WO2009155842A1 - 核糖核酸酶与毒蛋白膜转位结构域融合蛋白及其制备方法和用途 - Google Patents

核糖核酸酶与毒蛋白膜转位结构域融合蛋白及其制备方法和用途 Download PDF

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WO2009155842A1
WO2009155842A1 PCT/CN2009/072368 CN2009072368W WO2009155842A1 WO 2009155842 A1 WO2009155842 A1 WO 2009155842A1 CN 2009072368 W CN2009072368 W CN 2009072368W WO 2009155842 A1 WO2009155842 A1 WO 2009155842A1
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Prior art keywords
fusion protein
protein
tumor
amino acid
cell
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PCT/CN2009/072368
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English (en)
French (fr)
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王庆诚
沈如凌
孙瑞林
费俭
李俊
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上海南方模式生物科技发展有限公司
上海南方模式生物研究中心
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Publication of WO2009155842A1 publication Critical patent/WO2009155842A1/zh

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of DNA recombination technology and biomedicine. More specifically, the present invention relates to a fusion protein of a ribonuclease to a bacterial toxic protein (toxin) membrane translocation domain, a DNA sequence encoding the fusion protein, a vector containing the DNA sequence, a host cell containing the vector, and a gene A method of engineering the preparation of the fusion protein, and the use of the fusion protein in the treatment of tumors. Background technique
  • Ribonuclease (also known as: P30 protein, Ranpirnase, abbreviated as One) is a nuclease isolated from the oocytes and early embryos of the northern leopard frog (Rana pipiens) and belongs to the RNase A superfamily. Studies have shown that One enters mammalian cells by endocytosis, selectively degrades tRNA in the cytosol, inhibits protein biosynthesis and inhibits cell proliferation and induces apoptosis. In vitro experiments have shown that One has anti-proliferative and cytotoxic effects on a variety of tumor cells, such as: human malignant mesothelioma, non-small cell lung cancer, prostate cancer cells, pancreatic cancer cells and leukemia cells.
  • tumor cells such as: human malignant mesothelioma, non-small cell lung cancer, prostate cancer cells, pancreatic cancer cells and leukemia cells.
  • the object of the present invention is to provide a fusion protein of a ribonuclease and a bacterial virulence protein membrane translocation domain, a DNA sequence encoding the fusion protein, a vector containing the DNA sequence, a host cell containing the vector, and the fusion is prepared by genetic engineering The method of protein, and the application of the fusion protein in tumor therapy.
  • a fusion protein comprising:
  • a linker peptide consisting of 0-50 (preferably 5-30, more preferably 10-20) amino acids between (1) and (2).
  • the ribonuclease is located at the amino terminus of the fusion protein; the membrane translocation domain of the bacterial virulence protein is located at the carboxy terminus of the fusion protein.
  • the membrane translocation domain of the bacterial virulence protein is located at the amino terminus of the fusion protein
  • the ribonuclease is located at the carboxy terminus of the fusion protein.
  • the fusion protein consists essentially of (1), (3), linked together. More preferably, the fusion protein is composed of (1), (3), and (2).
  • sequence of the linker peptide includes at least one cleavage site sequence of the intracellular protease, thereby separating (1) and (2) after entering the cell.
  • the restriction enzyme site sequence is not present on the ribonuclease sequence or the membrane translocation domain sequence of the bacterial virulence protein.
  • the intracellular protease is a protease that is specifically located within the cell.
  • the intracellular enzyme is selected from, but not limited to, Furin, a matrix metalloproteinase, a prostate specific antigen, and a cathepsin.
  • the fusion protein is cleaved by an intracellular enzyme upon entry into the cell.
  • the cytotoxic protein is selected from, but not limited to, diphtheria toxin (DT) or Pseudomonas aeruginosa exotoxin A (PE).
  • DT diphtheria toxin
  • PE Pseudomonas aeruginosa exotoxin A
  • the membrane translocation domain of the diphtheria toxin is:
  • the ribonuclease is:
  • the linker peptide has the amino acid sequence shown at positions 105-120 of SEQ ID NO: 2 (preferably consisting of the amino acid sequence at positions 105-120 of SEQ ID NO: 2).
  • the fusion protein has the amino acid sequence shown in SEQ ID NO: 2.
  • nucleic acid molecule in a second aspect of the invention, is provided, the nucleic acid molecule encoding the fusion protein.
  • nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 1.
  • a vector comprising the nucleic acid molecule.
  • a genetically engineered cell comprising said vector; or said nucleic acid molecule integrated in said cell genome.
  • a method of producing the fusion protein comprising:
  • the use of the fusion protein is provided for the preparation of a composition for inhibiting tumor cell growth or treating a tumor.
  • the tumor is selected from (but not limited to): malignant mesothelioma; lung cancer; leukemia; malignant Lymphoma; myeloma; malignant melanoma; breast cancer; nervous system neoplasms; liver cancer; nasopharyngeal carcinoma; esophageal cancer; gastric cancer; colon cancer; prostate cancer; cervical cancer; oral cancer; salivary gland tumor; nasal cavity and paranasal sinus malignancy Laryngeal cancer; ear tumor; ocular tumor; thyroid neoplasm; mediastinal tumor; chest wall; pleural tumor; small intestine tumor; biliary tract tumor; pancreas and periampullary tumor; mesenteric and retroperitoneal tumor; renal tumor; adrenal tumor; Prostate cancer; testicular tumor; penile cancer; endometrial cancer;
  • composition for inhibiting tumor cell growth or treating a tumor comprising:
  • the composition is a pharmaceutical composition.
  • a method of inhibiting comprising treating a tumor cell with the fusion protein.
  • a method of inhibiting tumor cell growth or treating a tumor comprising: administering to the subject in need of inhibiting tumor cell growth or treating a tumor an effective amount of the fusion protein.
  • Figure 1 shows a schematic representation of the E. coli expression plasmid pET22b-ONC-DT 2Q3 . 390 containing the ONC-DT fusion protein gene.
  • Figure 2 shows the Onc-DT 2 of the present invention. 3. 39Q expression purification effect protein SDS-PAGE electropherogram. Wherein, lane 1. protein molecular weight standard;
  • Figure 3 shows the Ont-DT 2 measured by the MTT method. 3.39. Cytotoxicity effects on mouse myeloma cells SP2/0 and human leukemia cells K562. among them,
  • the inventors After long-term research and experiment, the inventors first converted the membrane of ribonuclease (One) gene and bacterial toxic protein.
  • the domain genes were fused together and highly expressed in E. coli, and the fusion protein of the ribonuclease-bacterial virulence protein membrane translocation domain was obtained.
  • the fusion protein retains the nuclease activity of One well, and improves the ability of One to enter the cell cytoplasm and the broad spectrum of anti-tumor, and has superior tumoricidal effect than One, on tumor cells (including some original The toxicity of One-insensitive tumor cells is greatly enhanced (the toxicity to different cells can be increased by 10-1000 times, respectively), which can be used as a new more effective and broad-spectrum anti-tumor drug.
  • ribonuclease and fusion protein of the diphtheria venom membrane translocation domain As used herein, the terms “ribonuclease and fusion protein of the diphtheria venom membrane translocation domain", “ONC-DT fusion protein” and the like are used interchangeably and refer to the ribonuclease amino acid sequence and the diphtheria venom membrane. A protein in which the amino acid sequence of the domain is fused, with or without a peptide sequence linked between the two.
  • the present invention provides a fusion protein comprising a One protein or a biologically active fragment thereof, and a bacterial virulence membrane translocation domain or a biologically active fragment thereof, which molecule can be used to inhibit tumors. More preferably, the fusion protein is an isolated protein which is not associated with other proteins, polypeptides or molecules, is a purified product of recombinant host cell culture or is a purified extract.
  • containing includes “comprising”, “consisting essentially of”, “consisting essentially of”, and “consisting of”; “mainly constituted by”, “consisting essentially of” and “consisting of” belonging to “contains” , the underlying concept of "with” or “including”.
  • One in the present invention refers to a polypeptide which can enter mammalian cells (especially tumor cells) by endocytosis, selectively degrades tRNA, or other RNA in the cytoplasm, thereby inhibiting protein synthesis and thereby inhibiting cell proliferation. And cause apoptosis.
  • mammalian cells especially tumor cells
  • endocytosis selectively degrades tRNA, or other RNA in the cytoplasm, thereby inhibiting protein synthesis and thereby inhibiting cell proliferation. And cause apoptosis.
  • One or a biologically active fragment thereof includes a portion of a conservative amino acid substitution sequence that does not affect its activity.
  • Proper replacement of amino acids is well known in the art, and the techniques can be readily implemented and ensure that the biological activity of the resulting molecule is not altered. These techniques have led to the recognition that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity. See Watson et al. Molecular Biology of The Gene, Fourth Edition, 1987, The Benjamin/Cummings Pub. Co. P224. Table 1 is some examples of such replacement. Table 1
  • any biologically active fragment of One can be applied to the present invention.
  • the biologically active fragment of One means that as a polypeptide fragment, after being linked to the toxic protein membrane translocation domain and forming a fusion protein, it still retains all or part of the function of the full length One.
  • the biologically active fragment retains at least 50% of the full-length One of the tumor suppressing activity.
  • the active fragment is capable of maintaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% inhibition of tumor activity.
  • One formed by substitution, deletion or addition of one or more (such as 1-30, preferably 1-20, better 1-10, further 1-5) amino acid residues Amino acid sequences are also included in the present invention.
  • the One formed by substitution, deletion or addition of one or more amino acid residues also has tumor suppressing activity.
  • the present invention may also employ a modified or modified One, for example, a One which may be modified or modified to prolong its half-life, improve its stability, or enhance its ability to kill tumor cells.
  • a modified or modified One or gene may be somewhat different from the naturally occurring One or gene, but can also kill tumor cells without causing other adverse effects or toxicity.
  • any biological activity that does not affect One's biological activity or gene Variations in function can be used in the present invention.
  • the amino acid sequence of the One may be substantially the same as the sequence shown by the 1-104th position in SEQ ID NO: 2.
  • the One has the sequence shown at positions 1-104 of SEQ ID NO: 2; more preferably, the One consists of the sequence shown at positions 1-104 of SEQ ID NO: 2.
  • Bacterial virulence protein membrane translocation domain may be substantially the same as the sequence shown by the 1-104th position in SEQ ID NO: 2.
  • the One has the sequence shown at positions 1-104 of SEQ ID NO: 2; more preferably, the One consists of the sequence shown at positions 1-104 of SEQ ID NO: 2.
  • the membrane translocation domain of the bacterial virulence protein of the present invention is a class of protein domains that act on endocytosis of toxins.
  • the bacterial toxic protein includes, but is not limited to, DT or PE.
  • membrane translocation domains of DT and PE are known to enhance endocytosis of a variety of molecules, it has not previously been known whether they have an effect on endocytosis of One.
  • the bacterial toxic protein is DT
  • DT is a bacterial exotoxin produced by diphtheria bacilli, which is composed of three domains, a N-terminal catalytic domain and a membrane translocation domain in the middle, C The end is a receptor binding domain.
  • the role of the membrane translocation domain is to help the catalytic domain penetrate the intracellular membrane into the cytoplasm and exert the cytotoxic effects of the catalytic domain.
  • the DT membrane translocation domain or a biologically active fragment thereof includes a portion of a conserved amino acid substitution sequence that may not affect its activity. See Table 1 for examples of some of the amino acid substitutions that can be used. There may be other similar alternatives that can often be determined empirically or based on known conserved sequences.
  • any biologically active fragment of the DT membrane translocation domain can be applied to the present invention.
  • the biologically active fragment of the DT membrane translocation domain means that as a polypeptide fragment, after linking to One and forming a fusion protein, it still retains all or part of the function of the complete DT membrane translocation domain.
  • the amino acid sequence of the DT membrane translocation domain formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention.
  • the DT membrane translocation domain formed by substitution, deletion or addition of one or more amino acid residues also has the function of helping One penetrate the tumor cell membrane.
  • the present invention may also employ a modified or modified DT membrane translocation domain, for example, which may be modified to increase its half-life, improve its stability, enhance its ability to penetrate cells, or enhance its specificity for penetrating cells.
  • DT membrane translocation domain for example, which may be modified to increase its half-life, improve its stability, enhance its ability to penetrate cells, or enhance its specificity for penetrating cells.
  • the modified or modified DT membrane translocation domain or gene may be different from the naturally occurring DT membrane translocation domain or gene, but may also help One penetrate the cell membrane without causing Other adverse effects or toxicity. That is, any biological activity that does not affect the DT membrane translocation domain or a variant of the biological function of the gene can be used in the present invention.
  • the amino acid sequence of the DT membrane translocation domain may be substantially identical to the sequence shown by positions 121 to 308 of SEQ ID NO: 2.
  • the DT membrane translocation domain has the sequence shown in positions 121-308 of SEQ ID NO: 2; more preferably, the DT membrane translocation domain is represented by SEQ ID NO: 2
  • the sequence shown in bits 121-308 is constructed. Connection
  • the One or its active fragment and the bacterial virulence membrane translocation domain or an active fragment thereof may be directly linked or linked by a polypeptide linker (linker peptide).
  • the One or an active fragment thereof and the bacterial virulence protein membrane translocation domain or an active fragment thereof are linked by a polypeptide linker (linker peptide) to form a fusion protein.
  • the linker comprises 0-50 amino acids; preferably 5-30 amino acids, more preferably 10-20 amino acids.
  • the sequence of the linker peptide includes at least one cleavage site sequence of an intracellular enzyme, and the cleavage site sequence is not present in the One sequence or the bacterial virulence protein membrane translocation domain sequence. on. Therefore, the fusion protein is not efficiently digested into the cell outside the cell, and when it enters the cell, One is separated from the bacterial virulence protein membrane translocation domain by the action of the intracellular enzyme.
  • the linker peptide is a linker peptide comprising a Furin cleavage site.
  • the extracellular environment is usually neutral or slightly alkaline, while Furin only works under acidic conditions.
  • Furin only works under acidic conditions.
  • the linker peptide has the amino acid sequence shown at positions 105-120 of SEQ ID NO: 2.
  • the One or an active fragment thereof is located at the amino terminus of the fusion protein; and the virulence protein membrane translocation domain or an active fragment thereof is located at the carboxy terminus (C-terminus) of the fusion protein.
  • the position of the two proteins may be interchanged, that is, the One or an active fragment thereof is located at the carboxy terminus of the fusion protein; the virulence protein membrane translocation domain or an active fragment thereof is located in the fusion The amino terminus of the protein.
  • the One and the bacterial virulence protein membrane translocation domain or their active fragments are directly ligated, for example, the bacterial virulence protein membrane translocation domain is directly linked to the coding gene of One, and the fusion expression is No amino acid linkers are added between the two.
  • the results of the present inventors show that the addition of a preferred linker of One to the bacterial virulence protein membrane translocation domain is better than that of the protein obtained without the linker.
  • the amino terminus (or carboxy terminus) of the fusion protein may also contain one or more polypeptide fragments as a protein tag.
  • Any suitable label can be used in the present invention.
  • the tag may be FLAG, HA, HA1, c-Myc, 6-His or 8-His. These tags can be used to purify the fusion protein.
  • Onc-DT fusion protein constructed by the present invention greatly enhances the toxicity of tumor cells, including some cells which were originally insensitive to One.
  • Onc-DT fusion protein can not only replace the traditional toxic protein to construct immunotoxin, because One itself has the ability to recognize tumor cells, and it can also become an independent anticancer drug.
  • Onc-DT fusion protein is more lethal to tumors and has a wider range of applications.
  • the invention provides an isolated nucleic acid encoding the fusion protein, or a complementary strand thereof.
  • Any suitable DNA structure encoding One or its active fragments is suitable for use in the present invention.
  • Any suitable DNA structure encoding a bacterial virulence protein membrane translocation domain or an active fragment thereof is also suitable for use in the present invention.
  • the sequences mentioned in the examples below are all applicable to the method of the invention.
  • the nucleic acid encoding the fusion protein has the nucleotide sequence shown in SEQ ID NO: 1.
  • the DNA sequence encoding the fusion protein of the present invention can be synthesized by whole sequence, or the DNA sequence encoding the amino acid of the translocation domain of One and bacterial virulence protein membrane can be obtained by PCR amplification, and then spliced together to form a fusion encoding the present invention.
  • the DNA sequence encoding the fusion protein of the present invention After obtaining the DNA sequence encoding the fusion protein of the present invention, it is ligated into a suitable expression vector and transferred to a suitable host cell. Finally, the fusion protein of the present invention is obtained by culturing the transformed host cells by isolation and purification.
  • the invention also provides a vector comprising a nucleic acid molecule encoding the fusion protein.
  • the vector may further comprise an expression control sequence operably linked to the sequence of the nucleic acid molecule to facilitate expression of the fusion protein.
  • operably linked refers to a condition in which portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence.
  • signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide, then the signal peptide (secretion leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, then it is operably linked to A coding sequence; if the ribosome binding site is placed at a position that enables translation, then it is operably linked to the coding sequence.
  • “operably linked to” means adjacent, and for secretory leader sequences means adjacent in the reading frame.
  • any suitable vector can be used, such as some vectors for cloning and expression of bacterial, fungal, yeast and mammalian cells, such as Pouwels et al., Cloning Vector: Laboratory Manual (Lasevier latest edition) describe.
  • Various carriers known in the art such as commercially available carriers can be used.
  • a commercially available vector is selected, and then a nucleotide sequence encoding a novel fusion protein of the present invention is operably linked to an expression control sequence to form a protein expression vector.
  • the vector is a prokaryotic vector, such as a pET vector.
  • Expression vectors include fusion protein DNA sequences ligated with appropriate transcriptional and translational regulatory sequences, such as genes derived from mammals, microorganisms, viruses or insects. Regulatory sequences can include transcriptional promoters, operons, enhancers, ribosome binding sites, or suitable sequences that control the initiation and termination of transcription and translation. When the fusion protein sequence requires the regulation of sequence functions, appropriate regulatory sequences are ligated. Thus, the promoter sequence is ligated to the front of the DNA sequence encoding the fusion protein. The ability to replicate within a host cell is usually controlled by the origin of replication. Screening genes for use in transformant recognition can also be added to expression vectors.
  • the coding sequence for a signal peptide of the non-native One or bacterial viral membrane translocation domain can be introduced into an expression vector.
  • the signal peptide (secretion leader) sequence can be fused to the fusion protein coding sequence such that the translated fusion protein can be secreted outside the cell.
  • the signal peptide enhances the host cell's secretion of the chimeric polypeptide to the extracellular.
  • the signal peptide can be cleaved off during secretion of the polypeptide from the cell.
  • recombinant cells containing a nucleic acid sequence encoding the fusion protein are also included in the present invention.
  • the term "host cell” includes prokaryotic cells and eukaryotic cells.
  • Common prokaryotic host cell package Escherichia coli, Bacillus subtilis, etc.; for example, E. coli, such as Escherichia coli HMS174 (DE3), or BL21 (DE3).
  • E. coli such as Escherichia coli HMS174 (DE3), or BL21 (DE3).
  • Common eukaryotic host cells include yeast cells, insect cells, and mammalian cells.
  • a prokaryotic cell is used as a host cell, and a fusion protein which retains good One antitumor activity and has good cell membrane permeability is obtained after expression, renaturation and purification.
  • the method comprises culturing a recombinant cell comprising a fusion protein encoding a nucleic acid.
  • the fusion protein comprises One or an active fragment thereof and a bacterial virulence protein membrane translocation domain or an active fragment thereof.
  • the method can include allowing the cell to express the encoded fusion protein and renaturation of the expressed fusion protein.
  • the method can further comprise isolation and/or purification of the renatured fusion protein.
  • the fusion protein obtained as described above can be purified to a substantially uniform property, for example, a single band on SDS-PAGE electrophoresis.
  • a commercially available ultrafiltration membrane can be used to separate the protein, for example, from Millipore, Pellicon, etc., and the expression supernatant is first concentrated.
  • the concentrate may be further purified by gel chromatography or by ion exchange chromatography. For example, anion exchange chromatography (DEAE, etc.) or cation exchange chromatography.
  • the gel matrix may be a matrix commonly used for protein purification such as agarose, dextran, polyamide, and the like.
  • the Q- or SP- group is an ideal ion exchange group.
  • the purified product may be further purified by hydroxyapatite adsorption chromatography, metal chelate chromatography, hydrophobic interaction chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). All of the above purification steps can utilize different combinations to ultimately achieve a substantially uniform protein purity.
  • the expressed fusion polypeptide can be purified using an affinity chromatography column containing a specific antibody, receptor or ligand of the One or bacterial virulence membrane translocation domain.
  • affinity column containing a specific antibody, receptor or ligand of the One or bacterial virulence membrane translocation domain.
  • the fusion polypeptide bound to the affinity column can be eluted by conventional methods such as high salt buffer, pH change, and the like.
  • the recombinant protein and the nucleic acid encoding the recombinant protein can be produced by an appropriate method, for example, chemical synthesis, recombinant expression, or a combination thereof. See “Current Protocols in Molecular Biology” by John Wiley & Sons, Inc, 2000, and “Molecular Coning: A Laboratory Manual", published by Cold Spring Harbor Laboratory Press, 1989.
  • the nucleic acid sequence encoding the fusion protein is prepared using PCR techniques, as described in Prodromou et al. (Protein Eng. 5(8): 827-29; 1992). Use of fusion protein
  • the fusion protein of One of the present invention and the bacterial virulence protein membrane translocation domain can be used to prepare a tumor suppressing composition.
  • the fusion protein of the invention not only has good tumor-killing biological activity, but also has stronger ability to enter the cytoplasm of tumor cells than single one, and thus has superior tumoricidal effect than One, and can be used for developing a more effective anti-tumor effect. Oncology drugs.
  • the fusion protein of the present invention has a broader spectrum of functions of inhibiting tumor cells than simple One. As can be seen from a specific example of the present invention, the fusion protein has an excellent inhibitory effect on myeloma cells, leukemia cells, neuroblastoma, and acute myeloid leukemia cells.
  • the "tumor" of the present invention may be of various types, for example, but may include, but is not limited to: malignant mesothelioma; lung cancer; leukemia; malignant lymphoma; myeloma; malignant melanoma; Cancer; Liver cancer; Nasopharyngeal carcinoma; Esophageal cancer; Gastric cancer; Colon cancer; Prostate cancer; Cervical cancer; Oral cancer; Salivary gland tumor; Nasal and paranasal sinus malignant tumor; Laryngeal cancer; Ear tumor; Eye tumor; Thyroid tumor; Mediastinal tumor; chest wall; pleural tumor; small intestine tumor; biliary tract tumor; pancreas and periampullary tumor; mesenteric and retroperitoneal tumor; renal tumor; adrenal tumor; bladder tumor; prostate cancer; testicular tumor; penile cancer; endometrial cancer; Ovarian malignancies; malignant trophoblastic tumors; vulvar and vaginal cancer; soft tissue
  • the fusion protein When used to inhibit tumors in a mammal, the fusion protein may be administered systemically, or locally, depending on factors such as the type of tumor, the site of growth, and the degree of progression. combination
  • the present invention also provides a composition
  • a composition comprising: (i) an effective amount (e.g., 0.0001 to 1000 uM) of a fusion protein of One of the present invention and a bacterial virulence protein membrane translocation domain; and (ii) a pharmaceutically acceptable carrier.
  • the composition is typically a pharmaceutical composition for inhibiting tumor cell growth or treating a tumor.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies;), ie, a substance having a reasonable benefit/risk ratio .
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • the term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A full description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).
  • the pharmaceutically acceptable carrier in the composition may contain liquids such as water, saline, glycerol and sorbitol.
  • liquids such as water, saline, glycerol and sorbitol.
  • auxiliary substances such as lubricants, glidants, wetting or emulsifying agents, P H buffering substances and stabilizers such as albumin and the like may also be present in these carriers.
  • compositions can be formulated into a variety of dosage forms suitable for mammalian administration, including, but not limited to, injections, capsules, tablets, emulsions, suppositories, and sprays.
  • a safe and effective amount of a fusion protein of the invention is administered to a mammal (e.g., a human), wherein the safe and effective amount is typically at least about 1 microgram per kilogram of body weight, and in most cases does not exceed about 10 mg / kg body weight, preferably the dose is about 1 microgram / kilogram body weight - about 1 mg / kg body weight.
  • a mammal e.g., a human
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • compositions of the invention can be used directly to kill tumor cells.
  • it can be used in combination with other therapeutic agents or adjuvants.
  • the main advantages of the invention are:
  • the fusion protein of One and the bacterial virulence protein membrane translocation domain was first provided and prepared, and it was confirmed that the fusion of the bacterial virulence protein membrane translocation domain with One well retained the nuclease activity of One, and significantly improved One's ability to enter the cytoplasm of the cell and the broad spectrum of anti-tumor, has a superior tumoricidal effect than One, on tumor cells (including The toxicity of some tumor cells that were originally insensitive to One is greatly enhanced.
  • the fusion protein of the present invention can inhibit the growth of various tumors, and is useful for treating many tumors, and is more broadly broad.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are intended to illustrate the invention and not to limit the scope of the invention. The following examples do not specify the specific experimental conditions, usually according to the conditions described in the conventional conditions, such as Sambrook et al., Molecular Cloning: Laboratory Manual, Third Edition (Cold Spring Harbor Laboratory Press, 2001), or according to the manufacturer. The suggested conditions.
  • Example 1 Onc-DT 2() 3 - 39 Synthesis of fusion gene and construction of E. coli expression plasmid
  • a 16-amino acid ligation peptide containing a furin cleavage site joins One and DT.
  • the DNA sequence of furin-DT is a whole gene synthesis, and One and furin- The DT sequences were ligated, and 25 One-terminal base sequences were added to the 5' end of the whole gene during the whole gene synthesis.
  • the whole gene synthesis sequence is shown in SEQ ID NO: 3.
  • the following pair of primers were designed and synthesized based on the gene sequences of One and DT:
  • Onc-DT-U (SEQ ID NO: 4):
  • Onc-DT-L (SEQ ID NO: 5):
  • Onc-DT-U is the coding sequence of the N-terminus of One mature protein
  • Onc-DT-L is the DT C-terminal coding sequence plus the reverse complement of the stop codon and Sail restriction site.
  • Onc-DT-U and Onc-DT-L were used as primers, and a mixture of One gene fragment and furin-DT gene fragment was used as a template.
  • the Onc-DT gene sequence was amplified by high-fidelity DNA polymerase and the product was gel-purified. After that, it was digested with Sail, and the insert of Onc-DT was recovered by 1% agarose gel electrophoresis.
  • the plasmid pET22b( + ) (; Novagen) was digested with Ball and Sail respectively, and the vector fragment of pET22b was recovered by 1% agarose gel electrophoresis.
  • the insert and the vector fragment were ligated with T4 DNA ligase to obtain pET22b-Onc-DT. 2 . 3 _ 39 .
  • the plasmid, a schematic representation of the recombinant plasmid is shown in Figure 1. Fusion protein of Example 2 Onc-DT 203 -39o embodiment
  • Example 3 Inclusion body collection and washing The collected cells were suspended in buffer SSB-A (20 mM Tris-HCl, 10 mM EDTA, pH 8.0), and the cells were disrupted by a sonicator; the inclusion bodies were collected by centrifugation at 4 ° C, 12000 rpm, 10 lOmin; WIBB ( Inclusion bodies were suspended by 20 mM Tris-HCl, 1 M guanidine hydrochloride, 65 mM DTT, 2 mM EDTA, pH 8.0, and the inclusion bodies were collected at 4 ° C, 12000 rpm, 1-10 min.
  • buffer SSB-A (20 mM Tris-HCl, 10 mM EDTA, pH 8.0
  • WIBB Inclusion bodies were suspended by 20 mM Tris-HCl, 1 M guanidine hydrochloride, 65 mM DTT, 2 mM EDTA, pH 8.0, and the inclusion bodies were collected at 4 ° C, 12000 rpm, 1
  • the inclusion bodies obtained from 1 L of the bacterial solution were dissolved in 6 ml of DIBB (20 mM Tris-HCl, 7 M guanidine hydrochloride, 65 mM DTT, 2 mM EDTA, pH 8.0), and after stirring with nitrogen, stirred overnight; 4 ° C, 12000 rpm X 15 Min, the supernatant was collected by centrifugation, and the protein was quantified to a concentration of 10 mg/ml ; 1 ml of the inclusion body solution was slowly diluted to 200 ml RB (0.5 M 10 Arginine-HCl, 20 mM Tris-HCl, 0.9 mM oxidized glutathione) , ImM EDTA, pH 9.0), 4°C,
  • the column was Q-Sepharose (2X2 cm, Pharmacia), equilibrated with 20 mM Tris-HCl, 1 mM EDTA, pH 9.0, flow rate 1 ml/min. 30 column volumes were eluted with 1 M NaCl and discarded, and the pH of the loading effluent was adjusted to 10.0 for use.
  • Elution method 0-100% WB elution 30 column volumes and collected step by step, the collection is equivalent to about 30% of the protein peak.
  • Myeloma SP2/0 cells purchased from the Chinese Academy of Sciences Shanghai Institute of Life Sciences cell bank cultured in RPMI 1640 (GIBICO) containing 10% calf serum, penicillin (100 units/ml), streptomycin (100 g/ml) In the medium.
  • RPMI 1640 GIBICO
  • penicillin 100 units/ml
  • streptomycin 100 g/ml
  • a 96-well plate add 90 ⁇ M medium (5 ⁇ 10 4 cells/ml) per well, and incubate for 1 hour in a C0 2 (5%) incubator.
  • Onc-DT 2 . 3.39 The results of the comparison of the cytotoxic effects of One and One on myeloma cells SP2/0 are shown in Fig. 3A. It can be seen that the Onc-DT 20 3-39o IC 50 is about 2 X 10" 8 mol/L; One IC 5 is greater than 8 X 10" 6 mol/L, indicating Onc-DT 2 . 3. The toxicity of 390 to tumor cells is clearly about 400 times greater than that of One.
  • Human leukemia cell K562 (purchased from the Chinese Academy of Sciences Shanghai Institute of Life Sciences cell bank;) cultured in DMEM (GIBICO) medium containing 10% calf serum, penicillin (100 units/ml), streptomycin (100 g/ml) in. 96-well plate culture, add 90 ⁇ M medium per well to C5 X 10 4 cells/ml), and incubate for 12 hours in a C0 2 (;5%) incubator. Add protein samples according to the set concentration gradient (One protein respectively) And Onc-DT 2()3 _ 39() protein) 10 ⁇ l, control group plus PBS, cultured for 72 hours, cell viability was measured by MTT assay.
  • Onc-DT 2 A comparison of the cytotoxic effects of 3 _ 39Q and One on human leukemia cell K562 is shown in Figure 3B. See Onc-DT 203-39 .
  • IC 5 It is about 2.5 X 10- 8 mol/L; the IC 50 of Onc is about 1 X l() - 6 mol/L; the description is Onc-DT 203 - 39 .
  • Toxic to tumor cells is about 40 times more potent than One.
  • Neuroblastoma SH-SY5Y (purchased from the Chinese Academy of Sciences Shanghai Institute of Life Sciences cell bank;) cultured in DMEM containing 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 g/ml) (GIBICO ) in the medium.
  • DMEM fetal bovine serum
  • penicillin 100 units/ml
  • streptomycin 100 g/ml
  • 96-well plate culture add 90 ⁇ M medium (5 ⁇ 10 4 cells/ml) per well, and culture for 12 hours in C0 2 (;5%;) incubator, add protein samples according to the set concentration gradient (; It was One and Onc-DT 2. 3 _ 39Q ) 10 ⁇ l , the control group was added with PBS, and after 72 hours of culture, the cell viability was measured by sputum method.
  • Onc-DT 203 -39o One and toxic effects on human neuroblastoma SH-SY5Y cells for comparison 3 C shown in FIG. Onc-DT 20 3-39o IC 5 .
  • 3. 390 is about 27 times more toxic to tumor cells than One.
  • Human acute myeloid leukemia cell line HL60 obtained from the Institute of Biochemistry and Cell Research, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences cultured in serum containing 10% calf, penicillin (100 units/ml), streptomycin (100 g/ml) In RPMI 1640 (GIBICO) medium.
  • RPMI 1640 GIBICO
  • 96-well plate culture add 90 ⁇ M medium (5 ⁇ 10 4 cells/ml) per well, and incubate for 12 hours in a C0 2 (5%) incubator, add protein samples according to the set concentration gradient (ONC and ⁇ 2 . 3 . 39 .) 10 ⁇ l, control group plus PBS, cultured for 72 hours, cell viability was measured by MTT assay.
  • the inventors also prepared a fusion protein containing a 6His-tag at the N-terminus.
  • the transformation and expression of the recombinant vector were the same as in Examples 2-4.

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Description

核糖核酸酶与毒蛋白膜转位结构域融合蛋白
及其制备方法和用途
技术领域
本发明属于 DNA重组技术和生物医药领域。 更具体地， 本发明涉及核糖核酸酶与细 菌毒蛋白 (毒素)膜转位结构域的融合蛋白， 编码该融合蛋白的 DNA序列， 含该 DNA序列 的载体， 含该载体的宿主细胞， 利用基因工程技术制备该融合蛋白的方法， 以及该融合蛋 白在肿瘤治疗方面的应用。 背景技术
核糖核酸酶 (Onconase 又称： P30 蛋白， Ranpirnase, 简称 One)是从北方豹蛙 (Rana pipiens)的卵母细胞和早期胚胎中分离得到的一种核酸酶， 属于 RNase A超家族。 研究表 明 One通过胞吞作用进入哺乳动物细胞， 在胞浆内选择性地降解 tRNA， 抑制蛋白质生物 合成进而抑制细胞增殖和引起细胞凋亡。 体外实验表明， One对多种肿瘤细胞具有抗增殖 和细胞毒性作用， 如： 人的恶性间皮细胞瘤， 非小细胞肺癌， 前列腺癌细胞、 胰腺癌细胞 和白血病细胞等。小鼠模型的体内实验证明， One可以抑制肿瘤生长，延长小鼠存活时间， 增强多种抗肿瘤药物的效果。 目前， One作为一种抗恶性间皮细胞瘤的药物已经进入三期 临床， 是一种具有良好前景的抗肿瘤药物。 最近， 美国和澳大利亚己批准 Onconase作为 非常见病药物 (Orphan Drug)投放市场。
然而，进一步的研究发现，不同肿瘤细胞对 One的敏感性不同 (Uldch Arnold & Renate Ulbrich-Hofmann. Natural and engineered ribonucleases as potential cancer therapeutics. Biotechnol Lett (2006) 28: 1615-1622)。 这大大限制了 One在肿瘤治疗中的适用范围。
因此， 本领域需要进一步开发具有更好的抑制肿瘤效果且广谱性更强的抗肿瘤药物。 发明内容
本发明的目的在于提供核糖核酸酶与细菌毒蛋白膜转位结构域的融合蛋白， 编码该 融合蛋白的 DNA序列， 含该 DNA序列的载体， 含该载体的宿主细胞， 利用基因工程制 备该融合蛋白的方法， 以及该融合蛋白在肿瘤治疗方面的应用。
在本发明的第一方面， 提供一种融合蛋白， 所述的融合蛋白包括：
(1) 核糖核酸酶 (One);
(2) 细菌毒蛋白的膜转位结构域；
(3) 位于 (1)和 (2)之间的由 0-50个 (较佳地为 5-30个， 更佳的为 10-20个)氨基酸构成 的连接肽。
在另一优选例中， 所述的核糖核酸酶位于融合蛋白的氨基端； 所述的细菌毒蛋白的 膜转位结构域位于融合蛋白的羧基端。
在另一优选例中， 所述的细菌毒蛋白的膜转位结构域位于融合蛋白的氨基端， 所述 的核糖核酸酶位于融合蛋白的羧基端。
在另一优选例中， 所述的融合蛋白基本上由（1)、 (3), 相连接而构成。 更佳地， 所 述的融合蛋白由（1)、 （3)、 （2)相连接而构成。
在另一优选例中， 所述的连接肽的序列中包括至少一个胞内蛋白酶的酶切位点序列， 从而在进入胞内后将 (1)和 (2)分离。
在另一优选例中， 所述的酶切位点序列不存在于核糖核酸酶序列或细菌毒蛋白的膜 转位结构域序列上。
在另一优选例中， 所述的胞内蛋白酶是特异性位于细胞内的蛋白酶。
在另一优选例中， 所述的胞内酶选自（但不限于)： Furin, 基质金属蛋白酶、 前列腺特 有抗原和组织蛋白酶。
在另一优选例中， 所述的融合蛋白在进入细胞后被胞内酶酶切。
在另一优选例中， 所述的细胞毒蛋白选自（但不限于 白喉毒素 (DT)或绿脓杆菌外毒 素 A(PE)。
在另一优选例中， 所述的白喉毒素的膜转位结构域是：
(a) 如 SEQ ID NO: 2中第 121-308位的氨基酸序列的蛋白； 或
(b) 将 (a)所限定的蛋白的氨基酸序列经过一个或多个氨基酸残基的取代、 缺失或添加 而形成的， 且具有 (a) 所限定的蛋白功能的由 (a)衍生的多肽； 和 /或
所述的核糖核酸酶是：
(al) 如 SEQ ID NO: 2中第 1-104位所示的氨基酸序列（较佳地由 SEQ ID NO: 2中第 1-104位的氨基酸序列构成)； 或
(bl) 将 (al)所限定的蛋白的氨基酸序列经过一个或多个氨基酸残基的取代、 缺失或添 加而形成的， 且具有 (al) 所限定的蛋白功能的由 (al)衍生的多肽； 和 /或
所述的连接肽具有 SEQ ID NO: 2中第 105-120位所示的氨基酸序列 (较佳地由 SEQ ID NO: 2中第 105-120位的氨基酸序列构成）。
较佳地， 所述的融合蛋白具有 SEQ ID NO: 2所示的氨基酸序列。
在本发明的第二方面， 提供一种核酸分子， 所述的核酸分子编码所述的融合蛋白。 在另一优选例中， 所述的核酸分子具有 SEQ ID NO: 1所示的核苷酸序列。
在本发明的第三方面， 提供一种载体， 它含有所述的核酸分子。
在本发明的第四方面， 提供一种基因工程化的细胞， 所述的细胞含有所述的载体； 或所述的细胞基因组中整合有所述的核酸分子。
在本发明的第五方面， 提供一种产生所述的融合蛋白的方法， 所述的方法包括：
(A) 培养所述的宿主细胞， 从而表达所述的融合蛋白；
(B) 分离出所述的融合蛋白。
在本发明的第六方面， 提供所述的融合蛋白的用途， 用于制备抑制肿瘤细胞生长或 治疗肿瘤的组合物。 所述的肿瘤选自（但不限于)： 恶性间皮细胞瘤； 肺癌； 白血病； 恶性 淋巴瘤； 骨髓瘤； 恶性黑色素瘤； 乳腺癌； 神经系统肿瘤； 肝癌； 鼻咽癌； 食管癌； 胃癌； 结肠癌； 前列腺癌； 宫颈癌； 口腔癌； 唾液腺肿瘤； 鼻腔与鼻旁窦恶性肿瘤； 喉癌； 耳部 肿瘤； 眼部肿瘤； 甲状腺肿瘤； 纵隔肿瘤； 胸壁； 胸膜肿瘤； 小肠肿瘤； 胆道肿瘤； 胰腺 与壶腹周围肿瘤； 肠系膜与腹膜后肿瘤； 肾脏肿瘤； 肾上腺肿瘤； 膀胱肿瘤； 前列腺癌； 睾丸肿瘤； 阴茎癌； 子宫内膜癌； 卵巢恶性肿瘤； 恶性滋养细胞肿瘤； 外阴癌与阴道癌； 软组织肿瘤； 骨肿瘤； 或皮肤及附件肿瘤。
在本发明的第七方面， 提供一种抑制肿瘤细胞生长或治疗肿瘤的组合物， 所述的组 合物含有：
(i) 有效量的所述的融合蛋白； 和
(ii) 药学上可接受的载体。
在另一优选例中， 所述的组合物是药物组合物。
另一方面， 还提供一种抑制（如体外抑制）肿瘤细胞生长的方法， 所述的方法包括利 用所述的融合蛋白处理肿瘤细胞。
另一方面， 还提供一种抑制肿瘤细胞生长或治疗肿瘤的方法， 所述方法包括： 给予 需要抑制肿瘤细胞生长或治疗肿瘤的对象有效量的所述的融合蛋白。
本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易见的。 附图说明
图 1显示了含 ONC-DT融合蛋白基因的大肠杆菌表达质粒 pET22b- ONC-DT2Q3.390的 示意图。
图 2显示了本发明 Onc-DT2。3.39Q的表达纯化效果蛋白 SDS-PAGE电泳图。 其中， 泳道 1.蛋白分子量标准；
泳道 2.诱导前菌体总蛋白；
泳道 3.诱导 3h后菌体总蛋白；
泳道 4.诱导后菌体包涵体；
泳道 5.复性并纯化后 Onc-DT2。3-390。
图 3显示了 MTT法测 Onc-DT2。3.39。对小鼠骨髓瘤细胞 SP2/0和人类白血病细胞 K562 的细胞毒性效果图。 其中，
A: Onc-DT2。3.39Q和 One对小鼠骨髓瘤细胞 SP2/0的细胞毒性作用比较。
B: Onc-DT2。3.39。和 One对人白血病细胞 K562的细胞毒性作用比较。
C: Onc-DT2Q3_39Q和 One对人神经母细胞瘤 SH-SY5Y的细胞毒性作用比较。
D: Onc-DT2。3_39Q和 One对人急性髓系白血病细胞株 HL60的细胞毒性作用比较。 具体实施方式
本发明人经过长期的研究和试验， 首次将核糖核酸酶 (One)基因和细菌毒蛋白的膜转 位结构域基因融合在一起， 并在大肠杆菌中进行了高效表达， 经纯化获得了核糖核酸酶- 细菌毒蛋白膜转位结构域的融合蛋白。所述融合蛋白良好地保留了 One的核酸酶活性， 又 提高了 One进入细胞胞质的能力和抗肿瘤的广谱性， 具有比 One更优异的杀肿瘤效果， 对肿瘤细胞 (；包括一些原来对 One 不敏感的肿瘤细胞)的毒性大为增强 (对不同细胞的毒性 可分别提高 10-1000倍；)， 从而可作为一种新的更有效更广谱的抗肿瘤药物。 含有核糖核酸酶和细菌毒蛋白膜转位结构域的融合蛋白
如本文所用，术语"核糖核酸酶和白喉毒蛋白膜转位结构域的融合蛋白"、 " ONC-DT 融合蛋白"等可互换使用， 都指由核糖核酸酶氨基酸序列和白喉毒蛋白膜转位结构域氨基 酸序列融合而成的蛋白， 其中在两者之间可以有或者没有连接肽序列。
本发明提供一种融合蛋白， 该蛋白包括 One蛋白或其生物活性片段， 和细菌毒蛋白 膜转位结构域或其生物活性片段， 该分子能用于抑制肿瘤。 更优选的， 所述的融合蛋白是 一种分离的蛋白， 与其它蛋白、 多肽或分子无联系， 是重组宿主细胞培养的纯化产物或作 为一种纯化的提取物。 如本文所用， 所述的 "含有"， "具有 "或 "包括"包括了 "包含"、 "主要由 ......构 成"、 "基本上由 ......构成"、 和 "由 ......构成"； "主要由 ......构成"、 "基本上由 ......构 成"和 "由 ... ...构成"属于 "含有" 、 "具有"或 "包括" 的下位概念。 核糖核酸酶
任何适合的 One均可用于制备本发明的融合蛋白。本发明中的 One是指一种多肽， 能 通过胞吞作用进入哺乳动物细胞 (特别是肿瘤细胞)， 在胞浆内选择性降解 tRNA, 或其它 RNA,从而抑制蛋白质合成， 并进而抑制细胞增殖和引起细胞凋亡。 One 对多种肿瘤细胞 具有抗增殖和细胞毒性作用。
One或其中的生物活性片段包括一部分保守氨基酸替代序列，所述经氨基酸替换的序 列并不影响其活性。适当替换氨基酸是本领域的公知的技术， 所述技术可以很容易地被实 施并且确保不改变所得分子的生物活性。 这些技术使人们认识到， 一般来说， 在一种多肽 的非必要区域改变单个氨基酸基本上不会改变生物活性。见 Watson等 Molecular Biology of The Gene, 第四版， 1987， The Benjamin/Cummings Pub. Co. P224。 表 1是这种替换的一 些示例。 表 1
Figure imgf000006_0001
可能还有其它的类似的可替换性， 这种可替换性往往可以根据经验确定或者根据己 知的保守序列进行确定。
任何一种 One的生物活性片段都可以应用到本发明中。 在这里， One的生物活性片段的 含义是指作为一种多肽片段，在与毒蛋白膜转位结构域连接并形成融合蛋白后，其仍然能保持 全长的 One的全部或部分功能。 通常情况下， 所述的生物活性片段至少保持 50%的全长 One 的抑制肿瘤活性。 在更优选的条件下， 所述活性片段能够保持 60%、 70%、 80%、 90%、 95%、 99%、 或 100%的抑制肿瘤活性。
经过一个或多个 (；如 1-30个， 较佳地 1-20个， 更佳的 1-10个， 进一步的 1-5个)氨基 酸残基的取代、缺失或添加而形成的 One的氨基酸序列也包括在本发明中。所述的经过一 个或多个氨基酸残基的取代、 缺失或添加而形成的 One也具有抑制肿瘤的活性。
本发明也可采用经修饰或改良的 One, 比如， 可采用为了延长其半衰期、改善其稳定 性或增强其杀伤肿瘤细胞能力而加以修饰或改良的 One。所述经过修饰或改良的 One或者 基因可以是与天然存在的 One或基因虽然具有一定的不同点，但也能杀伤肿瘤细胞，且不 会带来其它不良影响或毒性。也就是说，任何不影响 One的生物活性或者说是基因的生物 功能的变化形式都可用于本发明中。
在本发明的优选方式中， 所述的 One的氨基酸序列可以与 SEQ ID NO: 2中第 1-104 位所示的序列基本上相同。 优选的， 所述的 One具有 SEQ ID NO: 2中第 1-104位所示的 序列； 更优选的， 所述的 One由 SEQ ID NO: 2中第 1-104位所示的序列构成。 细菌毒蛋白膜转位结构域
本发明的细菌毒蛋白的膜转位结构域是一类对毒素的的内吞有作用的蛋白质结构 域。所述的细菌毒蛋白包括 (但不限于)： DT或 PE。尽管已知 DT与 PE的膜转位结构域有 助于增强多种分子的内吞， 然而以往并不知道它们对于 One的内吞是否有作用。
作为本发明的优选方式， 所述的细菌毒蛋白是 DT， DT是白喉杆菌产生的细菌外毒 素， 其由三个结构域组成， N端为催化结构域， 中间为膜转位结构域， C端为受体结合结 构域。膜转位结构域的作用为帮助催化结构域穿过胞内体质膜进入胞质， 发挥催化结构域 的细胞毒性作用。
DT膜转位结构域或其中的生物活性片段包括一部分保守氨基酸替代序列， 所述经氨 基酸替换的序列可能并不影响其活性。 可釆用的一部分氨基酸替换的示例见表 1。 可能还 有其它的类似的可替换性，这种可替换性往往可以根据经验确定或者根据己知的保守序列 进行确定。
任何一种 DT膜转位结构域的生物活性片段都可以应用到本发明中。 在这里， DT膜 转位结构域的生物活性片段的含义是指作为一种多肽片段，在与 One连接并形成融合蛋白 后， 其仍然能保持完整的 DT膜转位结构域的全部或部分功能。 经过一个或多个氨基酸残 基的取代、 缺失或添加而形成的 DT膜转位结构域的氨基酸序列也包括在本发明中。 所述 的经过一个或多个氨基酸残基的取代、 缺失或添加而形成的 DT膜转位结构域也具有帮助 One穿透肿瘤细胞膜的功能。
本发明也可采用经修饰或改良的 DT膜转位结构域，比如，可采用为了延长其半衰期、 改善其稳定性、 增强其穿透细胞的能力或增强其穿透细胞的特异性而改良的 DT膜转位结 构域。 所述经过修饰或改良的 DT膜转位结构域或者基因可以是与天然存在的 DT膜转位 结构域或基因虽然具有一定的不同点，但也能帮助 One穿透细胞膜， 且不会带来其它不良 影响或毒性。 也就是说， 任何不影响 DT膜转位结构域的生物活性或者说是基因的生物功 能的变化形式都可用于本发明中。
作为本发明的优选方式，所述的 DT膜转位结构域的氨基酸序列可以与 SEQ ID NO: 2 中第 121-308位所示的序列基本上相同。优选的，所述的 DT膜转位结构域具有 SEQ ID NO: 2中第 121-308位所示的序列； 更优选的， 所述的 DT膜转位结构域由 SEQ ID NO: 2中第 121-308位所示的序列构成。 连接 所述的 One或其活性片段和细菌毒蛋白膜转位结构域或其活性片段之间可以直接相 连接， 或者通过多肽连接子 (连接肽)连接。 作为本发明的一种优选的方式， 所述的 One或 其活性片段和细菌毒蛋白膜转位结构域或其活性片段通过多肽连接子 (连接肽)连接， 从而 形成融合蛋白。所述的连接子包括 0-50个氨基酸；较佳地为 5-30个氨基酸，更佳地为 10-20 个氨基酸。
作为本发明的优选方式， 所述的连接肽的序列中包括至少一个胞内酶的酶切位点序 列， 且该酶切位点序列不存在于 One序列或细菌毒蛋白膜转位结构域序列上。 因而， 融合 蛋白在细胞外不被酶切而高效地进入到细胞内， 当进入到细胞内后， 在胞内酶的作用下 One与细菌毒蛋白膜转位结构域分离。
作为本发明的一种特别优选的方式， 所述的连接肽是含有 Furin酶切位点的连接肽。 细胞外环境通常为中性或微碱性的， 而 Furin只有在酸性条件下才发挥作用， 当融合蛋白 进入胞内后， 可在酸性小泡中被 Furin酶切， 从而使得 One与毒蛋白膜转位结构域更易于 被释放到胞质中。 更优选的， 所述的连接肽具有 SEQ ID NO: 2中第 105-120位所示的氨 基酸序列。
作为一种优选的方式， 所述的 One或其活性片段位于融合蛋白的氨基端 端)； 所述 的毒蛋白膜转位结构域或其活性片段位于融合蛋白的羧基端 (C端)。 可选择地， 也可互换 两种蛋白所处的位置， 也即： 所述的 One或其活性片段位于融合蛋白的羧基端； 所述的毒 蛋白膜转位结构域或其活性片段位于融合蛋白的氨基端。
作为另一种方式， 将所述的 One和细菌毒蛋白膜转位结构域或它们的活性片段直接 连接， 比如把细菌毒蛋白膜转位结构域与 One的编码基因直接连接， 融合表达， 二者之间 不加氨基酸连接子。本发明人的结果显示， One与细菌毒蛋白膜转位结构域之间加上本发 明优选的连接子比不加连接子所获得的蛋白的抗肿瘤活性更佳。
此外，可选择地，所述的融合蛋白的氨基端 (或羧基端;)还可含有一个或多个多肽片段， 作为蛋白标签。任何合适的标签都可以用于本发明。例如，所述的标签可以是 FLAG, HA, HA1 , c-Myc, 6-His或 8-His等。 这些标签可用于对融合蛋白进行纯化。
实验结果证明， 本发明所构建的 Onc-DT融合蛋白对肿瘤细胞 （包括一些原来对 One 不敏感的细胞） 的毒性大为增强。 Onc-DT 融合蛋白不仅可以代替传统的毒蛋白构建免疫 毒素， 因为 One本身有识别肿瘤细胞的能力， 其本身也能成为一个独立的抗癌药物。较之 单独应用 One, Onc-DT融合蛋白对肿瘤的杀伤力更强， 适用范围更广。 核酸分子
另一方面， 本发明还提供了编码所述的融合蛋白的分离的核酸， 也可以是其互补链。 任何编码 One或其活性片段的合适的 DNA结构都适用于本发明。任何编码细菌毒蛋 白膜转位结构域或其活性片段的合适的 DNA结构也适用于本发明。 下文实例中提及的序 列都适用于本发明的方法。 作为本发明的优选方式，编码融合蛋白的核酸具有 SEQ ID NO: 1所示的核苷酸序列。 编码本发明融合蛋白的 DNA序列， 可以全序列人工合成， 也可用 PCR扩增的方法 获得编码 One和细菌毒蛋白膜转位结构域氨基酸的 DNA序列， 然后将其拼接起来， 形成 编码本发明融合蛋白的 DNA序列。 表达载体
在获得了编码本发明融合蛋白的 DNA序列之后， 将其连入合适的表达载体， 再转入 合适的宿主细胞。最后通过培养转化后的宿主细胞，通过分离纯化得到本发明的融合蛋白。
因此， 本发明还提供了包含编码所述融合蛋白的核酸分子的载体。所述的载体还可包 含与所述核酸分子的序列操作性相连的表达调控序列， 以便于所述融合蛋白的表达。
如本文所用， "操作性相连"或 "可操作地连于"指这样一种状况， 即线性 DNA序 列的某些部分能够影响同一线性 DNA序列其它部分的活性。 例如， 如果信号肽 DNA作 为前体表达并参与多肽的分泌， 那么信号肽 (分泌前导序列) DNA 就是可操作地连于多肽 DNA; 如果启动子控制序列的转录， 那么它就是可操作地连于编码序列； 如果核糖体结合 位点被置于能使其翻译的位置时，那么它是可操作地连于编码序列。一般"可操作地连于" 意味着相邻近， 而对于分泌前导序列则意味着在阅读框中相邻。
在本发明中， 任何合适的载体都可以使用， 比如一些用于细菌、 真菌、 酵母和哺乳 动物细胞的克隆和表达的载体， 如 Pouwels等， 克隆载体： 实验室手册 (Elsevier最新版) 中所描述的。 可选用本领域已知的各种载体如市售的载体。 比如， 选用市售的载体， 然后 将编码本发明新融合蛋白的核苷酸序列可操作地连于表达调控序列， 形成蛋白表达载体。 在本发明的一种实施方式中， 所述的载体为原核载体， 如 pET载体。
表达载体包括连接有合适的转录和翻译调控序列的融合蛋白 DNA序列，如来源于哺 乳动物、 微生物、 病毒或昆虫的基因。 调控序列可包括转录启动子、 操纵子、 增强子、 核 糖体结合位点或控制转录和翻译起始和终止的合适序列。在融合蛋白序列需要调节序列功 能的时候， 则连接合适的调控序列。 这样， 启动子序列被连接在编码融合蛋白 DNA序列 前端。在宿主细胞内的复制的能力通常由复制起始点控制。用于转化株识别的筛选基因也 可加入表达载体。
另外， 非天然的 One或细菌毒蛋白膜转位结构域的信号肽的编码序列可以引入表达 载体。 例如： 信号肽 (分泌引导物)序列可以和与融合蛋白编码序列融合， 从而使翻译的融 合蛋白可分泌到细胞外。信号肽可增强宿主细胞向胞外分泌嵌合多肽。信号肽在多肽从细 胞内分泌出来的过程中可被切去。 宿主细胞
此外， 含有编码所述融合蛋白的核酸序列的重组细胞也包括在本发明中。
在本发明中， 术语 "宿主细胞"包括原核细胞和真核细胞。 常用的原核宿主细胞包 括大肠杆菌、 枯草杆菌等； 例如可为大肠杆菌细胞 (E. coli)， 如大肠杆菌 HMS174(DE3)、 或 BL21(DE3)。 常用的真核宿主细胞包括酵母细胞、 昆虫细胞和哺乳动物细胞。
在本发明的优选实施方式中， 采用原核细胞作为宿主细胞， 经表达、 复性、 纯化后 获得保留了良好的 One抗肿瘤活性且细胞膜穿透性良好的融合蛋白。 生产融合蛋白的方法
生产融合蛋白的方法也已包括在本发明中。 所述方法包括培养含有融合蛋白编码核 酸的重组细胞。所述的融合蛋白包括 One或其有活性的片段以及细菌毒蛋白膜转位结构域 或其活性片段。所述方法可包括让细胞表达编码的融合蛋白， 以及使表达的融合蛋白的复 性。 在一个实例中， 所述方法还可包括复性的融合蛋白的分离和 /或纯化。
可将上述制备获得的融合蛋白纯化为基本均一的性质， 例如在 SDS-PAGE电泳上呈 单一条带。 例如， 当重组蛋白为分泌表达时， 可以采用商品化的超滤膜来分离所述蛋白， 例如 Millipore、 Pellicon等公司产品， 首先将表达上清浓缩。 浓缩液可采用凝胶层析的方 法进一步加以纯化， 或采用离子交换层析的方法纯化。 例如阴离子交换层析 (DEAE等)或 阳离子交换层析。 凝胶基质可为琼脂糖、 葡聚糖、 聚酰胺等常用于蛋白纯化的基质。 Q- 或 SP-基团是较为理想的离子交换基团。 最后， 还可用羟基磷灰石吸附层析， 金属螯合层 析， 疏水相互作用层析和反相高效液相色谱 (RP-HPLC)等方法对上述纯化产物进一步精制 纯化。 上述所有纯化步骤可利用不同的组合， 最终使蛋白纯度达到基本均一。
可利用含有 One或细菌毒蛋白膜转位结构域的特异性抗体、 受体或配体的亲和层析 柱对表达的融合性多肽进行纯化。 根据所使用的亲和柱的特性， 可利用常规的方法， 如高 盐缓冲液、 改变 pH等方法洗脱结合在亲和柱上的融合性多肽。
重组蛋白以及编码该重组蛋白的核酸可利用适当的方法制备， 例如， 化学合成、 重 组表达， 或其合并应用。 参见 John Wiley & Sons, Inc 2000年出版的 《Current Protocols in Molecular Biology》 ， 以及 Cold Spring Harbor Laboratory Pressl989年出版的 《Molecular Coning: A Laboratory Manual)) 。 在一个实例中， 编码融合蛋白的核酸序列是利用 PCR技 术制备的， 方法参见Prodromou等(Protein Eng. 5(8):827-29; 1992)论著。 融合蛋白的用途
本发明的 One与细菌毒蛋白膜转位结构域的融合蛋白可用于制备抑制肿瘤的组合物。 本发明的融合蛋白不仅具有良好的杀肿瘤生物活性，而且进入肿瘤细胞胞质的能力比单纯 的 One更强， 因而具有比 One更优异的杀肿瘤效果， 从而可用于开发一种更有效的抗肿 瘤药物。
本发明的融合蛋白比单纯的 One具有更为广谱的抑制肿瘤细胞的功能。 由本发明的 具体实例可见， 所述的融合蛋白对于骨髓瘤细胞、 白血病细胞、 神经母细胞瘤、 急性髓系 白血病细胞均具有优异的抑制效果。 因此， 本发明的 "肿瘤"可以是多种类型的， 例如可包括 (但不限于)： 恶性间皮细胞 瘤； 肺癌； 白血病； 恶性淋巴瘤； 骨髓瘤； 恶性黑色素瘤； 乳腺癌； 神经系统肿瘤； 肝癌； 鼻咽癌； 食管癌； 胃癌； 结肠癌； 前列腺癌； 宫颈癌； 口腔癌； 唾液腺肿瘤； 鼻腔与鼻旁 窦恶性肿瘤； 喉癌； 耳部肿瘤； 眼部肿瘤； 甲状腺肿瘤； 纵隔肿瘤； 胸壁； 胸膜肿瘤； 小 肠肿瘤； 胆道肿瘤； 胰腺与壶腹周围肿瘤； 肠系膜与腹膜后肿瘤； 肾脏肿瘤； 肾上腺肿瘤； 膀胱肿瘤； 前列腺癌； 睾丸肿瘤； 阴茎癌； 子宫内膜癌； 卵巢恶性肿瘤； 恶性滋养细胞肿 瘤； 外阴癌与阴道癌； 软组织肿瘤； 骨肿瘤； 或皮肤及附件肿瘤。
在用于抑制哺乳动物肿瘤时， 所述的融合蛋白可全身性给药， 或者局部给药， 具体 可视肿瘤的种类、 生长部位、 进展程度等因素决定。 组合物
本发明还提供一种组合物， 所述的组合物含有： （i) 有效量 （如 0.0001-lOOOuM) 的 本发明所述的 One与细菌毒蛋白膜转位结构域的融合蛋白； 和 (ii) 药学上可接受的载体。 所述的组合物通常是药物组合物， 用于抑制肿瘤细胞生长或治疗肿瘤。
如本文所用， "药学上可接受的" 的成分是适用于人和 /或哺乳动物而无过度不良副 反应 (如毒性、 刺激和变态反应;)的， 即具有合理的效益 /风险比的物质。 术语 "药学上可接 受的载体"指用于治疗剂给药的载体， 包括各种赋形剂和稀释剂。 该术语指这样一些药剂 载体： 它们本身并不是必要的活性成分， 且施用后没有过分的毒性。 合适的载体是本领域 普通技术人员所熟知的。在 Remington's Pharmaceutical Sciences (Mack Pub. Co. , N.J. 1991) 中可找到关于药学上可接受的载体的充分说明。在组合物中药学上可接受的载体可含有液 体， 如水、 盐水、 甘油和山梨醇。 另外， 这些载体中还可能存在辅助性的物质， 如润滑剂、 助流剂、 润湿剂或乳化剂、 PH缓冲物质和稳定剂， 如白蛋白等。
可将所述的组合物制成各种适合于哺乳动物给药的剂型， 所述剂型包括但不限于： 注射剂、 胶囊剂、 片剂、 乳剂、 栓剂和喷雾剂。
在使用时， 是将安全有效量的本发明所述的具有融合蛋白施用于哺乳动物（如人）， 其 中该安全有效量通常至少约 1微克 /千克体重， 而且在大多数情况下不超过约 10毫克 /千克 体重， 较佳地该剂量是约 1微克 /千克体重-约 1毫克 /千克体重。 当然， 具体剂量还应考虑 给药途径、 病人健康状况等因素， 这些都是熟练医师技能范围之内的。
本发明的组合物可直接用于杀伤肿瘤细胞。 此外， 还可同时与其它治疗剂或辅剂联 合使用。 本发明的主要优点在于：
(1) 首次提供且制备出 One与细菌毒蛋白膜转位结构域的融合蛋白，经证实细菌毒蛋 白膜转位结构域与 One的融合良好地保留了 One的核酸酶活性， 又显著提高了 One进入 细胞胞质的能力和抗肿瘤的广谱性， 具有比 One更优异的杀肿瘤效果， 对肿瘤细胞 (；包括 一些原来对 One不敏感的肿瘤细胞)的毒性大为增强。
(2) 本发明的融合蛋白可抑制多种肿瘤的生长， 对于治疗许多肿瘤均可用， 广谱性更 佳。 下面结合具体实施例， 进一步阐述本发明。 应理解， 所列举实施例仅用于说明本发 明而不用于限制本发明的范围。下列实施例中未注明具体实验条件的，通常按照常规条件， 例如 Sambrook等人， 分子克隆： 实验室手册第三版 (Cold Spring Harbor Laboratory Press, 2001 )中所述的条件， 或按照制造厂商所建议的条件。 实施例 1 Onc-DT2()3— 39。融合基因的合成以及大肠杆菌表达质粒的构建
为了便于 OnC-DT2()3— 39。从胞内体释放到胞质， 以一段 16个氨基酸的含 furin酶切位 点的连接肽将 One和 DT连接起来， furin-DT的 DNA序列为全基因合成， 为了便于 PCR 将 One与 furin-DT序列连接起来， 全基因合成时在 fudn 5'端添加了 25个 One末端碱基 序列。全基因合成序列见 SEQ ID NO: 3。根据 One和 DT的基因序列设计并合成了下列一 对引物：
Onc-DT-U (SEQ ID NO: 4)：
5' CCCAGGACTGGCTGACTTTCCA 3 '；
Onc-DT-L (SEQ ID NO: 5)：
5' GGGTCGACTTATTCCGGACCACCAGAAGC 3 Ό
其中， Onc-DT-U为 One成熟蛋白 N端的编码序列； Onc-DT-L为 DT C端编码序列 加终止密码子和 Sail酶切位点的反向互补序列。
以 Onc-DT-U和 Onc-DT-L为引物，含 One基因片断和 furin-DT基因片断的混合物为 模板，用高保真 DNA聚合酶 PCR扩增 Onc-DT基因序列，产物经凝胶纯化后用 Sail酶切， 1%琼脂糖凝胶电泳割胶回收得到 Onc-DT的插入片断。 将质粒 pET22b( + ) (； Novagen)分别 用 Ball和 Sail酶切， 1%琼脂糖凝胶电泳割胶回收得到 pET22b的载体片断， 将插入片断 和载体片断用 T4DNA连接酶连接得到 pET22b- Onc-DT2。3_39。质粒， 该重组质粒的示意图 见图 1。 实施例 2 Onc-DT203-39o融合蛋白的表达
将构建的表达质粒转化大肠杆菌 BL21(DE3)， 隔夜挑单克隆于 50ml LB培养基中， 于 37 °C培养过夜。 次日按 1 :20转接 1L TB培养基， 于 37 °C培养至 6()Q =2.0， 加 IPTG浓 度至 0.5mM诱导 3.5小时， 4 °C、 6000rpm X lOmin离心收集菌体， 菌体用于下一步的包涵 体收集和纯化。 实施例 3 包涵体收集和洗涤 将收集到的菌体悬浮在缓冲液 SSB— A (20 mM Tris-HCl, 10 mMEDTA, pH 8.0)中， 用超声波破碎仪破碎菌体； 4°C， 12000rpmX lOmin 离心，收集包涵体； WIBB (20 mM Tris-HCl, 1M 盐酸胍， 65mM DTT， 2mM EDTA, pH 8.0)悬浮包涵体， 搅拌过夜， 4°C， 12000rpmX lOmin收集包涵体。 实施例 4 蛋白变复性
将 1L菌液得到的包涵体溶解于 6ml DIBB (20 mM Tris-HCl, 7 M 盐酸胍， 65 mM DTT， 2mM EDTA, pH 8.0) 中， 充氮气后， 搅拌过夜； 4°C， 12000 rpmX 15 min, 离心 收集上清，将蛋白定量至浓度为 10mg/ml;将 1ml包涵体溶液缓慢稀释到 200 ml RB (0.5 M 10 Arginine-HCl, 20 mM Tris-HCl, 0.9 mM 氧化型谷胱甘肽， ImM EDTA, pH 9.0), 4°C，
12000 rpmX30 min离心弃沉淀； 在复性缓冲液 RB中 4°C复性 36〜48小时后， 用透析液 (0.5 M 尿素， 20 mM Tris-HCl， ImM EDTA, pH9.0)透析去盐。 蛋白溶液存于 4°C备用。 实施例 5 蛋白纯化
15 将复性好的蛋白溶液定量后上强阴离子交换柱。
(1)层析柱为 Q-Sepharose (2X2 cm, pharmacia), 用 20 mM Tris-HCl, 1 mM EDTA, pH 9.0平衡， 流速 1 ml/min。 用 1 M NaCl洗脱 30个柱体积并弃用， 将上样流出液 pH调 至 10.0备用。
(2)层析柱为 Q-Sepharose (2X2 cm， ?/ziW w"«'a)， 用 20 mM Tds-HCl， 1 mM EDTA, 20 pH 10.0平衡， 然后用 0.4MNacl， 流速 1 ml/min洗脱 20个柱体积， l MNaCl洗脱 20个 柱体积并分步收集， 考马斯亮兰 G250检测含有目的蛋白的样品。 收集含有蛋白的样品并 用透析液 20 mMTris-HCl， 1 mM EDTA, pH 10.0去盐后， 备用。
(3)将去盐后的样品溶液定量过强阴离子交换柱。 层析柱为预装柱 Mono Q(HR5/5， 1 \, pharmacia),流动相为 LB2 (20mM Tris-HCl, 1 mM EDTA, pH 10.0)和 WB2 (1 M aCl,
25 20 mM Tris-HCl, 1 mM EDTA, pH 10.0)， 流速 1 ml/min。
洗脱方法： 0-100%WB洗脱 30个柱体积并分步收集， 收集处相当于 30%左右处的蛋 白峰。
本发明 OnC-DT2()3— 39o蛋白的表达纯化效果蛋白凝胶图谱见图 2。
30 实施例 6 Onc-DT203-39o蛋白的细胞毒性测定
1. Onc-DT2«3_39()对骨髓瘤细胞 SP2/0的细胞毒性测定
骨髓瘤 SP2/0细胞 (购自中科院上海生命科学研究院细胞库)培养于含 10%小牛血清， 青霉素（100单位 /ml)， 链霉素（100 g/ml)的 RPMI 1640(GIBICO)培养基中。 96孔板培养， 每孔加 90 μΐ培养液 (5X104细胞 /ml)， C02(5%)培养箱中培养 1小时后， 按设定的浓度梯 35 度加蛋白样品 (分别是 One (制备方法参见： onconase 对 B16 黑色素瘤细胞的体内外生长 抑制作用； 沈如凌， 孙瑞林， 王庆诚， 欧伶， 费俭； 细胞生物学杂志， 2007 年 29 卷 6 期： 901-904 )和前述制备的 Onc-DT2Q3.39() (溶于 PBS中;) 10 μ1，对照组加 PBS，培养 72小时后， MTT法检测细胞存活率。
Onc-DT2。3.39。和 One对骨髓瘤细胞 SP2/0的细胞毒性作用比较结果如图 3 A所示。 可 见 Onc-DT203-39o的 IC50约为 2 X 10"8mol/L; One的 IC5。大于 8 X 10"6mol/L,说明 Onc-DT2。3.390 对肿瘤细胞的毒性显然比 One增强约 400倍。
2. One- DT203_390对人白血病细胞 K562的细胞毒性测定
人白血病细胞 K562(购自中科院上海生命科学研究院细胞库;)培养于含 10%小牛血 清， 青霉素（100 单位 /ml)， 链霉素 （100 g/ml)的 DMEM(GIBICO)培养基中。 96孔板培 养， 每孔加 90 μΐ培养液 C5 X 104细胞 /ml)， C02 (；5%)培养箱中培养 12小时后， 按设定的 浓度梯度加蛋白样品 （分别是 One蛋白和 Onc-DT2()3_39()蛋白） 10 μ1， 对照组加 PBS， 培养 72 小时后， MTT法检测细胞存活率。
Onc-DT2。3_39Q和 One对人白血病细胞 K562的细胞毒性作用比较如图 3B所示。 可见 Onc-DT203-39。的 IC5。约为 2.5 X 10-8mol/L; Onc的 IC50约为 1 X l()-6mol/L;说明 Onc-DT203-39。 对肿瘤细胞的毒性比 One增强约 40倍。
3. One- DT2Q3— 39。对人神经母细胞瘤 SH-SY5Y的细胞毒性测定
神经母细胞瘤 SH-SY5Y (购自中科院上海生命科学研究院细胞库；)培养于含 10%胎牛 血清， 青霉素（100 单位 /ml)， 链霉素 （100 g/ml)的 DMEM(GIBICO)培养基中。 96孔板 培养， 每孔加 90 μΐ培养液 （5 X 104细胞 /ml) , C02 (；5%；)培养箱中培养 12小时后， 按设定 的浓度梯度加蛋白样品 (；分别是 One和 Onc-DT2。3_39Q) 10 μ1， 对照组加 PBS , 培养 72 小时 后， ΜΤΤ法检测细胞存活率。
Onc-DT203-39o和 One对人神经母细胞瘤 SH-SY5Y的细胞毒性作用比较如图 3 C所示。 Onc-DT203-39o的 IC5。约为 3 X 10— 7mol/L; One的 IC5Q大于 8 X 10— 6mol/L;说明 Onc-DT2。3.390 对肿瘤细胞的毒性比 One增强约 27倍。
4. Onc-DT2()3_39()对人急性髓系白血病细胞株 HL60的细胞毒性测定
人急性髓系白血病细胞株 HL60 (获自中科院上海生命科学研究院生物化学与细胞所) 培养于含 10%小牛血清，青霉素（100 单位 /ml)，链霉素（100 g/ml)的 RPMI 1640(GIBICO) 培养基中。 96孔板培养， 每孔加 90 μΐ培养液 (5 X 104细胞 /ml)， C02(5%)培养箱中培养 12 小时后，按设定的浓度梯度加蛋白样品 （分别是 ONC和 ΟηοΟΤ2。3.39。)10μ1，对照组加 PBS， 培养 72小时后， MTT法检测细胞存活率。
OnC-DT2。3_39()和 One对人急性髓系白血病细胞株 HL60的细胞毒性作用比较如图 3D 所示。 可见 Onc-DT2。3-39Q的 IC5Q约为 3 X l Q-7mol/L; One 的 IC5。大于 l X l(r5mol/L; OnC-DT2Q3-39()对肿瘤细胞的毒性比 One增强约 33倍。 实施例 7 Onc-DT跨膜结构域融合蛋白变体
为了便于纯化， 本发明人还制备了 N端含有 6His-tag的融合蛋白。 重组载体的转化、 表达同实施例 2-4。
采用实施例 6 的方法对纯化后获得的融合蛋白进行细胞毒性测定， 结果发现带有 His-tag的融合蛋白对肿瘤细胞的毒性接近于前述 Onc-DT2。3_39()。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被单独引 用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员可 以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范 围。

Claims

权 利 要 求
1. 一种融合蛋白， 其特征在于， 所述的融合蛋白包括：
(1) 核糖核酸酶；
(2) 细菌毒蛋白的膜转位结构域；
(3) 位于 (1)和 (2)之间的由 0-50个氨基酸构成的连接肽。
2. 如权利要求 1 所述的融合蛋白， 其特征在于， 所述的连接肽的序列中包括至少 一个胞内蛋白酶的酶切位点序列， 从而在进入胞内后将 (1)和 (2)分离。
3. 如权利要求 2所述的融合蛋白， 其特征在于， 所述的胞内酶选自： Fudn、 基质 金属蛋白酶、 前列腺特有抗原和组织蛋白酶。
4. 如权利要求 1 所述的融合蛋白， 其特征在于， 所述的细胞毒蛋白选自： 白喉毒 素或绿脓杆菌外毒素 A。
5. 如权利要求 4所述的融合蛋白， 其特征在于， 所述的白喉毒素的膜转位结构域 是：
(a) 如 SEQ ID NO: 2中第 121-308位的氨基酸序列的蛋白； 或
(b) 将 (a)所限定的蛋白的氨基酸序列经过一个或多个氨基酸残基的取代、 缺失或添 加而形成的， 且具有 (a) 所限定的蛋白功能的由 衍生的多肽； 和 /或
所述的核糖核酸酶是：
(al) 如 SEQ ID NO: 2中第 1-104位所示的氨基酸序列； 或
(bl) 将 (al)所限定的蛋白的氨基酸序列经过一个或多个氨基酸残基的取代、 缺失或 添加而形成的， 且具有 al) 所限定的蛋白功能的由 (al)衍生的多肽； 和 /或
所述的连接肽具有 SEQ ID NO: 2中第 105-120位所示的氨基酸序列。
6. 一种核酸分子， 其特征在于， 所述的核酸分子编码权利要求 1所述的融合蛋白。
7. 如权利要求 6所述的核酸分子， 其特征在于， 所述的核酸分子具有 SEQ ID NO:
1所示的核苷酸序列。
8. 一种载体， 其特征在于， 它含有权利要求 7所述的核酸分子。
9. 一种基因工程化的细胞， 其特征在于，
所述的细胞含有权利要求 8所述的载体；
或所述的细胞基因组中整合有权利要求 7所述的核酸分子。
10. —种产生权利要求 1所述的融合蛋白的方法， 其特征在于， 所述的方法包括：
(A) 培养权利要求 9所述的宿主细胞， 从而表达权利要求 1所述的融合蛋白；
(B) 分离出所述的融合蛋白。
11. 权利要求 1所述的融合蛋白的用途， 其特征在于， 用于制备抑制肿瘤细胞生长 或治疗肿瘤的组合物。
12. 一种抑制肿瘤细胞生长或治疗肿瘤的组合物，其特征在于，所述的组合物含有： (i) 权利要求 1所述的融合蛋白； 和
(ϋ) 药学上可接受的载体。
13. 一种抑制肿瘤细胞生长或治疗肿瘤的方法， 其特征在于， 所述方法包括： 给予 需要抑制肿瘤细胞生长或治疗肿瘤的对象有效量的权利要求 1所述的融合蛋白。
PCT/CN2009/072368 2008-06-25 2009-06-22 核糖核酸酶与毒蛋白膜转位结构域融合蛋白及其制备方法和用途 WO2009155842A1 (zh)

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