接标签得到的蛋白。本发明还提供了所述融合蛋白在制备抑制肿瘤细胞增殖和/或治疗肿瘤的产品中的应用。

Description

融合蛋白IFN-ELP及其应用 技术领域
本发明属于生物医药领域，具体涉及融合蛋白IFN-ELP及其应用。
背景技术
干扰素-α2(IFN-α2)具有抗病毒复制、抗肿瘤增殖和免疫调节作用，已被成功用于治疗病毒性疾病(如乙型肝炎、丙型肝炎、尖锐湿疣等)和相关癌症(如白血病、肾癌、恶性黑色素瘤、多发性硬化症等)。但是，IFN经系统注射给药后容易被体内蛋白酶降解和肾排出，循环半衰期非常短，需要频繁给药以维持较高的血药浓度，从而导致严重毒副作用，同时给患者带来沉重的经济负担。用聚乙二醇(PEG)修饰IFN-α2，能有效改善其药代动力学，改善药物分布，提高其疗效。然而，目前的PEG化干扰素存在如反应产率低、结合位点和偶联化学计量难以控制、生物活性严重降低等弊端。现有技术中通过融合人血清白蛋白能够有效延长干扰素循环半衰期并有效控制修饰位点，但活性保持仅有1％，且临床试验效果并不明显。因此，研发反应条件温和、步骤简单、快速、高效的位点特异性修饰方法对干扰素及其他药用蛋白质尤为重要。
发明公开
本发明所要解决的技术问题是如何使蛋白质药物在生物体内的半衰期延长和/或活性保持率增加。
为解决上述问题，本发明首先提供了一种融合蛋白，所述融合蛋白是如下b1)或b2)的蛋白质：
b1)含有功能蛋白和弹性蛋白样多肽的融合蛋白；b2)将b1)的N端或C端连接标签得到的带标签蛋白质。
所述弹性蛋白样多肽为弹性蛋白样多肽甲或弹性蛋白样多肽乙。
所述弹性蛋白样多肽甲可为如下a1)—a8)中的任一种：a1)具有一个以上单拷贝乙的多肽，每个单拷贝乙中具有一个以上单拷贝甲；所述单拷贝甲的氨基酸序列为XGVPG，X代表一个脯氨酸残基以外的任意氨基酸残基；a2)所述a1)中，每个单拷贝乙中具有10个所述单拷贝甲；a3)所述a2)中，所述单拷贝乙如序列表序列2的第202位至第251位所示；a4)所述a1)中，所述弹性蛋白样多肽甲具有9个所述单拷贝乙；a5)所述a4)中，每个单拷贝乙中具有10个所述单拷贝甲；a6)所述a5)中，所述单拷贝乙如序列表序列2的第202位至第251位所示；a7)所述a6)中，所述弹性蛋白样多肽甲如序列表序列2的第202位至第651位所示；a8)所述a6)中，所述弹性蛋白样多肽甲如序列表序列2的第202位至第653位所示。
所述弹性蛋白样多肽乙可为如下a9)—a15)中的任一种)：a9)具有一个以上单拷贝丁的多肽，每个单拷贝丁中具有一个以上单拷贝丙；所述单拷贝丙的氨基酸序列为VPGVG；a10)所述a9)中，每个单拷贝丁中具有10个所述单 拷贝丙；a11)所述a10)中，所述单拷贝丁如序列表序列5的第202位至第251位所示；a12)所述a9)中，所述弹性蛋白样多肽乙具有9个所述单拷贝丁；a13)所述a12)中，每个单拷贝丁中具有10个所述单拷贝丙；a14)所述a13)中，所述单拷贝丁如序列表序列5的第202位至第251位所示；a15)所述a14)中，所述弹性蛋白样多肽乙如序列表序列5第202位至第651位所示。
上述任一所述功能蛋白可为如下c1)-c20)中的任一种：c1)干扰素-α2；c2)序列表的序列2第1位至第166位所示的干扰素-α2；c3)干扰素-α；c4)干扰素；c5)胰高血糖素样肽-1及其衍生物；c6)水蛭素；c7)胰岛素；c8)单克隆抗体；c9)血液因子；c10)生长激素；c11)白介素；c12)生长因子；c13)治疗性疫苗；c14)降钙素；c15)肿瘤坏死因子；c16)酶；c17)选自医药、农业、科研以及其它工业领域相关的蛋白、小肽或抗体；c18)集落刺激因子；c19)瘦素；c20)c1)-c19)任一所述功能蛋白经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的功能蛋白。
上述任一所述功能蛋白具体例子包括但不不限于：天冬酰胺酶、谷氨酸酶、精氨酸酶、精氨酸脱氨酶、腺苷脱氨酶核糖核酸酶、胞嘧啶脱氨酶、胰蛋白酶、胰凝乳蛋白酶、木瓜蛋白酶、表皮生长因子(EGF)、胰岛素样生长因子(IGF)、转化生长因子(TGF)、神经生长因子(NGF)、血小板衍生的生长因子(PDGF)、骨形态发生蛋白(BMP)、成纤维细胞生长因子、生长抑制素、生长激素、生长激素抑制素、甲状旁腺激素、凝血因子、肿瘤坏死因素、胃肠肽、血管活性肠肽(VIP)、肠促胰酶肽(CCK)、胃泌素、促胰液素、促红细胞生成素、荷尔蒙、抗利尿激素、奥曲肽、胰腺酶、超氧化物歧化酶、促甲状腺激素释放激素(TRH)、促甲状腺激素、促黄体生成激素、促黄体激素释放激素(LHRH)、组织型纤溶酶原激活剂、白细胞介素-1、白细胞介素-15、受体拮抗剂(IL-1RA)、胰高血糖素样肽-1(GLP-1)、生长素、粒单核细胞集落刺激因子(GM-CSF)、白细胞介素-2(IL-2)、腺苷脱氨酶、尿酸酶、天冬酰胺酶、人生长激素、天冬酰胺酶、巨噬细胞活化、绒毛膜促性腺激素、肝素、心房利钠肽、血红蛋白、逆转录病毒载体、松弛肽、环孢菌素、催产素、疫苗、单链抗体、锚蛋白重复蛋白、亲和体等。
上述任一所述融合蛋白中，所述弹性蛋白样多肽和所述功能蛋白的位置关系可为如下d1)、d2)或d3)：d1)所述弹性蛋白样多肽位于所述功能蛋白的上游或下游；d2)所述弹性蛋白样多肽融合于所述功能蛋白的C末端或N末端；d3)所述弹性蛋白样多肽插入所述功能蛋白中；所述弹性蛋白样多肽的插入位点可为远离所述功能蛋白的活性位点的位置或不干扰所述功能蛋白的活性位点的位置。
上述任一所述融合蛋白中，所述融合蛋白的氨基酸序列如序列表序列2的第1位至第651位或序列表序列2的第1位至第653位所示。
上述任一所述融合蛋白中，所述融合蛋白的氨基酸序列如序列表序列5所示。
上述任一所述融合蛋白在制备产品中的应用也属于本发明的保护范围；所述产品的功能为抑制肿瘤细胞增殖和/或治疗肿瘤。
所述抑制肿瘤细胞增殖具体可为抑制人Burkitt’s B淋巴瘤细胞和/或人卵巢癌细胞(OVCAR-3)增殖。
所述治疗肿瘤可为治疗由人Burkitt’s B淋巴瘤细胞和/或人卵巢癌细胞(OVCAR-3)引起的肿瘤。
本发明还提供了一种产品，所述产品的活性成分为上述任一所述融合蛋白。
所述产品可为药物。
编码上述任一所述融合蛋白的核酸分子也属于本发明的保护范围。
所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
上述核酸分子中，编码所述干扰素-α2的核苷酸序列可如序列表序列1的第5209位至第5706位所示；编码所述弹性蛋白样多肽的核苷酸序列可如序列表序列1的第5812位至第7161位或序列表序列1的第5812位至第7167位或序列表序列1的第5812位至第5961位所示。
上述核酸分子中，所述核酸分子可为如下e1)-e10)任一所示的DNA分子：e1)核苷酸序列含有序列表的序列1第5812位至第7161位所示的DNA分子；e2)核苷酸序列含有序列表的序列1第5812位至第7167位所示的DNA分子；e3)核苷酸序列含有序列表的序列1第5812位至第5961位所示的DNA分子；e4)核苷酸序列含有序列表的序列1第5209位至第5706位和序列表序列1的第5812位至第7161位所示的DNA分子；e5)核苷酸序列含有序列表的序列1第5209位至第5706位和序列表序列1的第5812位至第7167位所示的DNA分子；e6)核苷酸序列含有序列表的序列1第5209位至第5706位和序列表序列1的第5812位至第5961位所示的DNA分子；e7)核苷酸序列可如序列表的序列1第5209位至第7161位所示的DNA分子；e8)核苷酸序列可如序列表的序列1第5209位至第7167位所示的DNA分子；e9)与e1)-e8)中任一限定的核苷酸序列具有75％或75％以上同一性，且编码所述融合蛋白的DNA分子；e10)在严格条件下与e1)-e8)中任一限定的核苷酸序列杂交，且编码所述融合蛋白的DNA分子。
含有上述任一所述核酸分子的表达盒、表达载体、重组微生物或转基因细胞系也属于本发明的保护范围。
本发明还提供了一种制备半衰期延长和/或生物活性保持率增加的蛋白质药物的方法。本发明所提供的制备半衰期延长和/或生物活性保持率增加的蛋白质药物的方法，包括如下步骤：在功能蛋白中引入弹性蛋白样多肽，得到半衰期延长和/或生物活性保持率增加的蛋白质药物；所述弹性蛋白样多肽可融合于所述功能蛋白的C末端或N末端，或所述弹性蛋白样多肽位于所述功能蛋白的上游或下游，或所述弹性蛋白样多肽插入所述功能蛋白中；所述弹性蛋白样多肽的插入位点可为远离所述功能蛋白的活性位点的位置或不干扰所述功能蛋白 的活性位点的位置。
上述方法中，所述弹性蛋白样多肽为弹性蛋白样多肽甲或弹性蛋白样多肽乙。
所述弹性蛋白样多肽甲，可为如下a1)—a8)中的任一种：a1)具有一个以上单拷贝乙的多肽，每个单拷贝乙中具有一个以上单拷贝甲；所述单拷贝甲的氨基酸序列为XGVPG，X代表一个脯氨酸以外的任意氨基酸残基；a2)所述a1)中，每个单拷贝乙中具有10个所述单拷贝甲；a3)所述a2)中，所述单拷贝乙如序列表序列2的第202位至第251位所示；a4)所述a1)中，所述弹性蛋白样多肽甲具有9个所述单拷贝乙；a5)所述a4)中，每个单拷贝乙中具有10个所述单拷贝甲；a6)所述a5)中，所述单拷贝乙如序列表序列2的第202位至第251位所示；a7)所述a6)中，所述弹性蛋白样多肽甲如序列表序列2的第1位至第651位所示；a8)所述a6)中，所述弹性蛋白样多肽甲如序列表序列2的第1位至第653位所示。
所述弹性蛋白样多肽乙可为如下a9)—a15)中的任一种)：a9)具有一个以上单拷贝丁的多肽，每个单拷贝丁中具有一个以上单拷贝丙；所述单拷贝丙的氨基酸序列为VPGVG；a10)所述a9)中，每个单拷贝丁中具有10个所述单拷贝丙；a11)所述a10)中，所述单拷贝丁如序列表序列5的第202位至第251位所示；a12)所述a9)中，所述弹性蛋白样多肽乙具有9个所述单拷贝丁；a13)所述a12)中，每个单拷贝丁中具有10个所述单拷贝丙；a14)所述a13)中，所述单拷贝丁如序列表序列5的第202位至第251位所示；a15)所述a14)中，所述弹性蛋白样多肽乙如序列表序列5第202位至第651位所示。
上述方法中，所述功能蛋白是如下c1)-c20)中的任一种：c1)干扰素-α2；c2)序列表序列2的第1位至第166位所示的干扰素-α2；c3)干扰素-α；c4)干扰素；c5)胰高血糖素样肽-1及其衍生物；c6)水蛭素；c7)胰岛素；c8)单克隆抗体；c9)血液因子；c10)生长激素；c11)白介素；c12)生长因子；c13)治疗性疫苗；c14)降钙素；c15)肿瘤坏死因子；c16)酶；c17)选自医药、农业、科研以及其它工业领域相关的蛋白、小肽或抗体；c18)集落刺激因子；c19)瘦素；c20)c1)-c19)任一所述功能蛋白经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的功能蛋白。
上述任一所述干扰素可为干扰素-α、干扰素-β、干扰素-γ或干扰素-λ。
上述任一所述功能蛋白具体例子包括但不不限于：天冬酰胺酶、谷氨酸酶、精氨酸酶、精氨酸脱氨酶、腺苷脱氨酶核糖核酸酶、胞嘧啶脱氨酶、胰蛋白酶、胰凝乳蛋白酶、木瓜蛋白酶、表皮生长因子(EGF)、胰岛素样生长因子(IGF)、转化生长因子(TGF)、神经生长因子(NGF)、血小板衍生的生长因子(PDGF)、骨形态发生蛋白(BMP)、成纤维细胞生长因子、生长抑制素、生长激素、生长激素抑制素、甲状旁腺激素、凝血因子、肿瘤坏死因素、胃肠肽、血管活性肠肽(VIP)、肠促胰酶肽(CCK)、胃泌素、促胰液素、促红细胞生成素、荷尔蒙、抗利尿激素、奥曲肽、胰腺酶、超氧化物歧化酶、促甲状腺激素释放激素(TRH)、促甲状腺激素、促黄体生成激素、促黄体激素释放激素(LHRH)、组 织型纤溶酶原激活剂、白细胞介素-1、白细胞介素-15、受体拮抗剂(IL-1RA)、胰高血糖素样肽-1(GLP-1)、生长素、粒单核细胞集落刺激因子(GM-CSF)、白细胞介素-2(IL-2)、腺苷脱氨酶、尿酸酶、天冬酰胺酶、人生长激素、天冬酰胺酶、巨噬细胞活化、绒毛膜促性腺激素、肝素、心房利钠肽、血红蛋白、逆转录病毒载体、松弛肽、环孢菌素、催产素、疫苗、单链抗体、锚蛋白重复蛋白、亲和体等。
上述任一所述弹性蛋白样多肽的相变温度可高于人体内温度(如弹性蛋白样多肽甲的相转变温度为47.8℃)，也可以低于人体内温度(如弹性蛋白样多肽乙的相转变温度小于35℃)。
上述任一所述融合蛋白可高于人体内温度(如本发明的实施例中，IFN-ELP的相转变温度为45.3℃)，在人体内保持溶解状态，有效增加药物分子量，延长其体内半衰期，用于系统治疗(静脉、皮下、肌肉、腹腔给药等)。
上述任一所述融合蛋白也可低于人体内温度(如小于35℃)，在人体内聚集沉淀形成类似凝胶结构，“贮存”在人体内缓慢释放，可用于局部治疗(如病灶部位给药)和系统治疗(如皮下、肌肉、腹腔给药等)。
所述人体内温度为35～38℃。
为解决上述问题，本发明首先提供了一种融合分子，所述融合分子为含有功能分子和所述弹性蛋白样多肽的融合分子；所述功能分子为多核苷酸或核酸适配体或其它生物分子。
所述多核苷酸包括天然或人工合成的、单链或双链的多核苷酸或寡聚核苷酸，具体例子包括但不局限于：反义寡核苷酸、siRNA、anti-miR(靶基因如Bcl-2、V2R、EphA2、小窝蛋白1、TNF-α、MIF、GFPRAF-1、C-RAF、荧光素酶、血管内皮生长因子、SCV，FAS、INS2、Caspase-8或HBsAg)。
所述核酸适配体包括但不局限于：血管内皮细胞生长因子(VEGF)适配体、蓖麻毒素适配体、西葫芦毒蛋白适配体、凝血酶适配体、活化血浆蛋白C适配体、HIV-1逆转录酶适配体、HIV-1整合酶适配体、蛋白激酶C适配体、人嗜中性弹性蛋白酶适配体、L-选择素适配体、P-选择素适配体、耶尔森氏菌蛋白酪氨酸磷酸酶适配体、磷脂酶A2体血管生成素适配体、鼻病毒衣壳蛋白适配体等。
所述其它生物分子包括但不局限于：催产素、加压素、促肾上腺皮质激素、催乳激素、促黄体生成激素释放激素、黄体生成素释放激素、生长激素释放因子、促生长素抑制素、胰高血糖素、胃泌素、五肽胃泌素、四肽胃泌素、脑啡肽、内啡肽、血管紧张素、肾素、缓激肽、杆菌肽、多粘菌素、粘菌素、短杆菌酪肽、短杆菌肽及合成的类似物、变型和药理学活性片段物、单克隆抗体和可溶性疫苗等。
工业应用
以本发明公开的融合蛋白IFN-ELP作为蛋白质药物，可使生物活性保持率增加、体内半衰期延长、体内平均存留时间延长，并有效抑制肿瘤。同时，本发 明所提供的一种制备半衰期延长和/或生物活性保持率增加的蛋白质药物的方法为修饰蛋白药物以提高药物稳定性、改善药物代谢动力学和增强治疗功效的新方法。
附图说明
图1为反相转变循环技术(Inverse transition cycling，ITC)流程示意图。
图2显示了通过ITC纯化获得IFN-ELP。
图3为ELP合成及纯化过程。
图4为IFN合成及纯化过程。
图5为MALDI-TOF分析IFN-ELP和IFN的分子量。
图6为DLS分析IFN-ELP和IFN的水合半径。
图7为酶标仪分析IFN-ELP和ELP的相转变温度测定。
图8为CD分析IFN-ELP和IFN的二级结构。
图9为MTT法测定IFN-ELP和IFN的体外生物活性。
图10为利用DAS软件中房室消除模型分析IFN-ELP和IFN的药物代谢动力学参数。
图11为IFN-ELP和IFN在组织中的分布情况。
图12为IFN-ELP、ELP和IFN抑制肿瘤生长情况。
图13为裸鼠注射IFN-ELP、ELP、IFN或生理盐水后的生存曲线。
图14为裸鼠注射IFN-ELP、ELP、IFN或生理盐水后的肿瘤生长实物图。
图15为裸鼠注射IFN-ELP、ELP、IFN或生理盐水后的体重变化情况。
图16为裸鼠注射IFN-ELP、ELP、IFN或生理盐水后乳酸脱氢酶、肌酸激酶同工酶、谷丙转氨酶、谷草转氨酶、肌酐和尿素氮等生理指标变化情况。
图17为裸鼠注射IFN-ELP、ELP、IFN或生理盐水后红细胞、白细胞、血红蛋白和血小板等生理指标变化情况。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
质粒pET-25b(+)为生工生物工程(上海)股份有限公司产品。人Burkitt’s B淋巴瘤细胞和人卵巢癌细胞(OVCAR-3)均购自中国科学院肿瘤细胞库。RMPI-1640培养基为Gibco公司产品。雄性SD大鼠为北京维通利华实验动物技术有限公司产品；雄性SD大鼠在下文中简称大鼠。雌性无胸腺(Nude)裸鼠为 北京维通利华实验动物技术有限公司产品。雌性无胸腺(Nude)裸鼠在下文中简称裸鼠。
下述实施例中的TB培养基按照如下方法配置：向900mL水中加入蛋白胨12g、酵母提取物24g和甘油4mL，充分溶解后121℃高压灭菌15min，灭菌后的混合液冷却至60℃，然后加入100mL灭菌的含170mmol/LKH2PO4和0.72mol/L K2HPO4的水溶液。
下述实施例中，通过反相转变循环技术(Inverse transition cycling,ITC)提纯获得纯化的IFN-ELP。利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF)、酶标仪、动态光散射(DLS)、圆二色谱(CD)等分析手段表征IFN-ELP和IFN的分子量、相变温度、水合半径和二级结构等物理化学性能。选用人Burkitt’s B淋巴瘤细胞，测试IFN-ELP和IFN的体外生物活性，即其体外抗肿瘤细胞增殖的能力；使用大鼠模型，测试IFN-ELP和IFN在体内的药物代谢动力学，利用DAS 3.0药物代谢动力学分析软件计算出药物代谢动力学参数；建立裸鼠肿瘤模型，测试IFN-ELP和IFN的抗肿瘤效果和药物分布。
下述实施例中的定量实验，如无特殊说明均设置三次重复，结果取平均值。
实施例1、IFN-ELP和转肽酶Sortase的原核表达和纯化
一、重组质粒pET-25b-IFN-ELP的制备
人工合成重组质粒pET-25b-IFN-ELP(双链环形质粒)。重组质粒pET-25b-IFN-ELP的核苷酸序列如序列表序列1所示。重组质粒pET-25b-IFN-ELP中具有一个表达盒A。
表达盒A的核苷酸序列如序列表序列1的自5’末端第5121至7314位所示，其中自5’末端第5121至5140位为T7启动子，第5209至5706位为干扰素-α2的编码基因，第5728至5745位为转肽酶A的识别区域，第5812至7161位为弹性蛋白样多肽的编码基因，第7265至7314位为T7终止子。干扰素-α2在下文中简称IFN。弹性蛋白样多肽在下文中简称ELP。表达盒A表达的蛋白为IFN-ELP，在下文中简称IFN-ELP。
IFN的氨基酸序列如序列表序列2的第1位至第166位所示。ELP的氨基酸序列如序列表序列2的第202位至第651位所示。IFN-ELP的氨基酸序列如序列表序列2的第1位至第651位所示。
二、重组质粒pET-25b-Sortase的制备
人工合成序列表序列3所示的DNA分子，编码序列表序列4所示的转肽酶Sortase。
将pET-25b(+)的NdeI识别序列和EcoRI识别序列间的DNA替换为序列表的序列3所示的双链DNA分子，保持pET-25b(+)的其它序列不变，得到重组质粒pET-25b-Sortase。重组质粒pET-25b-Sortase表达序列表序列4所示的转肽酶Sortase。转肽酶Sortase在下文中简称为SrtA。
三、蛋白的表达和纯化
1、ELP-ELP的表达和纯化
将重组质粒pET-25b-IFN-ELP导入大肠杆菌感受态Rosetta-gami(DE3)pLysS(Novagen公司产品)，得到含有pET-25b-IFN-ELP的重组大肠杆菌，将该重组大肠杆菌命名为Rosetta/pET-25b-IFN-ELP。
按照下述步骤进行ELP-ELP的表达和纯化：
(1)将Rosetta/pET-25b-IFN-ELP单克隆接种于50mL含100μg/mL氨苄青霉素的TB培养基，37℃、250rpm振荡培养8小时得到培养菌液1；将培养菌液1以1：20(体积比)接种到1L含100μg/mL氨苄青霉素的TB培养基中，200rpm振荡培养5h，得到培养菌液2；向培养菌液2中加入IPTG，使IPTG在体系中的浓度为0.4mmol/L，18℃、200rpm振荡培养16h，得到培养菌液3。
(2)将上述步骤(1)得到的培养菌液3收集于离心瓶，以3000g离心力离心5min，收集菌体得到菌体1。
(3)用冰冷pH7.4、10mM PBS缓冲液重悬步骤(2)得到的菌体1，4℃超声破碎(超声功率50W，超声时间30min，5S破碎10S间隙)，得到菌体破碎液。
(4)将步骤(3)得到的菌体破碎液4℃、14000g离心15分钟，得到上清液1。
(5)向步骤(4)得到的上清液1加入2mL 10％(体积百分含量)聚乙烯亚胺水溶液，离心15分钟，得到上清液2。
(6)反相转变循环技术(Inverse transition cycling，ITC)第一次纯化：向步骤(5)得到的上清液2中加入NaCl，使NaCl在体系中的浓度为3mol/L，37℃充分溶解后，14000g离心15分钟，去上清A，得到沉淀1；用预冷的pH7.4、10mM PBS缓冲液溶解沉淀1，离心，得到上清液3。
(7)反相转变循环技术(Inverse transition cycling，ITC)第二次纯化：向步骤(6)得到的上清液3中加入NaCl，使NaCl在体系中的浓度为3mol/L，37℃充分溶解后，14000g离心15分钟，去上清B，得到沉淀2；用预冷的pH7.4、50mM Tris·HCl缓冲液溶解沉淀2，离心，得到上清液4。
上清液4中含有IFN-ELP，因此命名为IFN-ELP溶液。
将上清液4进行SDS-PAGE，测定IFN-ELP的纯度。用NanoDrop 2000分光光度计测定上清液4中IFN-ELP的浓度。反相转变循环技术(Inverse transition cycling，ITC)纯化原理见图1。
IFN-ELP的表达与纯化见图2(图2中，1为步骤(3)的菌体破碎液，2为步骤(6)的上清A，3为步骤(6)的沉淀1，4为步骤(7)的上清B，5为步骤(7)的沉淀2，6为蛋白标准品。
结果表明，通过ITC纯化后，上清液4中目的蛋白IFN-ELP的纯度＞95％，IFN-ELP的产率达到250mg/L培养菌液3。
2、SrtA的表达和纯化
(1)将上述步骤1中的重组质粒pET-25b-IFN-ELP替换为重组质粒 pET-25b-Sortase，其它同步骤1的(1)，得到培养菌液4。
(2)同步骤1的(2)。
(3)同步骤1的(3)。
(4)同步骤1的(4)，得到上清液,6。
(5)向步骤(4)得到的上清液6加入2mL 10％(体积百分含量)聚乙烯亚胺水溶液，离心15分钟，得到上清液7。
(6)将10mL上清液7经0.45μm滤膜过滤后上样至镍亲和层析柱(HisTrap HP 5mL，GE公司)，先用40mL平衡缓冲液(pH7.4、10mM PBS，500mM NaCl，5％甘油，10mM咪唑)洗脱，然后用20mL洗脱液(pH7.4、10mM PBS，500mM NaCl，5％甘油，500mM咪唑)进行洗脱，收集用洗脱液进行洗脱得到的过柱后溶液，然后将过柱后溶液经脱盐柱(HiPrep 26/10Desalting)除去咪唑，同时置换到pH7.4、50mM Tris·HCl缓冲液中，获得SrtA溶液(即含有SrtA的pH7.4、50mM Tris·HCl缓冲液)。
将SrtA溶液进行SDS-PAGE，测定SrtA的纯度。
用NanoDrop 2000分光光度计测定上述纯化样品中SrtA的浓度。
结果表明，SrtA溶液中SrtA的纯度＞95％，产率达到200mg/L培养菌液4。
实施例2、IFN和ELP的获得
获得IFN和ELP的步骤如下：
1、取实施例1的步骤三的2制备的100μM SrtA溶液10mL，加入CaCl2并使其浓度为10mM，得到溶液。
2、取实施例1的步骤三的1中制备的200μMIFN-ELP溶液10mL，向其中加入5mM三甘氨酸(sigma公司产品)，然后与10mL步骤1得到的溶液混合，室温反应过夜，得到混合液1。将混合液1用pH7.4、10mM PBS缓冲液经HiPrep26/10Desalting脱盐柱(GE公司产品)去除小分子杂质得到溶液，得到混合液2。
3、完成步骤2后，将混合液2在AKTA蛋白纯化系统(AKTA Purifier10，GE公司产品)上经阴离子交换层析(HiTrap Capto Q 5mL)纯化得到ELP溶液。
阴离子交换层析的参数：阴离子交换层析柱为HiTrap Capto Q 5mL；先用平衡缓冲液(pH4.5、20mM醋酸溶液)洗脱柱子，然后上样10mL混合液2，用洗脱液进行洗脱(洗脱过程：A液为pH4.5、20mM醋酸水溶液，B液为1M NaCl水溶液，洗脱液由A液和B液组成，使用流动相B逐渐递增、流动相A逐渐递减的线性洗脱条件，具体如下：在0-10min内，流动相中A液的体积百分含量由100％匀速降至0％。检测波长为280nm。收集洗脱体积60mL-65mL的过柱后溶液，即为ELP溶液。经HiPrep 26/10Desalting脱盐柱(GE公司产品)置换到含150mM NaCl的pH7.4、50mM Tris·HCl缓冲液中。
4、完成步骤2后，将混合液2在AKTA蛋白纯化系统(AKTA Purifier10，GE公司产品)上经阴离子交换层析(HiTrap Capto Q 5mL)纯化得到IFN溶液。
阴离子交换层析的参数：阴离子交换层析柱为HiTrap Capto Q 5mL；先用平衡缓冲液(pH7.4、20mM Tris·HCl)洗脱柱子，然后上样10mL混合液2，用洗脱液进行洗脱(洗脱过程：A液为pH7.4、20mM Tris·HCl水溶液，B液为1M NaCl水溶液，洗脱液由A液和B液组成，使用流动相B逐渐递增、流动相A逐渐递减的线性洗脱条件，具体如下：在0-10min内，流动相中A液的体积百分含量由100％匀速降至0％。检测波长为280nm。收集洗脱体积90mL-100mL的过柱后溶液，即为IFN溶液。经HiPrep 26/10Desalting脱盐柱(GE公司产品)置换到含150mM NaCl的pH7.4、50mM Tris·HCl缓冲液中。
实验结果见图3、图4。图3(图3中A为采用阴离子交换柱从混合液2中纯化ELP的色谱图，其中UV监测线性洗脱得到目标产物ELP；图3中B为纯化ELP过程中的SDS-PAGE分析结果，其中1为蛋白标准品，2为混合液1，3为混合液2，4为流穿液，5为收集洗脱体积43mL-50mL得到的洗脱蛋白IFN，6为收集洗脱体积52mL-58mL得到的洗脱蛋白IFN-ELP和IFN，7为收集洗脱体积60mL-65mL得到的洗脱蛋白ELP，8为收集洗脱体积70mL-80mL得到的洗脱蛋白SrtA，9为实施例1步骤1制备的上清液4)为ELP合成及纯化过程。图4(图4中A为采用阴离子交换柱从混合液2中纯化IFN的色谱图，其中UV监测线性洗脱得到目标产物IFN；图4中B为纯化IFN过程中的SDS-PAGE分析结果，其中M为蛋白标准品，1为实施例1步骤2制备的SrtA溶液，2为实施例1步骤1制备的上清液4，3为混合液1，4为收集洗脱体积0mL-40mL得到的流穿蛋白ELP和SrtA，5为收集洗脱体积90mL-100mL得到的洗脱蛋白IFN)为IFN合成及纯化过程。
实施例3、IFN-ELP的物理化学表征
1、分子量测定
用基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF)测定上述步骤获得的IFN-ELP、IFN和ELP的分子量，仪器为4800Plus MALDI-TOF/TOFTM分析仪(AB SCIEX)，具体测定方法及步骤参见仪器自带说明书。
结果表明(图5)，通过SrtA催化和阴离子交换柱(AEX)纯化获得IFN、ELP和IFN-ELP。IFN-ELP的分子量测定结果为58236.1，IFN的分子量测定结果为20338.1，均与理论值基本一致。
2、水合半径
用pH7.4、10mM PBS缓冲液溶解待测蛋白(IFN-ELP或IFN)，然后经0.22μm孔径滤膜过滤得到待测样品。将待测样品在Malvern Zetasizer Nano ZS90纳米粒径电位分析仪上用动态光散射(DLS)方法(Weiping Gao,et al.In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation.Proc Natl Acad Sci USA.2010Sep 21；107(38):16432-7.)测量待测蛋白(IFN-ELP或IFN)的水合半径。具体测量步骤参见仪器自带说明书。DLS测试使用的是Malvern  Zetasizer Nano-zs90。数据处理使用软件Zetasizer software 6.32。
结果表明(图6)，IFN的水合半径为2.2nm，IFN-ELP的水合半径为9.9nm。
3、相转变温度
运用浊度法测定相转变温度，具体步骤为：
用pH7.4、10mM PBS缓冲液溶解待测蛋白(IFN-ELP或ELP)至待测蛋白在体系中的浓度为1mg/mL，然后用酶标仪(Molecular Devices公司产品)测定其在35-55℃(以0.5℃/min线性递增)范围内的OD350nm值。当待测蛋白的OD350nm值达到上述温度范围内OD350值最大值的一半时的温度即为相转变温度。
结果表明(图7)，ELP的相转变温度为47.8℃，IFN-ELP的相转变温度为45.3℃。
4、二级结构
运用圆二色性色谱分析测得待测蛋白(IFN-ELP或IFN)的二级结构，具体步骤为：
用水溶解待测蛋白(IFN-ELP或IFN)至待测蛋白在体系中的浓度为0.18mg/mL，然后用圆二色光谱仪(Applied Photophysics有限公司)在200-260nm波长范围内进行紫外扫描分析。
结果表明(图8)，IFN-ELP和IFN在200-260nm波长范围内的圆二色性色谱分析都呈现出同样的208/222nm双峰曲线，均为典型的α螺旋结构。IFN-ELP与IFN的曲线重叠良好，表明在IFN上修饰ELP后二级结构没有明显影响。
实施例4、IFN-ELP的体外生物活性测定
采用MTT方法测定待测蛋白(IFN-ELP或IFN)的体外生物活性。具体步骤如下：
1、在含10％(体积百分比)FBS、50U/mL盘尼西林和50μg/mL链霉素的RMPI-1640培养基中培养人Burkitt’s B淋巴瘤细胞(简称Daudi B)获得细胞悬浮液。
2、取待测蛋白用含10％(体积百分比)FBS、50U/mL盘尼西林和50μg/mL链霉素的RMPI-1640培养基稀释，得到浓度不同的待测蛋白溶液。
3、取96孔细胞培养板，每孔加入50μL步骤1获得的细胞悬浮液(每孔约104个细胞)，然后加入步骤2配置的待测蛋白溶液(每孔50μL，使待测蛋白在孔中的浓度为1、2、5、10、20、50、100、1000或10000pg/mL)，5％CO2、37℃培养72h，然后加入MTT溶解液(Promega公司产品)，3h后，采用酶标仪检测490nm处的吸光值。
在96孔细胞培养板中设置阳性对照孔，每个阳性对照孔加入50μL步骤1获得的细胞悬浮液和50μL含10％(体积百分比)FBS、50U/mL盘尼西林和50μg/mL链霉素的RMPI-1640培养基。在96孔细胞培养板中设置阴性对照孔，每个阴性对照孔加入100μL含10％(体积百分比)FBS、50U/mL盘尼西林和50μg/mL链霉素的RMPI-1640培养基。采用酶标仪检测阴性对照孔和阳性对照孔490nm处的吸光值。
以待测蛋白在孔中的浓度为横坐标，相对活性为纵坐标，比较待测蛋白处 理后Daudi B细胞增殖的程度。相对活性根据下述公式计算：
相对活性(100％)＝(待测蛋白溶液在50nm处的吸光值-阴性对照在490nm处的吸光值)/(阳性对照在490nm处的吸光值-阴性对照在490nm处的吸光值)*100％。
实验结果见表1和图9。结果表明，IFN-ELP的半抑制浓度(IC50)为32.82pg/mL，相对活性为41％；IFN的半抑制浓度(IC50)为13.51pg/mL，相对活性为100％。结果表明，IFN经ELP修饰后并没有严重降低IFN体外生物的活性。
表1.IFN和IFN-ELP的体外抗细胞增殖活性测定
样品 IC50(pg/mL) 相对活性(％)
IFN 13.51 100
融合蛋白IFN-ELP 32.82 41
实施例5、IFN-ELP的药物代谢动力学测试
IFN-ELP或IFN的药物代谢动力学测试具体步骤如下：
1、将6只8周龄体重为250g左右的雄性SD大鼠随机分成两组(每组3只)，分别处理如下：
IFN组：尾静脉注射IFN，注射剂量为125μg/kg体重；
IFN-ELP组：尾静脉注射IFN-ELP，注射剂量为125μg/kg体重。
2、完成步骤1中的注射后开始计时，处理不同时间(IFN-ELP组时间点为1min，5min，15min，30min，1h，3h，6h，24h，48h，72h，96h，IFN组时间点为1min，5min，15min，30min，1h，3h，6h，24h)后的雄性SD大鼠分别用异氟烷麻醉，然后经眼内眦静脉用收集管取血0.3mL(收集管提前用肝素钠(江苏万邦生化医药股份有限公司产品)浸润并烘干)，室温静置1h，4℃、3000×g离心收集上层血浆，保存于-80℃低温冰箱。
3、完成步骤2后，用人IFN-α2ELISA试剂盒(PBL interferon source公司产品)测定步骤2中各雄性SD大鼠血浆中的IFN浓度。测定方法和步骤按自带说明书进行。
4、完成步骤3后，利用DAS软件进行数据分析。
利用DAS软件中房室消除模型分析IFN-ELP和IFN的药物代谢动力学参数，结果表明(表2和图10)：1、IFN的半衰期为0.3h，在给药几分钟后，血液中干扰素的浓度迅速下降到初始剂量的50％以下，给药24h后，干扰素残留浓度不足干扰素初始浓度的0.1％；IFN-ELP的半衰期为8.57h，其在血液中干扰素的浓度是逐渐减少的，给药72h后，干扰素残留浓度仍有干扰素初始浓度的10％以上。2、IFN-ELP的体内平均滞留时间(MRT0-∞)是IFN的体内平均滞留时间(MRT0-∞)的11.3倍。3、IFN-ELP的药时曲线面积(AUC)是IFN的药时曲线面积(AUC)的32.9倍。表明，与IFN相比，IFN-ELP的半衰期和体内平均存留时间明显延长，药时曲线面积显著增加，生物利用率显著提高。
表2.IFN和IFN-ELP的药物代谢动力学数据分析
参数 IFN IFN-ELP
T1/2(h) 0.30±0.03 8.57±3.88
MRT0-∞(h) 0.97±0.33 10.93±2.84
AUC(106pg/mL·h) 0.20±0.03 6.58±0.75
实施例6、IFN-ELP在组织中的分布情况
IFN-ELP或IFN在组织中的分布情况具体步骤如下：
1、将RMPI-1640培养基和BD Matrigel Matrix(Corning公司产品)等体积混得到混合物1。
2、将在含有10％FBS、50U/mL盘尼西林和50g/mL链霉素的RMPI-1640培养基中培养的人卵巢癌细胞(OVCAR-3)用胰蛋白酶消化剥离，经pH7.4、10mM PBS缓冲液洗涤后，用步骤1得到的混合物1重悬，得到重悬液。
3、将步骤2得到的重悬液接种与18只裸鼠左后肢股骨处背部皮下，接种量均为重悬液0.2mL(约5×106个细胞)/只，30天后6只裸鼠均形成大小约200mm3的实体瘤肿块。将6只裸鼠随机分成IFN组和IFN-ELP组，每组各9只。
4、向步骤4中IFN-ELP组的裸鼠尾静脉注射IFN-ELP，IFN组的裸鼠尾静脉注射IFN。注射剂量均为10μg/20g体重。
5、完成步骤4后，将IFN-ELP或IFN处理不同时间(2h、6h、或24h)后的裸鼠通过颈椎脱臼法处死，收集心脏、肾脏、肝脏、脾脏、肺脏、胰腺、胃、肌肉、小肠、血浆和肿瘤等组织器官。
6、将步骤5收集的组织器官(心脏、肾脏、肝脏、脾脏、肺脏、胰腺、胃、肌肉、小肠、血浆或肿瘤)用提取缓冲液破碎后，离心，得到上清提取液。提取缓冲液的溶质及其浓度为：1mM EDTA、0.5％Triton X-100、0.5％脱氧胆酸钠、1mM PMSF，1％(体积百分比)蛋白酶抑制剂混合物(Sigma-Aldrich公司产品)和1％磷酸酶抑制剂混合物(Sigma-Aldrich公司产品)；溶剂为pH7.4、10mM PBS缓冲液。
7、用人IFN-α2ELISA试剂盒(PBL interferon source公司产品)定量测定步骤6中上清提取液中的IFN的浓度，测定方法和步骤按自带说明书进行，进一步得到步骤5组织器官(心脏、肾脏、肝脏、脾脏、肺脏、胰腺、胃、肌肉、小肠、血浆或肿瘤)中的IFN的浓度。
结果表明(图11)，IFN-ELP组的裸鼠注射IFN-ELP处理不同时间(2h、6h、或24h)后，IFN在心脏、肾脏、肝脏、脾脏、肺脏、胰腺、胃、肌肉、小肠、血浆和肿瘤中均能够有效累积。尤其是在肿瘤中，IFN-ELP组肿瘤中干扰素的浓度和IFN组肿瘤中干扰素的浓度相比，注射2h为29倍，注射6h为66倍，注射24h为21倍。表明，IFN-ELP能够有效利用增强通透和滞留效应，使干扰素在肿瘤组织中聚集、在血液循环中半衰期延长，从而提高干扰素在体内的生物利用度和抗肿瘤功效。
实施例7、IFN-ELP体内抗肿瘤活性测试
IFN-ELP体内抗肿瘤活性测试具体步骤如下：
1、取实施例6中经步骤1和2制备的重悬液，接种于26只裸鼠左后肢股骨处背部皮下，接种量为重悬液0.2mL(约5×106个细胞)/只，30天后，26只裸鼠均形成大小约40mm3的实体瘤肿块。
2、将26只裸鼠随机分成四组，分别为IFN-ELP组(7只)、IFN组(7只)、ELP组(6只)和生理盐水组(6只)。
3、向步骤2中IFN-ELP组的裸鼠尾静脉注射IFN-ELP，IFN组的裸鼠尾静脉注射IFN，ELP组的裸鼠尾静脉注射ELP，注射剂量均为60μg/20g体重。向步骤2中的生理盐水组的裸鼠尾静脉注射生理盐水，注射等体积生理盐水。
各组均为每三天注射一次，直至生理盐水组、IFN组和ELP组的裸鼠全部死亡。在本实验中裸鼠死亡包括自然死亡和安乐处死，安乐处死是指裸鼠肿瘤生长超过1000mm3或体重下降超过15％，被注射巴比妥钠类药物处死。
上述步骤3的实验过程中，每三天测量各组裸鼠的体重和肿瘤体积；每15天通过眼球取血，测量乳酸脱氢酶、肌酸激酶同工酶、谷丙转氨酶、谷草转氨酶、肌酐、尿素氮、红细胞、白细胞、血小板、血红蛋白等基本生理指标。
实验结果表明(图12、图13和图14)，实验期间，生理盐水组和ELP组的裸鼠的肿瘤体积逐渐增大，注射第39天生理盐水组和ELP组的裸鼠肿瘤体积均超过1000mm3，生存中值仅为34.5天和36天；实验期间，IFN组的裸鼠的肿瘤体积也逐渐增大，注射第45天IFN组的裸鼠肿瘤体积也超过1000mm3，生存中值为42天，没有体现明显的抗肿瘤活性；实验期间，IFN-ELP组的裸鼠的肿瘤体积基本没有变化，生存中值为87天。表明，IFN-ELP能够有效地抑制肿瘤的生长，具有非常好的体内抗肿瘤活性。
实验结果还表明，实验期间，IFN-ELP组的裸鼠体重略有增加(图15)，表明IFN-ELP对裸鼠没有明显的副作用。
实验结果表明(图16和图17)，实验期间，与生理盐水组、IFN组和ELP组的裸鼠相比，IFN-ELP组的裸鼠的乳酸脱氢酶、肌酸激酶同工酶、谷丙转氨酶、谷草转氨酶、肌酐、尿素氮、红细胞、白细胞、血小板、血红蛋白等基本生理指标没有显著差别。表明IFN-ELP不会对裸鼠体内器官造成明显毒性。
以本发明所提供的融合蛋白IFN-ELP作为蛋白质药物，可使生物活性保持率增加、体内半衰期延长、体内平均存留时间延长，并有效抑制肿瘤。同时，本发明所提供的一种制备半衰期延长和/或生物活性保持率增加的蛋白质药物的方法为修饰蛋白药物以提高药物稳定性、改善药物代谢动力学和增强治疗功效的新方法。

Claims (16)

  1. 一种融合蛋白，是如下b1)或b2)的蛋白质：
    b1)含有功能蛋白和弹性蛋白样多肽的融合蛋白；
    所述弹性蛋白样多肽为弹性蛋白样多肽甲或弹性蛋白样多肽乙；
    所述弹性蛋白样多肽甲是如下a1)—a8)中的任一种：a1)具有一个以上单拷贝乙的多肽，每个单拷贝乙中具有一个以上单拷贝甲；所述单拷贝甲的氨基酸序列为XGVPG，X代表一个脯氨酸残基以外的任意氨基酸残基；a2)所述a1)中，每个单拷贝乙中具有10个所述单拷贝甲；a3)所述a2)中，所述单拷贝乙如序列表序列2的第202位至第251位所示；a4)所述a1)中，所述弹性蛋白样多肽甲具有9个所述单拷贝乙；a5)所述a4)中，每个单拷贝乙中具有10个所述单拷贝甲；a6)所述a5)中，所述单拷贝乙如序列表序列2的第202位至第251位所示；a7)所述a6)中，所述弹性蛋白样多肽甲如序列表序列2的第202位至第651位所示；a8)所述a6)中，所述弹性蛋白样多肽甲如序列表序列2的第202位至第653位所示；
    所述弹性蛋白样多肽乙可为如下a9)—a15)中的任一种)：a9)具有一个以上单拷贝丁的多肽，每个单拷贝丁中具有一个以上单拷贝丙；所述单拷贝丙的氨基酸序列为VPGVG；a10)所述a9)中，每个单拷贝丁中具有10个所述单拷贝丙；a11)所述a10)中，所述单拷贝丁如序列表序列5的第202位至第251位所示；a12)所述a9)中，所述弹性蛋白样多肽乙具有9个所述单拷贝丁；a13)所述a12)中，每个单拷贝丁中具有10个所述单拷贝丙；a14)所述a13)中，所述单拷贝丁如序列表序列5的第202位至第251位所示；a15)所述a14)中，所述弹性蛋白样多肽乙如序列表序列5第202位至第651位所示；
    b2)将b1)的N端或C端连接标签得到的带标签蛋白质。
  2. 如权利要求1所述融合蛋白，其特征在于：所述功能蛋白是如下c1)-c20)中的任一种：c1)干扰素-α2；c2)序列表的序列2第1位至第166位所示的干扰素-α2；c3)干扰素-α；c4)干扰素；c5)胰高血糖素样肽-1及其衍生物；c6)水蛭素；c7)胰岛素；c8)单克隆抗体；c9)血液因子；c10)生长激素；c11)白介素；c12)生长因子；c13)治疗性疫苗；c14)降钙素；c15)肿瘤坏死因子；c16)酶；c17)选自医药、农业、科研以及其它工业领域相关的蛋白、小肽或抗体；c18)集落刺激因子；c19)瘦素；c20)c1)-c19)任一所述功能蛋白经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的功能蛋白。
  3. 如权利要求1所述融合蛋白，其特征在于：所述功能蛋白具体例子包括但不不限于：天冬酰胺酶、谷氨酸酶、精氨酸酶、精氨酸脱氨酶、腺苷脱氨酶核糖核酸酶、胞嘧啶脱氨酶、胰蛋白酶、胰凝乳蛋白酶、木瓜蛋白酶、表皮生长因子、胰岛素样生长因子、转化生长因子、神经生长因子、血小板衍生的生 长因子、骨形态发生蛋白、成纤维细胞生长因子、生长抑制素、生长激素、生长激素抑制素、甲状旁腺激素、凝血因子、肿瘤坏死因素、胃肠肽、血管活性肠肽、肠促胰酶肽、胃泌素、促胰液素、促红细胞生成素、荷尔蒙、抗利尿激素、奥曲肽、胰腺酶、超氧化物歧化酶、促甲状腺激素释放激素、促甲状腺激素、促黄体生成激素、促黄体激素释放激素、组织型纤溶酶原激活剂、白细胞介素-1、白细胞介素-15、受体拮抗剂、胰高血糖素样肽-1、生长素、粒单核细胞集落刺激因子、白细胞介素-2、腺苷脱氨酶、尿酸酶、天冬酰胺酶、人生长激素、天冬酰胺酶、巨噬细胞活化、绒毛膜促性腺激素、肝素、心房利钠肽、血红蛋白、逆转录病毒载体、松弛肽、环孢菌素、催产素、疫苗、单链抗体、锚蛋白重复蛋白或亲和体。
  4. 如权利要求1所述融合蛋白，其特征在于：所述融合蛋白中，所述弹性蛋白样多肽和所述功能蛋白的位置关系为如下d1)、d2)或d3)：
    d1)所述弹性蛋白样多肽位于所述功能蛋白的上游或下游；d2)所述弹性蛋白样多肽融合于所述功能蛋白的C末端或N末端；d3)所述弹性蛋白样多肽插入所述功能蛋白中；所述弹性蛋白样多肽的插入位点为远离所述功能蛋白的活性位点的位置或不干扰所述功能蛋白的活性位点的位置。
  5. 如权利要求1所述融合蛋白，其特征在于：所述融合蛋白的氨基酸序列如序列表序列2的第1位至第651位或序列表序列2的第1位至第653位所示。
  6. 权利要求1至5中任一所述融合蛋白在制备产品中的应用；所述产品的功能为抑制肿瘤细胞增殖和/或治疗肿瘤。
  7. 一种产品，其活性成分为权力要求1至6中任一所述融合蛋白。
  8. 编码权利要求1至5任一所述融合蛋白的核酸分子。
  9. 如权利要求8所述的核酸分子，其特征在于：
    编码所述干扰素-α2的核苷酸序列如序列表序列1的第5209位至第5706位所示；编码所述弹性蛋白样多肽的核苷酸序列如序列表序列1的第5812位至第7161位或序列表序列1的第5812位至第7167位或序列表序列1的第5812位至第5961位所示。
  10. 如权利要求8所述的核酸分子，其特征在于：所述核酸分子为如下e1)-e10)任一所示的DNA分子：
    e1)核苷酸序列含有序列表的序列1第5812位至第7161位所示的DNA分子；e2)核苷酸序列含有序列表的序列1第5812位至第7167位所示的DNA分子；e3)核苷酸序列含有序列表的序列1第5812位至第5961位所示的DNA分子；e4)核苷酸序列含有序列表的序列1第5209位至第5706位和序列表序列1的第5812位至第7161位所示的DNA分子；e5)核苷酸序列含有序列表的序列1第5209位至第5706位和序列表序列1的第5812位至第7167位所示的DNA分子；e6)核苷酸序列含有序列表的序列1第5209位至第5706位和序列表序列1的第5812位至第5961位所示的DNA分子；e7)核苷酸序列如序列表的序列1第5209位至 第7161位所示的DNA分子；e8)核苷酸序列如序列表的序列1第5209位至第7167位所示的DNA分子；e9)与e1)-e8)中任一限定的核苷酸序列具有75％或75％以上同一性，且编码所述融合蛋白的DNA分子；e10)在严格条件下与e1)-e8)中任一限定的核苷酸序列杂交，且编码相应所述蛋白的DNA分子。
  11. 含有权利要求8至10任一所述核酸分子的表达盒、表达载体、重组微生物或转基因细胞系。
  12. 一种制备半衰期延长和/或生物活性保持率增加的蛋白质药物的方法，包括如下步骤：在功能蛋白中引入弹性蛋白样多肽，得到半衰期延长和/或生物活性保持率增加的蛋白质药物；所述弹性蛋白样多肽融合于所述功能蛋白的C末端或N末端，或所述弹性蛋白样多肽位于所述功能蛋白的上游或下游，或所述弹性蛋白样多肽插入所述功能蛋白中；所述弹性蛋白样多肽的插入位点为远离所述功能蛋白的活性位点的位置或不干扰所述功能蛋白的活性位点的位置；
    所述弹性蛋白样多肽为弹性蛋白样多肽甲或弹性蛋白样多肽乙；
    所述弹性蛋白样多肽甲，是如下a1)—a8)中的任一种：a1)具有一个以上单拷贝乙的多肽，每个单拷贝乙中具有一个以上单拷贝甲；所述单拷贝甲的氨基酸序列为XGVPG，X代表一个脯氨酸残基以外的任意氨基酸残基；a2)所述a1)中，每个单拷贝乙中具有10个所述单拷贝甲；a3)所述a2)中，所述单拷贝乙如序列表序列2的第202位至第251位所示；a4)所述a1)中，所述弹性蛋白样多肽甲具有9个所述单拷贝乙；a5)所述a4)中，每个单拷贝乙中具有10个所述单拷贝甲；a6)所述a5)中，所述单拷贝乙如序列表序列2的第202位至第251位所示；a7)所述a6)中，所述弹性蛋白样多肽甲如序列表序列2的第202位至第651位所示；a8)所述a6)中，所述弹性蛋白样多肽甲如序列表序列2的第202位至第653位所示；
    所述弹性蛋白样多肽乙可为如下a9)—a15)中的任一种)：a9)具有一个以上单拷贝丁的多肽，每个单拷贝丁中具有一个以上单拷贝丙；所述单拷贝丙的氨基酸序列为VPGVG；a10)所述a9)中，每个单拷贝丁中具有10个所述单拷贝丙；a11)所述a10)中，所述单拷贝丁如序列表序列5的第202位至第251位所示；a12)所述a9)中，所述弹性蛋白样多肽乙具有9个所述单拷贝丁；a13)所述a12)中，每个单拷贝丁中具有10个所述单拷贝丙；a14)所述a13)中，所述单拷贝丁如序列表序列5的第202位至第251位所示；a15)所述a14)中，所述弹性蛋白样多肽乙如序列表序列5第202位至第651位所示。
  13. 如权利要求12所述的方法，其特征在于：所述功能蛋白是如下c1)-c20)中的任一种：c1)干扰素-α2；c2)序列表序列2的第1位至第166位所示的干扰素-α2；c3)干扰素-α；c4)干扰素；c5)胰高血糖素样肽-1及其衍生物；c6)水蛭素；c7)胰岛素；c8)单克隆抗体；c9)血液因子；c10)生长激素；c11)白介素；c12)生长因子；c13)治疗性疫苗；c14)降钙素；c15)肿瘤坏死因子；c16)酶；c17)选自医药、农业、科研以及其它工业领域相关的蛋白、 小肽或抗体；c18)集落刺激因子；c19)瘦素；c20)c1)-c19)任一所述功能蛋白经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的功能蛋白。
  14. 如权利要求12所述的方法，其特征在于：所述功能蛋白具体例子包括但不不限于：天冬酰胺酶、谷氨酸酶、精氨酸酶、精氨酸脱氨酶、腺苷脱氨酶核糖核酸酶、胞嘧啶脱氨酶、胰蛋白酶、胰凝乳蛋白酶、木瓜蛋白酶、表皮生长因子、胰岛素样生长因子、转化生长因子、神经生长因子、血小板衍生的生长因子、骨形态发生蛋白、成纤维细胞生长因子、生长抑制素、生长激素、生长激素抑制素、甲状旁腺激素、凝血因子、肿瘤坏死因素、胃肠肽、血管活性肠肽、肠促胰酶肽、胃泌素、促胰液素、促红细胞生成素、荷尔蒙、抗利尿激素、奥曲肽、胰腺酶、超氧化物歧化酶、促甲状腺激素释放激素、促甲状腺激素、促黄体生成激素、促黄体激素释放激素、组织型纤溶酶原激活剂、白细胞介素-1、白细胞介素-15、受体拮抗剂、胰高血糖素样肽-1、生长素、粒单核细胞集落刺激因子、白细胞介素-2、腺苷脱氨酶、尿酸酶、天冬酰胺酶、人生长激素、天冬酰胺酶、巨噬细胞活化、绒毛膜促性腺激素、肝素、心房利钠肽、血红蛋白、逆转录病毒载体、松弛肽、环孢菌素、催产素、疫苗、单链抗体、锚蛋白重复蛋白或亲和体。
  15. 一种融合分子，为含有功能分子和权利要求1中所述弹性蛋白样多肽的融合分子；所述功能分子为多核苷酸或核酸适配体或其它生物分子。
  16. 如权利要求15所述的融合分子，其特征在于：
    所述多核苷酸包括天然或人工合成的、单链或双链的多核苷酸或寡聚核苷酸，具体例子包括但不局限于：反义寡核苷酸、siRNA或anti-miR；
    所述核酸适配体包括但不局限于：血管内皮细胞生长因子适配体、蓖麻毒素适配体、西葫芦毒蛋白适配体、凝血酶适配体、活化血浆蛋白C适配体、HIV-1逆转录酶适配体、HIV-1整合酶适配体、蛋白激酶C适配体、人嗜中性弹性蛋白酶适配体、L-选择素适配体、P-选择素适配体、耶尔森氏菌蛋白酪氨酸磷酸酶适配体、磷脂酶A2体血管生成素适配体或鼻病毒衣壳蛋白适配体；
    所述其它生物分子包括但不局限于：催产素、加压素、促肾上腺皮质激素、催乳激素、促黄体生成激素释放激素、黄体生成素释放激素、生长激素释放因子、促生长素抑制素、胰高血糖素、胃泌素、五肽胃泌素、四肽胃泌素、脑啡肽、内啡肽、血管紧张素、肾素、缓激肽、杆菌肽、多粘菌素、粘菌素、短杆菌酪肽、短杆菌肽及合成的类似物、变型和药理学活性片段物、单克隆抗体或可溶性疫苗。
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US11224662B2 (en) 2012-02-13 2022-01-18 University Of Southern California Methods and therapeutics comprising ligand-targeted ELPs
CN114796516A (zh) * 2022-04-20 2022-07-29 台州学院 一种多功能蛋白抗肿瘤纳米药物及制备方法和应用
US11464867B2 (en) 2018-02-13 2022-10-11 University Of Southern California Multimeric elastin-like polypeptides

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108383912A (zh) * 2018-02-06 2018-08-10 清华大学 人工融合蛋白及其用途
CN108822219B (zh) * 2018-07-06 2020-08-18 中国科学院微生物研究所 一种融合干扰素及其在制备粘膜免疫增强剂中的应用
CN108578709B (zh) * 2018-07-09 2021-05-04 清华大学 温敏性长效缓释药物载体及其应用
CN110101868B (zh) * 2019-05-24 2021-03-23 北京大学 一种环境刺激响应性蛋白质高分子偶联物自组装体及其制备方法与应用
CN111603551B (zh) * 2020-04-16 2022-04-22 北京大学 融合蛋白ifn-elp(v)在制备预防或治疗胶质母细胞瘤的药物中的应用
CN113735977A (zh) * 2020-05-28 2021-12-03 江苏康缘瑞翱生物医药科技有限公司 rhFGF21融合蛋白、编码其的多核苷酸、包含其组合物及其用途
CN112029000A (zh) * 2020-08-25 2020-12-04 江苏农牧科技职业学院 鸭干扰素α融合蛋白在制备抗鸭1型肝炎病毒的药物中的用途

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101384272A (zh) * 2005-12-20 2009-03-11 杜克大学 用于递送具有增强的药理性质的活性剂的方法和组合物
CN101578373A (zh) * 2006-09-06 2009-11-11 费斯生物制药公司 融合肽治疗组合物
CN102924603A (zh) * 2011-08-09 2013-02-13 哈药集团技术中心 人干扰素与靶向肽的融合蛋白及其制备
CN103237808A (zh) * 2010-12-03 2013-08-07 阿达梅德公司 抗癌融合蛋白
CN103313730A (zh) * 2010-11-01 2013-09-18 佩普蒂梅德股份有限公司 用于治疗癌症的肽靶向系统的组合物

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101384272A (zh) * 2005-12-20 2009-03-11 杜克大学 用于递送具有增强的药理性质的活性剂的方法和组合物
CN101578373A (zh) * 2006-09-06 2009-11-11 费斯生物制药公司 融合肽治疗组合物
CN103313730A (zh) * 2010-11-01 2013-09-18 佩普蒂梅德股份有限公司 用于治疗癌症的肽靶向系统的组合物
CN103237808A (zh) * 2010-12-03 2013-08-07 阿达梅德公司 抗癌融合蛋白
CN102924603A (zh) * 2011-08-09 2013-02-13 哈药集团技术中心 人干扰素与靶向肽的融合蛋白及其制备

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11224662B2 (en) 2012-02-13 2022-01-18 University Of Southern California Methods and therapeutics comprising ligand-targeted ELPs
US10961317B2 (en) 2012-08-10 2021-03-30 University Of Southern California CD20 scFv-ELPs methods and therapeutics
WO2016094627A1 (en) 2014-12-10 2016-06-16 S-Aima Holding Company, Llc Generation of hemoglobin-based oxygen carriers using elastin-like polypeptides
EP3229845A4 (en) * 2014-12-10 2018-07-11 S-Aima Holding Company, LLC Generation of hemoglobin-based oxygen carriers using elastin-like polypeptides
US11124559B2 (en) 2014-12-10 2021-09-21 University Of Southern California Generation of hemoglobin-based oxygen carriers using elastin-like polypeptides
US11464867B2 (en) 2018-02-13 2022-10-11 University Of Southern California Multimeric elastin-like polypeptides
CN111019962A (zh) * 2019-12-18 2020-04-17 南京理工大学 一种sod-elp融合蛋白及其制备方法
CN114796516A (zh) * 2022-04-20 2022-07-29 台州学院 一种多功能蛋白抗肿瘤纳米药物及制备方法和应用

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